IMPROVEMENT OF A SANDWICH ELISA ASSAY TO … 3-Nucleos(t)ide Probes (109... · improvement of a sandwich elisa assay to enhance quantification capabilities of lna oligonucleotides

IMPROVEMENT OF A SANDWICH ELISA ASSAY TO ENHANCE QUANTIFICATION CAPABILITIES OF LNA OLIGONUCLEOTIDES

Nanna Albæk,1* Jacob Ravn,1 Henrik Frydenlund Hansen,1 Troels Koch,1 Christoph Rosenbohm1

1Santaris Pharma A/S, Kogle Allé 6, DK-2970 Hørsholm, Denmark. * Correspondence to: [email protected]

ABSTRACT

The refinement of an oligonucleotide sandwich ELISA

assay to quantify a specific LNA oligonucleotide has

been accomplished. The improvement of the ELISA as-

say for the specific compound involved synthesis of

modified nucleosides and a new design of the ELISA

probes using the diaminopurine and 2-thiothymine nu-

cleobases.

INTRODUCTION

In the development of drug candidates it is desirable to

evaluate the distribution of the drug in tissue and serum. For

the quantification of LNA oligonucleotides an oligonucleo-

tide sandwich ELISA assay is used. The assay relies on the

selective hybridization of the target oligonucleotide to a set

of capture and detection probes. The probes are Biotin- and

Digoxigenin-labelled full LNA phosphordiester oligonu-

cleotides of approximately half the length of the target oli-

gonucleotide.

In some cases a high background has been observed. This

is most likely due to affinity between the capture and the

detection probe of the specific ELISA assay or due to

selfcomplementarity of the target oligonucleotide resulting

in a poor detection (Figure 1).

Figure 1: A: Oligonucleotide sandwich ELISA assay, B: Selfcomplementarity of the gapmer LNA oligonucleotide or affini-ty between capture and detection probes can give a high back-ground signal.

RESULTS AND DISCUSSION

In order to break the recognition between the capture and

the detection probes 2,6-diaminopurine (D) and 2-

thiothymidine (T2s

) monomers were introduced. This allows

the capture and detection probes to bind to the LNA oli-

gonucleotide but diminishes the affinity between the capture

and detection probes (Figure 2).

Figure 2: Diaminopurine (D) and 2-thiothymine (T2S) recognizes T and A respectively and have a lower affinity towards each other.

We have used both DNA and LNA 2,6-diaminopurine

and 2-thiothymidine monomers to screen sets of capture and

detection probes. The DNA monomers are commercially

available. The LNA monomers have previously been report-

ed in the literature1-3

and they were made via a slightly mod-

ified synthesis route.

A change of ion strength in the buffer systems used for

the assay also added to the development of a more sensitive

assay probably by disturbing the selfcomplementarity of the

LNA oligonucleotide.

CONCLUSION

The modifications of this specific oligonucleotide ELISA

assay resulted in a significant improvement of the detection

limits for the LNA oligonucleotide. The results from this

work provide a larger tool box when developing ELISA

assays for other LNA oligonucleotides.

REFERENCES

1. Koshkin, A. A. J. Org. Chem, 2004, 69, 3711-3718.

2. Rosenbohm, C., Pedersen, D. S., Frieden, M., Jensen,

F.R., Arent, S., Larsen, S., Koch, T. Bioorg. Med.

Chem., 2004, 12, 2385-2396.

3. WO2004024314

109

PHOTOPHYSICAL PROPERTIES OF SINGLE AND DOUBLE STRANDED DNA CONTAINING 6-PHENYLPYRROLOCYTOSINE (PHPC)

Fereshteh Azizi and Masad Damha*

1Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, Canada H3A 0B8

*Correspondence to: [email protected]

ABSTRACT

The effect of surrounding base pairs on the fluores-cent properties of phenylpyrrolocytosine (PhpC) was studied in both single- and double-stranded structures containing all 16 possible neighbouring XY pair combi-nations (i.e., X-PhpC-Y). The data indicate that the nature and orientation of nucleoside neighbours sur-rounding dPhpC affect its fluorescent properties.

INTRODUCTION

The possibility to detect emission at the single molecule

level makes fluorescence one of the most sensitive analyti-

cal techniques available today.[1] In this regard fluorescent

base analogues are particularly interesting. Of the common-

ly used fluorescent nucleoside analogues, those containing

6-Phenylpyrrolocytosine (PhpC)[2] are of interest due to

their ability to form stable Watson-Crick base pairs with

guanine with minimal disturbance to the overall duplex

structure.[2, 3] Due to its red-shifted absorbance, PhpC can

be selectively excited in the presence of natural nucleosides

[3]. Incorporation of PhpC into an oligonucleotide results in

a significant reduction of fluorescence intensity, relative to

the free ribonucleoside [3]. Oligonucleotides containing

PhpC have shown fluorescence quenching upon duplex

formation. These properties, together with its high quantum

yield and sensitivity to its micro environment makes PhpC a

potential site-specific probe for studying nucleic acid struc-

ture and dynamics.

In this study, we exam-

ine the properties of

PhpC when incorporated

into different DNA se-

quence environments.

Specifically, we investi-

gated the effect of sur-

rounding nucleotides on

the quantum yield of

PhpC within 16 single-

and double-stranded

DNA sequences. This library covers all possible nearest

neighbour variations around the PhpC residues (Table 1).


The data indicates that (a) the average fluorescence quan-

tum yield for single-strands is greater than for their corre-

sponding double-strands; (b) in a single strand, a neighbor-

ing adenine causes a significant increase in PhpC fluores-

cent intensity, whereas guanine most effectively quenches

the fluorescence; c) the orientation of neighbors around

PhpC (X-PhpC-Y vs Y-PhpC-X) significantly affects PhpC

quantum yield, e.g. the quenching effect of guanine is high-

er when it is placed at the 3'-side of PhpC.

Table 1. Oligonucleotides synthesized for this study. C = PhpC

CONCLUSION

In conclusion, the fluorescence quantum yield of PhpC

within DNA structures is highly affected by the nature of

nearest neighbours and the orientation of these bases around

PhpC.

REFERENCES

1. Ha,T. Curr. Opin. Struct. Biol. 2001, 11, 287–292.

2. (a) R. H. E. Hudson, A. Ghorbani-Choghamarani, Synlett

2007, 870-873. (b) R. H. E. Hudson, A. K. Dambenieks, R.

D. Viirre, Synlett 2004, 2400-2402. (c) F. Wojciechowski, R.

H. E. Hudson, J. Am. Chem. Soc. 2008, 130, 12574-12575.

3. Wahba, A. S., Esmaeili, A., Damha, M. J., and Hudson,

R. H. E. Nucleic Acids Res. 2010, 38, 1048–1056.

4. Wahba, A. S., Azizi, F., Deleavey, G. F., Brown, C.,

Robert, F., Carrier, M., Kalota, A., Gewirtz, A. M., Pelletier,

J., Hudson, R. H. E., Damha, M. J., ACS Chem. Biol. 2011,

6, 912-919.

110

DNA APTAMER-BASED VIABILITY IMPEDIMETRIC SENSOR FOR VIRUSES

Mahmoud Labib,1 Anna S. Zamay,

1,2 Darija Muharemagic,

1 Alexey V. Chechik,

1 John C. Bell,

3,4 and

Maxim V. Berezovski 1*

1Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5, Canada;

2Institute of Mo-

lecular Medicine and Pathological Biochemistry, Krasnoyarsk State Medical University, 1 P. Zheleznyaka Street, Kras-noyarsk 660022, Russia;

3Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University

of Ottawa, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada; dJennerex Inc., 450 Sansome Street, 16

th floor, San

Francisco, California 94111, USA.

ABSTRACT

The development of Aptamer-based Viability Impedi-

metric Sensor for viruses (AptaVISens-V) is presented.

Highly specific DNA aptamers to intact vaccinia virus

were selected using cell-SELEX technique and integrat-

ed into impedimetric sensors via self-assembly onto a

gold microelectrode. Remarkably, this aptasensor is

highly selective and can successfully detect viable vaccin-

ia virus particles (down to 60 virions in µL) and distin-

guish them from nonviable viruses in a label-free elec-

trochemical assay format. It also opens a new venue for

the development of a variety of viability sensors for de-

tection of many microorganisms and spores.

INTRODUCTION, RESULTS AND DISCUSSION,

CONCLUSION

Standard methods used to assess the presence of micro-

organisms (bacteria and viruses) and to determine whether

they are viable or not involve the use of specific enrichment

media to separate, identify and count viable cells. This pro-

cess is time-consuming and requires bacterial growth media

or cell cultures in case of viruses. Regular spectrophotomet-

ric assays with dye exclusion or metabolic transformation

by live cells cannot be applied on viruses due to their very

small sizes (< 1µm) and compact structures.

In this work [1], DNA aptamers specific to live vaccinia

virus (VACV) were selected based on Cell-SELEX from a

80nt DNA library containing a 40nt random region: 5′-

CTC CTC TGA CTG TAA CCA CG -N40- GC ATA GGT

AGT CCA GAA GCC-3. Taking advantage of successive

rounds of positive and negative selection, we were able to

produce highly specific and selective aptamers that do not

bind to plasma proteins and can distinguish between viable

and nonviable (heat or protease-treated) viruses, for the first

time. These aptamers were further integrated into impedi-

metric aptasensors by self-assembling a hybrid of a thiolated

partial complementary single-stranded DNA and a VACV-

specific aptamer to detect VACV and test its viability, as

shown in Figure 1.

It was observed that the binding between 60 PFU µL−1

of

VACV and the immobilized anti-VACV aptamer pool

caused a decrease in the interfacial resistance (R) of the sen-

sor. This can be attributed to a conformational change in the

aptamers after binding to VACV which allows the external

redox mediator, [Fe(CN)6]3−/4−

, to penetrate more freely to

the electrode surface. Incubation of the aptasensor with a

nonviable heat-treated 60 PFU/µL VACV caused only

13.8% decrease in R. The same number of virus particles

was treated with 1.0 µL of 20 mg/mL proteinase K and

caused a 7.1% increase in R. The selectivity of the aptasen-

sor was tested using 60 PFU/µL vesicular stomatitis virus

(VSV) instead of VACV and it resulted in 11.7% decrease

in R. Furthermore, an aptasensor prepared using the native

DNA library, instead of the anti-VACV aptamer, caused

only a 15% increase in R. This indicates that VACV binds

weakly to the nonspecific DNA, without changing its con-

formation, causing only a small increase in the interfacial

resistance.

This work represents the proof of principle for the first

Aptamer-based Viability Sensor for Viruses (AptaVISens-

V) taking advantage of the tunable specificity of aptamers,

which enables the sensor to distinguish between viable and

nonviable virus. It also opens a new venue for the develop-

ment of a variety of viability sensors for detection of many

microorganisms and spores. This is particularly important in

sterility tests and for validating the efficiency of sterilization.

In addition, the developed aptamers can be used for large

scale purification of VACV since they are significantly

cheaper (~1000 times) and more stable then antibodies.

Moreover, the developed aptamers can be used to enhance

the in vivo survival of the oncolytic VACV by protecting

the virus from neutralizing antibodies.

REFERENCES

1. Labib, M.; Zamay, A.S.; Muharemagic, D.; Chechik,

A.V.; Bell, J.C.; Berezovski, M.V. Analytical Chemis-

try, 2012, in press.

Figure 1. Scheme of AptaVISens-V. (a) An anti-vaccinia

aptamer on a gold microelectrode surface. (b) Binding of the

viable virus to the immobilized aptamer causes a decrease in

impedance. (c) However, binding of the nonviable virus causes

a negligible change in impedance.

111

REACTIVITY STUDIES OF 2,6-DITRIAZOLYLPURINE NUCLEOSIDES WITH

NUCLEOPHILES

Armands Kovaļovs, Irina Novosjolova, Ērika Bizdēna* and Māris Turks

Faculty of Material Science and Applied Chemistry, Riga Technical University, 14/24 Azenes str., Riga LV1007, Latvia. *Correspondence to: Email address [email protected]

ABSTRACT

Reaction of 2,6-ditriazolylpurine nucleosides with nu-

cleophiles is mild and efficient route to C6 derivatization

of purine base. To explore scope and limitations of the

method, we studied reactivity of various N- and S-

nucleophiles as well as kinetics for selected reactions.


CONCLUSION

New methods for the synthesis of C6 purine derivatives have been intensively developed for decades. Search for new anticancer and antiviral agents, adenosine receptors agonists and antagonists prompted an renewed interest in purine chemistry, resulting in numerous synthetic method-ologies [1,2]. Purines are excellent scaffolds for construc-tion of bioprobes. To the best of our knowledge, di-(1,2,3-triazol-1-yl)purines were unexploited for the synthesis of C6 substituted purines.

We used click chemistry for the synthesis of series of 2,6-ditriazolylpurine nucleosides 1 from corresponding 2,6-diazidopurine derivatives. The observation that treatment of 1 with ammonia and amines gave fluorescent compounds, promted us to more detailed investigation of this reaction.

6-Methylamino-2-(1,2,3-triazol-1-yl)purine ribonucleo-

sides were prepared earlier by a different method and inves-tigated as selective adenosine A3 receptor agonists or antag-onists [2].

We report here reactions of 2,6-ditriazolylpurine nucleo-sides 1 with primary and secondary amines and hydrazines. The nucleophilic aromatic substitutions at C6 with amines, such as methyl- and dimethylamine, pyrrolidine, piperidine and other low molecular weight amines proceed smoothly at ambient temperature in water, water-THF or water-MeCN. Reaction times are from 30 min to 2 h. During these reac-tion conditions acetyl protecting groups were simultaneous-ly removed from sugar moiety. Dipropylamine, dibenzyla-

mine, morpholine required longer reaction time and/or ele-vated (40-50 0C) temperatures, and deprotection of sugar was carried out separately with NH3/EtOH or CH3NH2/H2O. All obtained N6-modified 2-triazolylpurine derivatives 2 possess fluorescent properties. The only exception is 6-hydrazino derivative.

Further, we extended investigation to the reactions of amino acid esters as nucleophiles. Reaction of 1 with pro-line methyl ester is rather slow, however, we obtained prod-uct with bright blue fluorescence in UV light. Reactivity studies of 1 with amino acids are still in progress.

A number of 6-thioalkylated purines have interesting bio-logical activity. For example, 6-mercaptopurine ribonucleo-side is inhibitor of de novo purine biosynthesis.

To explore suitability of 2,6-ditriazolpurines as substrates for the synthesis of 6-thioalkylated purine nucleosides, we investigated reactions of 1 with thiols. Dodecanethiol was chosen for reaction kinetics studies. 6-Dodecylthio-2-triazolylpurine ribonucleoside 3 was obtained in 60-65% yield.

In conclusion, we have demonstrated the versatility of

2,6-ditriazolylpurines as reactive intermediates suitable for C6 modifications.

REFERENCES

1. a) Lagisetty, P., Russon, L.M., Lakshman, M.K. Angew.Chem. Int.Ed., 2006, 45, 3660-3663 and refer-ences therein; b) Guo, H-M., Wu, J., Niu, H-Y., Wang, D.C., Zhang, F., Qu, G-R. Bioorg.Med.Chem.Lett., 2010, 20, 3098-3102; c) Veliz, E.A., Beal, P. J.Org.Chem., 2001, 66, 8592-8598; d) Lakshman, M. K., Choudhury, A., Bae, S., Rochttis, E., Pradhan, P., Kumar, A. Eur. J. Org. Chem., 2009, 152-159.

2. Cosyn, L., Palaniappan, K.K., Kim, S-K., Duong, H.T., Gao, Z-G., Jacobson, K.A., Van Calenbergh, S. J. Med.

Chem. 2006, 49, 7373-7383.

112

ELABORATION OF NEW NITROGEN MUSTARDS ANALOGUES

Benjamin Boëns,* Tan-Sothea Ouk, Rachida Zerrouki.

Laboratoire de Chimie des Substances Naturelles, EA1069, 123, Avenue Albert Thomas, 87060, Limoges, France

* Correspondence to: [email protected]

ABSTRACT

This presentation describes the syntheses of new alkylat-

ing agents, whose the design is based on click chemistry.

INTRODUCTION

Nitrogen mustards, such as chlorambucil, melphalan or

cyclophosphamide, are still among the most useful clinical

agents in anticancer treatments. Their alkylating activity

relies on the bis(2-chloroethyl)amine function, which con-

duces to the formation of an interstrand cross-linking in

DNA. Although these drugs are efficient against cancer cells,

their toxicity towards healthy tissues is a major issue, bring-

ing a lot of harmful and well-known side effects. Expecting

to decrease this high toxicity, we have chosen to design

some new alkylating agents, based on the “click” chemistry

concept. We chose a simple synthetic pathway to provide

various nitrogen mustards (Scheme 1).

Scheme 1. Strategy of synthesis.


First, we synthesized the N-propargyl-diethanolamine (1),

key compound in this strategy. Diethanolamine was reacted

with propargyl bromide in presence of K2CO3 in DMF at

80 °C. The monopropargylated compound was obtained in

66% yield. This synthesis has proved to be efficient on a

multigram scale (up to 11 g).

“Click” reactions were then conducted using five azide

compounds (Scheme 2).

Scheme 2. Azide compounds used in click chemistry.

Benzylazide is a commercial compound and the other

ones were synthesized according to the literature.

The CuAAC reaction was conducted under microwave ir-

radiations and “click” compounds were obtained in good

yields (Table 1).

Table 1. Results with click reactions.

Conditions: a) NaAsc (0.1 equiv), CuSO4,5H2O (0.01 equiv), H2O/tBuOH (1/1; v/v; 6

mL), ratio alkyne:azide, 0.95:1, MW 3 min, 80 °C, 300W ; b) SOCl2 (3 equiv), Pyrdine

(3 equiv), CHCl3, US activation 1h30.

Chlorination were then proceeded in chloroform with

pyridine (3 equiv) and thionyl chloride (3 equiv) with ultra-

sonic activation and provided final nitrogen mustards in

good yields (Scheme 3).

Scheme 3. Final nitrogen mustards.

Cytotoxicity assays (MTT) were then conducted on vari-

ous cancer cell lines: JURKAT (T cell lymphoma), K562

(chronic myeloid leukemia), U266 (myeloma) and A431

(vulvar epidermoid carcinoma).

RN3 2 3 4 5 6

Yield of CuAACa 78% 100% 71% 78% 64%

Yield of chlorinationb 61% 50% 57% 76% 47%

113

mailto:[email protected]

PYRROLOCYTIDINE AS A VERSATILE SCAFFOLD FOR FLUOROPHORE DEVELOPMENT

Kirby Chicas,1* Robert H.E. Hudson

1

1Department of Chemistry, The University of Western Ontario, London, Ontario, CANADA N6A 5B7


ABSTRACT

Pyrrolocytidine (pC) nucleoside analogues have prov-

en utility as highly fluorescent base discriminating fluor-

ophores. Herein, we describe the effect of polyaromatic

hydrocarbon (PAH) substitution at the 6-position of the

pyrrole on the base discriminating nature of pC. We

also explore the limits of substituent size and describe

the development of new applications for the pC scaffold.

We also report a new C analogue outside of the pC

framework: 5-(pyren-1-ylethynyl)cytidine.

INTRODUCTION

Recently, the Hudson group has shown the utility of 6-

phenylpyrrolocytidine as a fluorescent reporter group for

nucleic acid structures. When incorporated into oligonucleo-

tides, PhpC is able to fluorimetrically report the state of hy-

bridization and able to discriminate between matched and

mismatched targets [1],[2]. The phenyl and cytidine moie-

ties alone are nonfluorescent, but together they act as a

fluorophore, hence PhpC is intrinsically fluorescent. This

property gives PhpC the ability to communicate changes in

the state of hybridization and changes in the local environ-

ment of the nucleobase. To vary the fluorescence properties

of the cytidine analogues, the nature of the aromatic group

on the pyrrole ring can be changed (Figure 1).

Figure 1. - Cytidine analogues of interest


To this effect, pyrrolocytidine analogues modified with

either fluorene (FlpC) or pyrene (PypC) were prepared. The

pyrrolocytidines were accessed by one of two synthetic

routes utilizing either 5-iodocytidine [3] or 5-iodouridine [1].

The 5-iodocytidine route may be conveniently modified to

prepare the 5-ethynylpyrene derivative (PyEthpC) as well

[4]. The fluorescence of the nucleosides has been evaluated

(Table 1):

Table 1 – Fluorophore Efficiencies

Nucleoside Quantum Yield

(EtOH)

Quantum Yield

(Water)

FlpC ND ND

PypC 0.60 0.018

PyEtpC 0.55 0.003

Pyrene is well known for its desirably high quantum

yield (0.65) and fluorescence lifetime in EtOH at 293 K [2].

Pyrene has furthermore been known to have dramatic solva-

tochromatic properties. Fluorene is also well known for its

high quantum yields and excellent wavelength-tunability.

Photophysical characterization of the pyrene modified C

analogues has shown favourable quantum yields, high molar

absorptivities, and striking solvatochromicity.

Our studies have shown not only favourable photophysi-

cal properties of the free nucleosides, but dramatic solvato-

chromatic sensitivity allowing entry into quencher free mo-

lecular beacons (MBs). Furthermore, an advantage of this

design is the possible use of the excimer / monomer emis-

sion of pyrene-labelled nucleosides to prepare positive re-

sponse MBs [6].

CONCLUSION

The facile synthesis and unique photophysical properties

of the pyrene and fluorene C analogues permit us to readily

access to simple MB platforms for application in studying

nucleic acid interactions.

REFERENCES

1. Hudson, R. H. E.; Ghorbani-Changhamarani, A.; Syn-

lett, 2007, 870-873

2. Wahba, A. S.; Esmaeili, A.; Damha, M. J.; Hudson, R.

H. E. Nucleic Acids Res. 2010, 38 (3), 1048-1056.

3. Wojciechowski, F.; Hudson, R. H. E. Org. Lett. 2009,

11 (21), 4878-4881.

4. Hudson, R. H. E.; Dambenieks, A. K.; Viirre, R. D.;

Synlett, 2004, 2400 – 2402.

5. Valeur, B., Molecular Fluorescence: Principles and

Applications. Valeur. B., Wiley-VCH: New York, 2002,

p. 200.

6. Seo, Y. J., Hwang, G. T., Kim, B. H. Tetrahedron Lett.

2006, 47, 4037–4039.

114

HOMO-DNA AS A REPORTING UNIT FOR SNP DETECTION

Camille Désiron 1*, Matthias Stoop

1 and Christian J. Leumann

1

1 Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.


ABSTRACT

Homo-DNA is a DNA homolog that can form anti-

parallel duplexes with itself but does not pair with natu-

ral nucleic acids. Those properties have been used to

design a new tool for single nucleotide polymorphism

detection on DNA and RNA targets with high sensitivity.

INTRODUCTION

Although homo-DNA is composed of a six-membered

ring sugar moiety (Figure 1) it can still form Watson-Crick

and reverse-Hoogsteen duplexes in an anti-parallel fashion

with itself. [1] However, it has been shown that homo-DNA

does not cross pair with natural nucleic acids. [1, 2]

In the context of single nucleotide polymorphism (SNP)

detection two key events are necessary: the sensing of the

mismatch and the generation of a signal. For example, the

annealing of a probe to a molecular beacon induces a fluo-

rescent signal due to the opening of its stem part that is

equipped with a fluorophore-quencher couple. [3] In order

to enhance sensitivity, templated chemistry has been used

by several research groups. [4, 5]


In a previous study we showed that homo-DNA can be

used as a template for the bioorthogonal Staudinger reduc-

tion of azides (Figure 2). [6] Based on the particular base-

pairing properties of homo-DNA, we decided to split the

sensing activity from the reporting activity by using chimer-

ic DNA:homo-DNA oligomers (Figure 3). The DNA part of

the chimera serves as sensing unit while the homo-DNA

part is used as reporting unit. Signal generation is achieved

when the complementary strands of homo-DNA - each bear-

ing either a rhodamine azide or a triphenylphosphine unit -

base-pair to the chimeric units bound to the target (Figure 3).

Indeed, we were able to show that the presence of the target

is a prerequisite for the appearance of fluorescence and that

mismatch discrimination was possible.

CONCLUSION

We successfully applied this assay to detect SNP in DNA

and RNA targets. Fluorescence enhancement allowed live

monitoring of the reaction and showed a good mismatch

discrimination at room temperature.

REFERENCES

1. Hunziker, J., Roth, H. J., Bohringer, M., Giger, A.,

Diederichsen, U. Gobel, M. Krishnan, R., Jaun, B.

Leumann, C., Eschenmoser, A. Helv. Chim.Acta., 1993,

76, 259-352.

2. Crey-Desbiolles, C., Ahn, D.-R., Leumann, C. J. Nucl.

Acids Res., 2005, 33, e77.

3. Tyagi, S., Kramer, F. R. Nat. Biotechnol., 1996, 14,

303-308.

4. Grossmann, T. N., Seitz O. J. Am. Chem. Soc., 2006,

128, 15596-15597.

5. Pianowski, Z., Gorska, K., Oswald, L., Merten, C. A.,

Winssinger, N. J. Am. Chem. Soc., 2009, 131, 6492-

6497.

6. Stoop, M., Leumann, C. J. Chem. Commun., 2011, 47,

7494-7496.

Figure 1. DNA and homo-DNA backbone comparison

Target

Rhodamine azide

TPP

Rhodamine

TPP oxide

DNA / RNA

DNA

Homo-DNA

Figure 3. Design of the fluorescent SNP detection assay; TPP: triphenylphosphine.

Figure 2. Staudinger reduction of a rhodamine azide by a tri-phenylphosphine.

115

SYNTHESIS AND FLUORESCENT PROPERTIES OF BENZOPYRROLOCYTIDINE

NUCLEOSIDES: A NOVEL INTRINSICALLY FLUORESCENT TRICYCLIC ANALOGUE

Adam A.H. Elmehriki,1*

Mojimr Suchy1 and Robert H.E. Hudson

1


*Correspondence to: <[email protected]>

ABSTRACT

A novel intrinsically fluoroesent cytidine analogue has

been prepared. The fused tricyclic structure of the ben-

zopyrrolocytosine moiety, described herein through the

synthesis of benzopyrrolocytidine (benzopC) and deox-

ybenzopyrrolocytidine (deoxybenzopC). BenzopC is

structurally similar to both phenylpyrrolocytosine

(PhpC)1 and phenoxazine (tCO)

2, two highly fluorescent

cytosine analogues. The notable photophysical proper-

ties, demonstrated by both the benzopC and deoxyben-

zopC, suggest that this tri-cyclic system represents a

highly promising parent structure for modification.

INTRODUCTION

The tricylic cytosine analogue, phenoxazine, has found

utility in nucleic acid chemistry as a bright fluorescence

label. Inspired by this, we sought to prepare an analogue of

6-phenylpyrrolocytosine in which the phenyl group was

fused to the pyrrole ring, thus providing a tricyclic structure

similar to tCo whilst retaining some structural similarity to

PhpC. This modification retains the Watson-Crick face

while extending the aromatic system of the parent pC and

potentially yielding new and useful fluorescent properties,

Figure 1.


We have established a synthetic methodology for the

preparation of benzopC and deoxybenzopC from uridine

and deoxyuridine, respectively. The notable florescent prop-

erties, including high quantum yields (Φ = 0.76, 0.84 in

EtOH), large Stokes shift (123 nm) and reasonable molar

absorptivity (ε = 6543, 7231 M-1

cm-1

, at 322 nm) have

prompted investigation into their incorporation into oligo-

nucleotides.

Figure 1. Structure of PhpC (1), tCO (2), deoxy(benzopC) (3)

and benzopC (4).

Figure 2. Fluoresence spectrum of 3’,5’-O-diacetyldeoxybenzopC: excitation spectrum (black line); emission spectrum (grey line).

CONCLUSION

The synthesis of benzopC and deoxybenzopC nucleoside

analogues has been achieved and their photophysical prop-

erties described.

REFERENCES

1. (a)Hudson, R. H. E.; Dambenieks, A. K.; Viirre, R. D.

Synlett 2004, 13, 2400–2402. (b) Hudson, R. H. E.;

Ghorbani-Choghamarani, A. Synlett, 2007, 6, 870–873.

2. Lin, K. Y.; Matteucci, M. D. J. Am. Chem. Soc. 1998,

120, 8531-8532.

116

Investigation of Ire1p endonuclease activity in the S. cerevisiae unfolded protein response using a fluorescent oligonucleotide.

Andrew Frazer,1* John Zhao,2 James Tucker,2 David Timson3 and Joseph Vyle1

1School of Chemistry and Chemical Engineering, Queen's University Belfast, David Keir Building, Stranmillis Road,

Belfast BT9 5AG, Northern Ireland, United Kingdom, 2 School of Chemistry, The University of Birmingham, Edgbaston,

Birmingham B15 2TT, United Kingdom and 3School of Biological Sciences, Queen’s University Belfast, Medical Biolo-

gy Centre, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland, United Kingdom. * Correspondence to:[email protected]

ABSTRACT

The unfolded protein response (UPR) is a eukaryotic stress response that mediates the response to unfavoura-ble protein folding conditions in the endoplasmic reticu-lum (ER). In Saccharomyces cerevisiae Ire1p is the main signal transduction protein in the UPR. Here we de-scribe the development of a fluorescence-based approach to the in vivo monitoring of Ire1p activity.

The Saccharomyces cerevisiae inositol-requiring enzyme

1 (Ire1p) is an ER transmembrane signalling protein that

monitors the protein folding environment in the ER in a

pathway known as the unfolded protein response. Misfold-

ed proteins in the lumen of the ER interact with the luminal

domain of Ire1p to promote oligomerisation of the protein.

The cytoplasmic domain of Ire1p contains a Ser/Thr kinase

and a kinase extension nuclease that possesses endonuclease

activity. Oligomerisation of Ire1p initiates signal transduc-

tion by allowing the cytoplasmic domain kinase to trans-

autophosporylate its neighbouring subunit. Phosphorylation

subsequently activates the endonuclease activity of the pro-

tein.1

Upon activation, the endonuclease activity of Ire1p

leads to the splicing of an intron from the Hac1 pre-mRNA,

lifting the translational repression caused by the long-range

interaction between the intron and the 5'-UTR.2 The Ire1p

cytoplasmic domain targets splice sites in the Hac1 pre-

mRNA that are both located within the loop of a stem-loop

secondary structure and contain the consensus motif 5'-

CXGXXGX-3'.3 Following ligation of the exons, the ma-

ture Hac1 mRNA is translated, generating a bZip transcrip-

tion factor (Hac1p) that binds to the UPR elements present

in the promoter regions of the target genes. The genes that

are under the regulation of Hac1p encode a wide range of

proteins, including chaperones and those involved in ER-

associated protein degradation, cell growth and differentia-

tion.4

In order to monitor the activation of Ire1p directly

in vivo, we have developed an approach that utilises a fluo-

rescently-tagged short oligonucleotide analogue of the target

Hac1 pre-mRNA stem-loop. The fluorescent probe has a

similar design to that of a molecular beacon, with a 3'-

quencher moiety positioned proximally to a 5'-fluorophore

(fig. 1). Upon splicing by Ire1p, the fluorophore is liberat-

ed from the quencher and a fluorescence signal is emitted.

This approach has been used previously in in vitro to inves-

tigate small molecule activators and inhibitors of Ire1p and

its homologues.5,6

This in vivo approach will allow for the

investigation of UPR activation under a wide range of cellu-

lar stress conditions.

In this poster we will describe the optimisation of

the transfection of yeast with the short oligonucleotide

probe through the analysis of several different techniques,

alongside complementary in vitro biochemical studies in-

vestigating Ire1p activation.

Figure 1. Activation of the fluorescent probe by the endonuclease

activity of Ire1p.

REFERENCES

1. Ron, D., Walter, P. Nature Rev, 2007, 8, 519-29.

2. Ruegsegger, U., Leber, J. H. and Walter, P. Cell, 2001,

107, 103-14.

3. Kawahara I., Haruta K., Kojima C., Tanaka Y. Nucleic

Acids Symp Ser, 2009, 53, 269-70.

4. Mori K., Ogawa N., Kawahara T., Yanagi H., Yura T.B.

J Biol Chem, 1998, 273, 9912-20.

5. Wiseman, R.L., Zhang, Y., Lee, K.P.K., et al. Mol Cell,

2010, 38, 291-304.

6. Volkmann, K., Lucas, J.L., Vuga, D., et al. J Biol Chem,

2011, 286, 12743-55.

117

SELECTION OF DNA APTAMERS FOR OXIDIZED GUANINE LESIONS

Pranjali Ghude* and Cynthia J. Burrows

Department of Chemistry, The University of Utah, Salt Lake City, Utah 84112, United States.

* Correspondence: [email protected]

ABSTRACT

Selection of a DNA aptamer for the hydantoin lesions

by an in vitro method is described herein. The biotinyl-

ated guanine lesions, especially 8-oxo-7,8-dihydro-2’-

deoxyguanosine (8-oxo-dG) and spiroiminodihydantoin

(dSp), are synthesized for immobilization on streptavidin

affinity matrix. The ssDNA pool is generated with N25

randomized regions as a starting point for selection.


CONCLUSION

Guanine being the most readily oxidized nucleobase, un-

dergoes two electron oxidation to form 8-oxo-guanine (8-

oxo-G). This 8-oxo-G undergoes further oxidization to form

spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh),1

that are highly mutagenic hydantoin lesions.2,3

Figure 1. Oxidation of guanine to mutagenic lesions

Detection of oxidized lesions is essential to study the de-

velopment of human diseases. These lesions are formed in

cells because of oxidative stress. Most base oxidation prod-

ucts are detected by nuclease digestion followed by LC-MS.

However, these lesions are not good substrates for nuclease

digestion, and generate a large number of dA, dC, dT and

dG nucleosides, as compared to small number of modified

nucleosides. Hence, nucleic acid aptamers are an alternative

approach, as they benefit from higher chemical stability.

Hydantoin lesions are biotinylated at the 5’-hydroxyl po-

sition of the deoxyribonucleosides or at the N2 positions of

the nucleobase. Coupling of these lesions to biotin aids in its

attachment to the streptavidin affinity matrix. The end prod-

ucts of the synthesis are purified and characterized by LC-

MS. A 65-mer single-stranded DNA pool with N25 random

Figure 2. Synthetic scheme for biotinylation of hydantoin lesions.

ized positions is used as a starting point for selection. The

chemically synthesized pool is PCR amplified and the ssD-

NA is generated by lambda exonuclease digestion of the 5’-

phospohorylated antisense strand. The ssDNA pool is 12%

denaturing PAGE purified and extracted.

SELEX experiments are currently being performed on

these oxidized lesions. Ultimately, these aptamers could

help identify hydantoins formed in cell extracts, either in

intact DNA, in fragments or as products of base excision

repair.

REFERENCES

1. Luo, W.; Muller, J. G.; Rachlin, E.; Burrows, C. J. Org.

Lett. 2000, 2, 613-616.

2. Henderson, P. T.; Delaney, J. C.; Muller, J. G.; Neeley,

W. L.; Tannenbaum, S. R.; Burrows, C. J.; Essigmann, J. M.

Biochemistry 2003, 42, 9257-9262.

3. Ghude, P.; Schallenberger, M. A.; Fleming, A.

M.; Muller, J. G.; Burrows, C. J.; Inorganica Chimica Acta

2011, 369, 240-246.

118

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A CONFORMATIONALLY UNAMBIGUOUS ISOINDOLINE-DERIVED EPR PROBE FOR

DISTANCE MEASUREMENTS IN NUCLEIC ACIDS

Dnyaneshwar B. Gophane,1* Snorri Th. Sigurdsson

1

1Science Institute, University of Iceland, Dunhaga 3, 107 Reykjavik, Iceland. * Correspondence to: [email protected]

ABSTRACT

A conformationally unambiguous isoindoline-derived

nitroxide spin label (Im

T) was prepared and incorpo-

rated into oliogonucleotides. The spin label moiety is

attached to a uracil base by a single bond which lies on

the same axis as the nitroxide bond. Thus, rotation

around the single bond does not cause a displacement of

the nitroxide relative to the nucleic acid.


CONCLUSION

The knowledge of the molecular structure and conforma-

tional dynamics of biopolymers are important in understand-

ing the function of DNA, RNA and proteins. X-ray crystal-

lography, high resolution NMR spectroscopy [1] and in

some cases fluorescence resonance energy transfer (FRET)

[2] have been used for distance determination in DNA.

Though such techniques can provide valuable structural

information, they still have some drawbacks. For example

NMR is restricted by the size of the molecule and provides

mostly short range distance constraints. X-ray crystallog-

raphy is dependent on the availability of well diffracting

crystals. FRET can be used to measure long-range distances,

but has limited accuracy.

EPR spectroscopy has been increasingly used to investi-

gate the organizational and dynamic properties of DNA,

using persistent nitroxide spin probes [3]. Spin labels that

are conjugated to biopolymers with a tether that has some

flexibility decreases the accuracy of the EPR-based distance

measurements between labels. In contrast, rigid spin labels,

like Ç (Figure 1) do not move independently of the biopol-

ymer to which they are attached [4]. The complete rigidity

of Ç makes is advantageous for the determination orienta-

tions of structural elements in nucleic acid [5]. However, for

simple distance measurements one needs to deconvolute the

orientational effect of Ç, which currently requires non-

trivial analysis.

We report here a conformationally unambiguous [6] iso-

indoline-derived nitroxide spin label (Im

T, Figure 1). Alt-

hough there is rotation around the single bond attaching the

imidazole to the base, its rotation is around an axis which

goes through the N-O bond of the nitroxide. Therefore, rota-

tion around the single bond does not cause a displacement

of the nitroxide relative to the nucleic acid, making this a

promising probe for determination of accurate distances

within oligonucleotides.

Figure 1. Rigid nucleosides Ç (a) and ImT (b) and base pairing of the latter with A (c).

The phosphoramidite of the spin-labelled nucleoside Im

T

was prepared and used for incorporation of Im

T into DNA

by solid-phase synthesis. The incorporation of Im

T was con-

firmed by MALDI-Tof, enzymatic digestion and EPR spec-

troscopy. The EPR spectra of DNA duplexes containing Im

T

were very broad, indicating limited mobility of the probe in

DNA. Comparing the EPR spectra of four duplexes, in

which Im

T was paired with either A, T, G or C, yielded four

distinguishable spectra. The spin label that paired with A

had the lowest mobility while the Im

T-T pair had the highest

mobility. Addition of Hg2+

ions, that are known to form

metallo base-pairs in T-T mismatch is yielded an EPR spec-

trum similar to the Im

T-A duplex.

REFERENCES

1. Lukin,M., De los Santos,C. Chem. Rev. 2006, 106,

607–686.

2. Norman,D.G., Grainger,R.J., Uhrin,D., Lilley,D.M.J.

Biochemistry, 2000, 39, 6317–6324.

3. Shelke, S.A., Sigurdsson, S.T. Eur. J. of Org. Chem.

2012, 12, 2291-2301.

4. Barhate, N., Cekan, P., Massey, A. P., Sigurdsson, S. T.

Angew. Chem. Int. Ed. 2007, 46, 2655-2658.

5. Schiemann, O., Cekan, P., Margraf, D., Prisner, T.F.

and Sigurdsson, S.T. Angew. Chem. Int. Ed. 2009, 48,

3292-3295.

6. Sajid, M., Jeschke, G., Wiebcke, M. and Godt, A.

Chemistry - European Journal, 2009, 15, 12960-12962.

119

SINGLE LABELLED DNA FIT-PROBES FOR QPCR AND RNA DETECTION IN CELL MEDIA

Felix Hövelmann,1 Lucas Bethge

1 and Oliver Seitz

1*

1Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, * Correspondence to: Email address

[email protected]

ABSTRACT

We developed fluorogenic oligonucleotide probes

which contain a single ‘thiazole orange base’ as reporter

dye. The so-called DNA FIT-probes facilitate the

detection of DNA and RNA in complex biological

samples.

INTRODUCTION

Fluorogenic hybridization probes enable the real-time

detection of DNA during PCR and allow the imaging of

RNA in biological samples like cell lysate or live cells.

Conventional probes such as the Molecular Beacons rely on

the distant-dependent interaction of two chromophores.

Fluorescence signalling is observed not only upon

hybridization but any change in distance like unspecific

binding or degradation. We have introduced the single

labelled PNA FIT-probes, in which the intercalator dye

thiazole orange (TO) occupies the position of a ‘canonical’

nucleobase. [1]

The ‘TO base’ serves as a local probe, which

reports hybridization by increases of TO emission. PNA

FIT-probes have been successfully used in qPCR analysis

and in the imaging of viral mRNA in live infected cells.[2]

However, PNA probes are, at current; more costly than

DNA probes. Furthermore, PNA is not adapted to molecular

biology methods commonly used to modify DNA. To

overcome these disadvantages we set out to develop DNA

FIT-probes.

Figure 1. Concept of forced intercalation DNA-probes.


Previous studies on PNA-based FIT-probes have shown the

critical influence of the linker that connects the ‘TO base’

with the PNA scaffold. A hybridization induced turn-on of

TO emission can only be achieved when very short linkers

are used. In the development of DNA-based FIT-probes we

explored the acyclic serinol-TO (1L) and a new, bioisosteric

carbacyclic TO-nucleoside (1).[3]

We found that the duplex

stability is not significantly disturbed by the introduction of

the TO-nucleotides. Most importantly, DNA FIT-probes

provide enhancements of TO emission upon hybridization

with both complementary DNA and RNA (Fig. 2A). Exper-

iments with cell lysates revealed that DNA FIT-probes

overcome two major drawbacks pertinent to molecular bea-

con probes: 1. false positive signaling caused by degrada-

tion; 2. false positive signaling due to unspecific binding in

cell media. To enable the simultaneous, yet spectrally re-

solved detection of two different RNA targets we developed

a BO-containing nucleoside. The combined use of TO and

BO probes allowed the simultaneous, sequence specific de-

tection of two different RNA-targets (Fig. 2B).

Figure 2. A) Fluorescence spectra of 1L-TO labelled FIT-probe

(ex. 485 nm). B) Simultaneous fluorescence readout of BO and TO

probes in cell lysate upon addition of neuraminidase RNA (7min)

and actin RNA (14 min).

For sequence specific DNA detection during real-time

PCR we introduced a 3’-cap on DNA FIT-probes. The

probes did not perturb the PCR process as inferred by the up

to 91% PCR-efficiency. A H1N1-neuraminidase-specific

DNA FIT-probe enabled the sensitive and unambiguous

detection of viral transcripts in cell lysate over a linear range

of at least 7 orders of magnitude.

REFERENCES

1. O. Köhler, D. V. Jarikote, and O. Seitz, Chembiochem,

2005, 6, 69-77.

2. a) S. Kummer, A. Knoll, E. Socher, L. Bethge, A.

Herrmann, and O. Seitz, Angew. Chem. Int. Ed., 2011,

50, 1931-1934.; b) A. G. Torres, M. M. Fabani, E.

Vigorito, D. Williams, N. Al-Obaidi, F. Wojciechowski,

R. H. E. Hudson, O. Seitz, and M. J. Gait, Nucleic

Acids Res., 2012, 40, 2152-2167.

3. L. Bethge, I. Singh, and O. Seitz, Org. & Biomol.

Chem., 2010, 8, 2439-2448.

120

ISOLATION OF NEW DNA APTAMERS FOR PANCREATIC CANCER CELLS

Mamoru Hyodo,1* Mst Naznin Ara1, Yusuke Takaya1 and Hideyoshi Harashima1,2

1Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-Ku, Sapporo, Japan and 2 Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences,

Hokkaido University, Kita 12, Nishi 6, Kita-Ku, Sapporo, Japan. * Correspondence to: Email address for [email protected]

ABSTRACT

We applied cell-SELEX method to pancreatic cancer cells to generate DNA aptamers. We completed the selec-tion and identified sequences and confirmed that one of these aptamers showed good affinity and selectivity to pancreatic cells.

INTRODUCTION, RESULTS AND DISCUSSION, CONCLUSION

Pancreatic cancer ranked fourth among cancer-caused death. It is difficult to detect at early stages and 5-years sur-vival rate of pancreatic cancer is less than 5% [1]. For the treatment of pancreatic cancer, the device to detect and tar-get pancreatic cancer cells is highly demanded. Aptamers are new class of molecules comparable to antibody in terms of its binding affinity and selectivity. Aptamers were firstly reported by Ellington and Turck group, indipendently [2,3]. Aptamers have the potential for the use of diagnosis, therapy, biomarker identification, biosensor and the ligand for drug delivery system.

We applied cell-SELEX method that was explored by Tan group [4]. Cell-SELEX is the method to investigate aptamers using cells directly as a target. It has beneficial points about the representation of membrane proteins as a native form and the identification of new biomarkers. For this research, we selected PANC-1 cells as the model of pancreatic cancer cells. We employed one modification through this selection, the use of temperature-detachable culture dishes named RepCell available from CellSeed. It coated with special polymer and could detach cells by cool-ing dishes. Compared to trypsin, we could reduce the risk to degrade cell surface proteins.

Figure 1. Schematic representation of cell-based SELEX.

After 5 rounds of selection, random ssDNA pool was ap-plied for cloning, transformed to E. coli and sequenced each random sequence. We picked some sequence up and checked the binding affinity with PANC-1 cells via flow cytometry. Fortunately, one of the sequence 'PANC-20' showed good binding affinity. Next, we tried the binding assay of PANC-20 aptamer with NIH3T3 which is the nega-tive target cell in our selection and we could show that it didn't bind.

Figure 2. Binding assay of PNAC-aptamer 20 to PANC-

1 cell. Red: non treated, Green: 0 cycle library, Blue: PANC-aptamer 20.

We have investigated aptamers to pancreatic cancer cells

via cell-SELEX method. We employed the temperature-detachable culture dishes to keep cell surface proteins intact. We applied 5 rounds of selection and checked the enrich-ment of binding affinity. We confirmed the binding affinity and specificity of PNAC-20 aptamer to PANC-1 and other cells.

REFERENCES

1. Hidalgo. M. N. Engl. J. Med., 2010, 362, 1065-1078.

2. Ellington, A. D., Szostak, J. W. Nature, 1990, 346, 818-822.

3. Tuerk, C., Gold, L. Science, 1990, 249, 505-510.

4. Shagguan, D., Li, Y., Tang, Z., Cao, Z. C., Chen, H. W., Mallikaratchy, P., Sefah, K., Yang, C. J., Tan, W. Proc. Natl. Acad. Sci. USA, 2006, 103, 11838-11848.

121

FUNCTIONALIZED MRNA CAP ANALOGUES AND THEIR CONJUGATES – MOLECULAR

TOOLS FOR INVESTIGATION OF PROTEINS INVOLVED IN MRNA METABOLISM

Marcin Warminski, Zofia Tomasiewicz, Krystian Ubych, Joanna Kowalska, Edward Darzynkiewicz, Jacek Jemielity*

Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland *Correspondence to: Email address [email protected]

ABSTRACT

Synthetic mRNA cap analogues functionalized with an

amino- or carboxy- group attached via linker to various

positions of the dinucleoside 5’, 5’ triphosphate structure

have been synthesized. An efficient method for their con-

jugation with biotin, fluorescent dyes, or nanoparticles is

also presented. The resulting conjugates have been de-

signed for investigation of several gene expression pro-

cesses, in which the mRNA cap structure is involved.

INTRODUCTION,

The 5’ end of eukaryotic mRNA is modified by a

distinctive structure called 5’ cap. It consists of 7-

methylguanine attached by an unusual 5’,5’-triphosphate

linkage to the first transcribed nucleotide. In cells cap is

bound by numerous cap-binding proteins and thus plays an

important role in a variety of cellular processes associated

with mRNA metabolism and regulation of gene expression,

including maturation, transport, degradation and initiation of

translation. Thus synthetic mRNA cap analogues are invalu-

able tools for investigation of cap dependent processes [1].


We synthesized a series of cap analogues containing

linkers varying in length and hydrophobicity and functional-

ized with either amine or carboxylic group. Some of them

have been additionally modified in the triphosphate bridge

with either bisphosphonate or imidodiphosphate moiety to

stabilize them against specific cap related pyrophosphatases

[2-4]. The analogues are suitable for labelling with biotin,

fluorophores and for covalent binding to macromolecules

and nanomaterials. Hence, they are intended to serve as pre-

cursors of versatile tools for studying mRNA fate in the

cells, localization of cap molecules in vivo or for medical

diagnostic applications.

The linkers are attached at one of three different po-

sitions: 2’/3’-OH groups of 7-methylguanosine (m7Guo),

2’/3’-OH groups of second nucleoside (Guo) via carbamate

moiety or N6 exocyclic amine group of adenosine as a se-

cond nucleoside (Ado). The selection of those modification

positions is based on the finding that their effect on associa-

tion with particular cap-binding proteins is generally slight.

The analogs containing an amino group have been labelled

with biotin, carboxyfluorescein or carboxy-X-rhodamine.

For this purposes we developed a method for efficient con-

jugation of in situ generated NHS active esters of appropri-

ate labels with aliphatic amino functionalized dinucleotide

cap. We believe that the adenine-labelled derivatives are

especially useful for studies on Decapping Scavenger

(DcpS), whereas the m7G-carbamates should be appropria-

ble for studying interactions between cap and eukaryotic

Initiation Factor 4E (eIF4E), crucial for cap-dependent initi-

ation of translation and hence for the total rate of protein

synthesis. The latter analogues are incorporated into mRNA

during transcription in vitro, and hence, pave the way for

obtaining variously 5’ end labelled, yet biologically active

mRNAs. Linkers attached at the ribose of the second nucle-

oside enabled us to prepare conjugates with macromolecules

and nanomaterials without affecting association with cap-

binding proteins to a significant extent. We believe that

modified caps and their conjugates described herein will

benefit a wide range of biotechnological applications.

REFERENCES

1. Jemielity, J., Kowalska, J., Rydzik, A.M.

Darzynkiewicz, E. New J. Chem., 2010, 34, 829-844.

2. Kalek, M., Jemielity, J., Darzynkiewicz, Z.M., Bojarska,

E., Stepinski, J., Stolarski, R., Davis, R.E.,

Darzynkiewicz, E.; Bioorg. Med. Chem. 2006, 14,

3223-3230.

3. Rydzik, A.M., Kulis, M., Lukaszewicz, M., Kowalska,

J, Zuberek, J., Darzynkiewicz, Z.M., Darzynkiewicz, E.,

Jemielity, J. Bioorg. Med. Chem. 2012, 20, 1699-1710.

4. Su, W., Slepenkov, S., Grudzien-Nogalska, E.,

Kowalska, J., Kulis, M., Zuberek, J., Lukaszewicz, M.,

Darzynkiewicz, E., Jemielity, J., Rhoads, R.E. RNA

2011, 17, 978-988.

n=1, 3 or 5

linkers =

X=Y=O, CH2 or NH

linker

linker linker

= dyes, biotin, nanoparticle, protein

m=1, 2 or 5

Figure 1. General structure of mRNA cap analogue conjugates

122

FLUORESCENCE TURN-ON SENSOR FOR TRIPLEX FORMATION UTILIZING BENZOFURAN-MODIFIED PYRIMIDINES

Takashi Kanamori1, Hiroki Ohzeki

2, Hirosuke Tsunoda

2, Akihiro Ohkubo

2, Mitsuo Sekine

1,2* and

Kohji Seio2*

1Educational Academy of Computational Life Sciences, Tokyo Institute of Technology and

2Department of Life Science,

Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama, 226-8501, Japan. *Correspondence to: Email address [email protected], [email protected]

ABSTRACT

Benzofuran derivative-modified deoxycytidines (dCBF

)

and deoxyuridines (dUBFs

) were synthesized and their

fluorescence properties were studied. We incorporated

them into triplex DNAs aiming for development of turn-

on sensor for indicating triplex formation. It was found

that the intensity of fluorescence drastically enhanced

when duplex having UAr

formed triplex with TFO which

contained abasic site as a pair for UAr

.

INTRODUCTION

We have studied the triplex formation between DNA du-

plex containing 5-aryl modified deoxycytidine (CAr

) and

TFO containing propylene-linker (C3) as a partner for CAr

.

Especially, triplex DNA incorporating G-PPIR•C3 triad

1a,b

(Fig.1) could form thermally stable triplex due to high

stacking interaction.

Interestingly, it was observed that fluorescence intensity

derived from PPIR moderately increased upon triplex for-

mation. Inspired by this observation, we tried to develop

fluorescence turn-on sensor, which sensitively indicates

triplex formation. This fluorescence enhancement of PPI

seemed to be due to solvatochromic effect. In order to de-

velop more sensitive fluorescent sensor, we focused on con-

formational change of chromospheres. Seits and co-

workers2a,b

previously reported fluorescent PNA probes

(FIT probes) using thiazole orange as a base surrogate. Sri-

bastsan3a

and Tor3b

reported furan or benzofuran-modified

pyrimidines. These examples show that conformationally

flexible fluorophores intercalated into nucleobases and

fluorophores located coplanar, then fluorescence strongly

increases. Therefore, to develop turn-on fluorescent sensor,

we tried to induce conformational change upon triplex for-

mation.


For such purposes, modified nucleoside dCBF

and dUBFs

were synthesized and incorporated into duplex DNAs

(Fig.2). We introduced substituents such as methyl group

into the 3-position of benzofuran ring expecting methyl

group twists benzofuran ring and uracil ring.

We measured the fluorescence spectra of triplex DNAs

incorporating these fluorescence nucleosides and it was

turned out that in the case of dUBFs

, fluorescence drastically

enhanced upon triplex formation.

CONCLUSION

We could achieve the development of fluorescence turn-

on sensor, which sensitively indicates triplex formation.

Further researches utilizing this technic are undergoing aim-

ing for powerful biological sensors.

REFERENCES

1. (a) Mizuta, M., Banba, J., Kanamori, T., Tawarada, R.,

Ohkubo, A., Sekine, M., Seio, K., J. Am. Chem. Soc.,

2008, 130, 9622-9623. (b) Kanamori, T., Masaki, Y.,

Mizuta, M., Tsunoda, H., Ohkubo. A., Sekine, M., Seio,

K., Org. Biomol. Chem., 2012, 10, 1007-1013.

2. (a) Seitz, O., Bergmann, F., Heindl, D., Angew. Chem.

Int. Ed. 1999, 39, 2203-2206. (b) Kummer, S., Knoll,

A., Socher, E., Bethge, L., Herrmann, A., Seitz, O., An-

gew. Chem. Int. Ed., 2011, 50, 1931-1934.

3. (a) Tanpure, A.A., Srivatsan, S.G., Chem. Eur. J., 2011,

17, 12820-12827. (b) Sinkeldam, R.W., Wheat, A.J.,

Boyaci, H., Tor, Y., Chemphyschem, 2011, 12, 567-570.

Figure 2. Chemical structures of fluorescent pyrimidine deriva-

tives used in this study.

Figure 1. Structure of G-PPIR•C3 triad.

123

QUENCHER FREE MOLECULAR BEACON DESIGN BASED ON PYRENE-UNA EXCIMER

FLUORESCENCE EMMISION

Kasper K. Karlsen* and Jesper Wengel

Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark. *Correspondence to: [email protected]

ABSTRACT

A pyrene-UNA (monomer X, Figure 1) modified ON,

capable of generating pyrene excimer fluorescence when

single stranded, was used in the development of a

quencher-free molecular beacon (QF-MB). Based on

explorations concerning the optimal placement and

number of incorporations of monomer X, molecular

beacons was synthesized, and their ability to detect a

target was investigated.


CONCLUSION

Recently, we discovered the occurrence of pyrene

excimer formation in a 21-mer DNA oligonucleotide (ON)

modified with three pyrene labeled 2´-piperazino UNA

monomers (monomer X, Figure 1).[1]

Interestingly, no

excimer emission was present for when ONX was

hybridized to its complementary DNA strand (Figure 1). We

have investigated the possibility of applying this unforeseen

feature of ONX in the construction of a novel molecular

beacon (MB), as will be described in the following.

Figure 1. Steady-state fluorescence spectra of single strand and

duplex ONX: 5´-TGCACXGTAXGTCTGXACCAT.

MBs were introduced in 1996 by Tyagi and Kramer as

nucleic acid hairpin structures used for detection of nucleic

acid sequences.[2]

Classical MBs are ONs with terminal

conjugations of a fluorophore and a quencher, one at each

end of the strand.[3]

Two drawbacks are associated with this

construction, one being increased background noise or false

positive signals upon degradation, and another being that

further terminal functionalization is very difficult. In this

light, quencher-free molecular beacons (QF-MBs) have

emerged, and as the name indicates, the QF-MBs contains

no quencher and therefore are often not associated with the

same drawback as classical MBs.[4]

Two pyrene molecules, one being in an exited state and one

in the ground state, are able to form a complex called an

excimer.[5]

The relaxation of pyrene excimer is accompanied

by fluorescence emission with a λmax around 480 nm and a

relatively long fluorescence lifetime (30-60 ns) compared to

the autofluorescence of cellular extracts (~7 ns).[6]

Pyrene

excimer fluorescence can therefore be selectively detected

in biological assays if fluorescence is measured after the

cellular autofluorescence has decayed. Due to these

photophysical properties, pyrene excimer emission is a

valuable tool within diagnostic applications, and therefore

we decided to investigate the possibilities of creating a QF-

MB based on ONX (Figure 2).

The pyrene excimer forming sequence was studied with

respect to the number and specific placement of the pyrene-

UNA modification X. We found that the excimer formation

was sequence dependent, and thus decided to use the

original sequence (ONX) as part of the stem sequence for

new QF-MB (UNA-MB). As a target for the UNA-MB,

swine origin influenza A virus (H1N1) was chosen, since

this target is both highly relevant and the sequence had

previously been detected by a MB as reported by Ge et al.[7]

The current version of UNA-MB can detect the H1N1 target

sequence with a ~7-fold fluorescence intensity increase at

492 nm, but we hope to improve this by further modifying

the construction of the MB.

Figure 2. Schematic representation of the UNA-MB based on

the sequence of ONX.

REFERENCES

1. Karlsen, K. K., Pasternak, A., Jensen, T. B., Wengel,

J., ChemBioChem 2012, 13, 590-601.

2. Tyagi, S., Kramer, F. R., Nat. Biotechnol. 1996, 14,

303-308.

3. Wang, K. M., Tang, Z. W., Yang, C. Y. J., Kim, Y.

M., Fang, X. H., Li, W., Wu, Y. R., Medley, C. D.,

Cao, Z. H., Li, J., Colon, P., Lin, H., Tan, W. H.,

Angew. Chem. Int. Ed. 2009, 48, 856-870.

4. Venkatesan, N., Seo, Y. J., Kim, B. H., Chem. Soc.

Rev. 2008, 37, 648-663.

5. Winnik, F. M., Chem. Rev. 1993, 93, 587-614.

6. Kolpashchikov, D. M., Chem. Rev. 2010, 110, 4709-

4723.

7. Ge, Y. Y., Cui, L. B., Qi, X., Shan, J., Shan, Y. F., Qi,

Y. H., Wu, B., Wang, H., Shi, Z. Y., J. Virol. Methods

2010, 163, 495-497.

124

HIGH-AFFINITY DNA-TARGETING USING SIMPLE MIMICS OF N2’-INTERCALATOR-FUNCTIONALIZED 2’-AMINO-α-L-LNA

Saswata Karmakar,1* Dale C. Guenther,

1 Sujay P. Sau,

1 Brooke A. Anderson,

1 Rie L. Rathje,

1 Sanne

Andersen1 and Patrick J. Hrdlicka

1

1Department of Chemistry, University of Idaho, PO Box 442343, Moscow, ID 83844-2343, USA. * Correspondence to:

[email protected]

ABSTRACT

Synthetic routes toward a series of N2’-intercalator-

functionalized 2’-amino-α-L-LNA mimics have been de-

veloped, and hybridization properties of ONs modified

with these simple monomers were studied. Several of the

monomers display extraordinary thermal affinity to-

ward complementary DNA and were subsequently ex-

plored as “Invader LNA” mimics for mixed-sequence

targeting of double-stranded DNA (dsDNA) using elec-

trophoretic mobility shift assays.


CONCLUSION

N2’-Pyrene-functionalized 2’-amino-α-L-LNAs (locked

nucleic acids) display extraordinary affinity toward com-

plementary DNA targets (ΔTm up to + 20 °C per modifica-

tion) due to favorable preorganization of the pyrene moie-

ties for hybridization-induced intercalation [1]. These build-

ing blocks have been utilized to a) develop probes for detec-

tion of single nucleotide polymorphisms (SNPs) [2], b) gen-

erate pyrene arrays in duplex cores [3] and c) develop novel

strategies for sequence-unrestricted targeting of double

stranded DNA (i.e., the Invader LNA approach) [4]. Their

synthesis is, unfortunately, very challenging (∼20 steps,

<3% overall yield), which has prevented full exploration of

these applications. Access to synthetically more readily ac-

cessible structural and functional mimics would accordingly

be very desirable.

Here we describe synthetic routes to and hybridization

characteristics of a series of O2’-intercalator-functionalized

uridine (Y-Z) and N2’-intercalator-functionalized 2’-N-

methyl-2’-aminouridine monomers (Q-V), which we sur-

mised to be likely mimics of N2’-pyrene functionalized 2’-

amino-α-L-LNA (Fig. 1) [5].

These specific targets were selected as they allowed us to

systematically evaluate the influence of linker chemistry

(monomer Q vs S), linker length (monomer S vs V) and

aromatic surface area (monomer Y vs Z) on DNA-

hybridization properties. Our study revealed that several of

these monomers indeed are close functional mimics of N2’-

intercalator-functionalized 2’-amino-α-L-LNA benchmark

monomer L. Next, we evaluated these monomers as poten-

tial “Invader LNA” mimics for mixed-sequence targeting of

double-stranded DNA (dsDNA). Toward this end, we have

developed an electrophoretic assay where Invaders are tar-

geted toward double-stranded stem regions of model stem-

loop DNA targets. Several easily accessible and potent In-

vader LNA mimics were identified during the course of this

study. Moreover, valuable insight into the design of opti-

mized Invader probes has been gained. Invaders were com-

pared at a single concentration (200-fold excess of 34 nM

target) to obtain a kinetic profile (time course) and to deter-

mine the concentration dependence of Invader-mediated

dsDNA-recognition (KD). The invasion potential increases

progressively with the number of “energetic hotspots” in

Invaders. For example, Invader probes containing four se-

quential hotspots possess a KD of ~340nM, which means

that 50% dsDNA recognition occurs at that concentration.

In conclusion, synthetically easily accessible mimics of

N2’-Pyrene-functionalized 2’-amino-α-L-LNAs have been

identified, which allows for full exploration of the “Invader

approach” toward sequence-unrestricted targeting of dsDNA.

We expect this to pave the way for enabling applications in

fundamental research, diagnostics, therapeutics, and materi-

als science.

REFERENCES

1. Kumar, T. S., Madsen, A. S., Østergaard, M. E., Sau, S.

P., Wengel, J., Hrdlicka, P. J. J. Org. Chem., 2009, 74,

1070-1081.

2. Kumar, T. S., Wengel, J., Hrdlicka, P. J. ChemBioChem.

2007, 8, 1122-1125.

3. Kumar, T. S., Madsen, A. S., Østergaard, M. E.,

Wengel, J., Hrdlicka, P. J. J. Org. Chem. 2008, 73,

7060-7066.

4. Sau, S. P., Kumar, T. S., Hrdlicka, P. J. Org. Biomol.

Chem., 2010, 8, 2028-2036.

5. Karmakar, S., Anderson, B. A., Rathje, R. L., Andersen,

S., Jensen, T., Nielsen, P., Hrdlicka, P. J. J. Org. Chem.,

2011, 76, 7119-7131.

Figure 1. Benchmark monomer L, O2’-intercalator-functionalized uridine monomers Y/Z and N2’-intercalator-functionalized 2'-N- methyl-2'-aminouridine monomers Q/S/V.

125

Synthesis and hybridization properties of oligonucleotides modified with 5-(1-aryl-1,2,3-triazol-4-yl)-2’-deoxyuridines

Mamta Kaura1* and Patrick J. Hrdlicka

1

1Department of Chemistry, University of Idaho, PO Box 442343, Moscow, ID 83844-2343, USA.* Correspondence to:

[email protected]

ABSTRACT

The hybridization properties of oligonucleotides modi-

fied with three different 5-(1-aryl-1,2,3-triazol-4-yl)-2’-

deoxyuridine monomers - differing in the size of the ar-

omatic unit on the 1-position of the triazole ring – are

reported. Napthyl and pyrenyl units appear to be too

large to facilitate efficient aromatic stacking in the ma-

jor groove of nucleic acid duplexes.


CONCLUSION

The use of nucleic acids to generate self-assembling

chromophore arrays is an area of high current interest,

which is fuelled by the promise for materials with unique

photophysical properties [1]. Oligonucleotides (ONs) modi-

fied with C5-functionalized pyrimidine monomers have

been studied in particular detail toward this end [2], as

chromophores point into the spacious major groove upon

duplex formation. Traditionally, the chromophore has been

attached directly to the nucleobase or via an alkynyl linker,

but other linker strategies are emerging. The Nielsen group

recently demonstrated that ONs modified with consecutive

5-(1-phenyl-1,2,3-triazol-4-yl)-2’-deoxyuridines display

interesting RNA hybridization characteristics, presumably

due to formation of aromatic arrays in the major groove [3].

While the influence of phenyl substituents on aromatic

stacking has been studied [3, 4], there are no reports on how

the size of the aromatic units influence stacking efficiency

and hybridization characteristics. To address this shortcom-

ing, we synthesized a small series of C5-functionalized 2’-

deoxyuridines that carry three different aromatic units on

the 1-position of the triazole ring (Fig. 1), incorporated the-

se monomers into ONs and studied their hybridization prop-

erties with matched/mismatched DNA/RNA targets.

Monomer X, R = phen-1-ylMonomer Y, R = napth-1-ylMonomer Z, R = pyren-1-yl

O

PO O-

1

N

NH

O

O

NNNR

OO

Figure 1. C5-functionalized triazole-linked 2’-deoxyuridines.

The corresponding phosphoramidites of target monomers

X-Z were obtained via a short synthetic route that features a

CuI catalyzed [3+2] azide-alkyne cycloaddition reaction as

the key step. The building blocks were incorporated one,

two or four times into a 9-mer ON via machine-assisted

solid-phase oligonucleotide synthesis; B1: 5’-d(GTG TBT

TGC; B2: 5’-d(GTG TBB TGC); B3: 5’-d(GTG BBB BGC).

Thermal denaturation temperatures (Tm’s) of duplexes be-

tween modified ONs and complementary DNA/RNA targets

were determined at high ionic strength.

Singly modified ONs display markedly lower thermal af-

finity toward complementary DNA or RNA than unmodi-

fied reference duplexes, especially if modified with mono-

mers carrying large aryl groups. As previously reported [3],

sequential incorporation of two or four X monomers com-

pletely reverts this trend and leads to highly stabilized du-

plexes with complementary RNA in particular (ΔTm = +

4 °C/modification). Interestingly, X3 displays the most effi-

cient thermal discrimination of mismatched RNA targets. A

similar, albeit less pronounced trend, is observed upon se-

quential incorporation of Y monomers, which carry the

larger napth-1-yl unit. Duplexes with two or four sequential

incorporations of monomer Z [5], did not display any transi-

tions. To conclude, the bulkier napthyl and pyrenyl units of

monomers Y and Z appear to be too large (or hydrophobic)

to facilitate aromatic stacking in the major groove of nucleic

acid duplexes.

REFERENCES 1. Kashida, H., Asanuma, H. Phys. Chem. Chem. Phys.,

2012, 14, 7196-7204

2. Nguyen, N.T., Brewer, A., Stulz, E. Angew. Chem. Int.

Ed. 2009, 48, 1974-1977

3. Andersen, N. K., Chandak, N., Brulikova, L., Kumar,

P., Jensen, M. D.; Jensen, F.; Sharma, P. K., Nielsen, P.

Bioorg. Med. Chem. 2010, 18, 4702-4710.

4. Kumar, P., Chandak, N., Neilson, P., Sharma, P.K.

Bioorg. Med. Chem. 2012, doi:

10.1016/j.bmc.2012.04.036

5. Østergaard, M.E., Guenther, D.C., Kumar, P., Baral, B.,

Deobald, L., Paszczynski, A.J., Sharma, P.K., Hrdlicka,

P.J. Chem. Commun., 2010, 46, 4929-4931

126

CHARGE TRANSFER DYNAMICS IN DNA AT THE SINGLE MOLECULE LEVEL

Kiyohiko Kawai,1* Atsushi Maruyama,

2 Tetsuro Majima

1*

11The Institute of Scientific and Industrial Research (SANKEN), Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-

0047, Japan, 2Institute for Materials Chemistry and Engineering, Kyushu University, Motooka 744-CE11, Nishi-ku, Fu-kuoka 819-0395, Japan. [email protected], [email protected]

ABSTRACT

Charge separation, charge transfer, and charge re-

combination process in DNA was monitored by blinking

of the fluorescence emitted from each single fluorophore.

INTRODUCTION

Photo-induced charge-transfer quenching results in the

formation of a non-emissive radical-ion state of the fluoro-

phore and charge injection into a bio-molecule. An injected

charge can migrate along the biomolecule, and subsequent

charge-recombination makes the reaction reversible. The

charge-migration dynamics along the biomolecule are re-

flected in a change in the lifetime of the charge separated

state (), thus the measurement of would provide us with

additional unique and fruitful information around the fluo-

rescent dye. However, the is usually determined by the

transient absorption measurement, which is labor-intensive

and requires a significant amount of sample (>1 nmol); thus

it is essentially incompatible with live-cell imaging and

high-throughput applications. Looking at each single fluor-

ophore, charge-separation and -recombination causes a

blinking of the fluorescence, and here the duration of the

dark state “off time” is supposed to correlate with the (Fig.

1). In this study, we clearly demonstrated that charge-

separation and -recombination dynamics in DNA in the time

range of microsecond to tens of microseconds can be probed

by fluorescence correlation spectroscopy (FCS).

Figure 1. A schematic representation for charge separation, charge migration, and charge recombination in DNA.


ATTO 655 (ATTO) was selected as a fluorophore to

measure the charge-transfer dynamics in DNA by FCS. One

G was replaced with deazaguanine (Z) to behave as a posi-

tive-charge trap. Charge-recombination dynamics were in-

vestigated by conventional transient absorption measure-

ment as well as FCS. In addition to the diffusion component,

an additional relaxation process was observed by FCS (FCS)

which correlated well with the measured from the transient

absorption measurements (TA) and was attributed to the

on/off dynamics of the single ATTO due to the formation of

the non-emissive charge-separated state. FCS varied reflect-

ing the DNA sequence and the presence of a mismatch

which allows the read-out of DNA sequence information

including data on single-nucleotide polymorphisms (SNPs).

The present method enables the automatic measurement and

analysis of more than 100 samples within 1 h using less than

10 fmol (0.5 nM 20 L) of sample, which is over 100,000

times less than that typically required for transient absorp-

tion measurements. This makes possible the high-

throughput screening needed in DNA diagnosis.

CONCLUSION

The present report clearly demonstrated that single-

molecule-level fluorescent measurement is a powerful tool

for examining the charge-transfer dynamics in the time

range of microsecond to milliseconds. Since FCS can be

measured in living cells, the investigation of the charge-

transfer dynamics may provide unique information around

the fluorescent dye in living cells.

REFERENCES

1. Kawai, K., Hayashi, M., Majima, T. J. Am. Chem. Soc.

2012, 134, 4806-4811.

2. Kawai, K., Matsutani, E., Maruyama, A., Majima, T. J.

Am. Chem. Soc. 2011, 133, 15568-15577.

3. Kawai, K., Kodera, H., Majima, T. J. Am. Chem. Soc.

2010, 132, 14216-14220.

4. Kawai, K., Kodera, H., Majima, T. J. Am. Chem. Soc.

2010, 132, 627-630.

5. Kawai, K., Kodera, H., Osakada, Y., Majima, T. Nature

Chem. 2009, 1, 156-159.

Off-time

FCS

~

3'

X

ATTO 655

Chargeseparation

Charge migration& recombination

ATTO 655

•

hn

Single-molecule-levelfluorescencemeasurement

•+

•+

•+

on!

off

on!

~5'

127

SULFUR-CONTAINING PHOSPHONATE MONOMERS FOR OLIGONUCLEOTIDE SYNTHESIS

Ondřej Kostov, Eva Zborníková and Ivan Rosenberg *

Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, 166 10, Prague 6, Czech Republic. * Correspondence to: Email address [email protected]

ABSTRACT

A series of novel compounds, suitably protected (i)

nucleoside-5'-S-methylphosphonates and (ii) nucleoside-

-5'-O-methylphosphonothioates, was prepared as

monomers for solid phase synthesis of modified

oligonucleotides. In addition we have examined the

synthetic potential of nucleoside-5'-O-methyl-(H)-

phosphinates for phosphonate oligonucleotide assembly.


CONCLUSION

A partial replacement of the phosphodiester C3'-O-P-O-

-C5´ bonds in oligodeoxynucleotides with the isopolar

nonisosteric phosphonate C3'-O-P-C-O-C5´ internucleotide

linkage led, surprizingly, to the significant increasing the

hybridization ability with a complementary RNA strand.1,2

Furthermore, the hybrid duplex containing DNA strand

modified with C3'-O-P-C-O-C5´ bonds is capable to elicit

RNase H activity.3 Moreover, the introduction of this type

of phosphonate linkage into 2',5' oligoadenylates gave rise

potent agonists of RNase L.4

These findings prompted us to start a study on the

synthesis and usefulness of the suitably protected

nucleoside-5'-S-methylphosphonates,5 5'-O-

methylphosphono-thioates, and 5'-O-methyl-(H)-

phosphinates. The replacement of the 5'-bridging and P=O

non-bridging oxygen atoms with more nucleophilic, bulky

and lipophilic sulfur atom was expected to influence

significantly the physico-chemical and biological properties

of the corresponding oligonucleotides. We developed and optimized two methods for synthesis

of the target nucleoside 5´-S-methylphosphonates 1

(Scheme 1). The reaction of 2 with synthon 3b gave the

monomer 1 directly. The synthesis of oligonucleotides using

monomer 1 was realized only by the phosphotriester chem-

istry because this method does not use the oxidation step

which led to the formation of S-oxides. Nevertheless also

these sulfoxides and sulfons are in focus of our attention.

To introduce the phosphonothioate C3'-O-P(=S)-C-O-

-C5´ linkage into the modified oligonucleotides, we elabo-

rated synthesis of (H)-phosphinate synthon 6 (starting from

tosylate 7 according to the procedure in ref.6). The alkyla-

tion of 8 provided smoothly desired nucleoside-5'-O-

methyl-(H)-phosphinate 5 (Scheme 2). This key synthon

was examined as (i) monomer for solid phase synthesis of

oligonucleotides using H-phosphonate chemistry, and (ii) an

intermediate for the transformation into phosphonoamidite

monomers applicable in the phosphoramidite chemistry.

Scheme 2: (i) a. LiAlH4/TMSCl, THF, -78 °C to r.t., b. H2O2, H2O/THF; (ii) NaH, DMF, -40 °C to r.t.

In conclusion, the synthesis of new nucleoside phospho-

nate monomers suitable for the introduction of two types of

sulfur-containing isopolar, non-isosteric phosphonate-based

internucleotide linkages has been elaborated.

Support by the grant 202/09/0193 (Czech Science Foun-

dation), Research center KAN200520801 (Acad. Sci. CR)

under the Institute research project RVO: 61388963, is

gratefully acknowledged.

REFERENCES

1. Rosenberg,I. (2004) In Frontiers in Nucleosides and

Nucleic Acids; Schinazi,R.F. and Liotta, D.C. (eds.);

IHL Press: Tucker, GA, pp. 519–548.

2. Rosenberg Ivan et al. Manuscript in preparation

3. Šípová Hana et al Manuscript in preparation

4. Páv, O., Panova, N., Snášel, J., Zborníková, E., Rosen-

berg, I. Bioorganic & Medicinal Chemistry Letters,

2012, 22, 181–185.

5. Kóšiová, I., Buděšínský, M., Panova, N., Rosenberg, I.

Org. Biomol. Chem., 2011, 9, 2856-2860

6. Barral, K., Priet, S., Céline, M., Sire, J., Neyts, J., Bal-

zarini, J., Canard, B., Alvarez, K., European J. of Med.

Chem., 2010, 45, 849-856.

Scheme 1: (i) tBuOK, DMF, argon; (ii) 4-methoxy-1-oxido- -2-pyridylmethanol, MSNT, AcCN; (iii) 60% aq. pyridine

128

SYNTHESIS OF NUCLEOTIDES BEARING FLUOROPHOSPHATE MOIETY AND THEIR NON-HYDROLYZABLE ANALOGS - USEFUL PROBES FOR NMR STUDIES

Joanna Kowalska, Marek R. Baranowski, Renata Kasprzyk, Agnieszka Osowniak, Jacek Jemielity

Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, Warsaw, Poland, e-mail: [email protected]

ABSTRACT

A comprehensive method for the synthesis of nucleo-

side mono-, di-, tri- and teraphosphates bearing a termi-

nal fluorophosphate moiety and their non-hydrolyzable

analogs is reported.

INTRODUCTION

Fluorophosphate analogs of nucleotides are known for their

use in studying substrate properties of various phosphotrans-

ferases and phosphohydrolases. Replacing one of the oxy-

gen atoms within the phosphate moiety by fluorine elimi-

nates the negative charge, but the produced analogue, due to

the small size and high electronegativity of fluorine, may

putatively still be involved in hydrophilic interactions im-

portant for biomolecules.1

Hence, nucleoside fluorophos-

phates have been identified as unnatural enzyme substrates,

receptor agonists, and, in the cases they proved to be enzy-

matically resistant, as enzymatic inhibitors. This, in combi-

nation with the fact that fluorine atom allows selective and

sensitive studies by means of 19

F NMR spectroscopy, makes

nucleoside fluorophosphates an interesting target for effi-

cient chemical synthesis.


Here, we report a method enabling straightforward access

to fluorophosphate analogs of nucleoside mono-, di- and tri-

and even tetraphosphates (Fig. 1). The method consists of

three complementary synthetic approaches, each based on

phosphorimidazolide chemistry. First approach, which is

particularly useful for the synthesis of nucleoside fluo-

romonophosphates, but could be also applied for fluorodi-

phosphates and their analogs, employs substituting the im-

idazole leaving group of an activated nucleotide with fluo-

ride anion (Fig. 1A). Second approach, applicable mainly

for the synthesis of nucleoside β-fluorodiphosphates and γ-

fluorotriphosphates, encompasses coupling an appropriate

nucleoside phosphorimidazolide with triethylammonium

monofluorophosphate (MPF) as an nucleophile (Fig. 1B).

The third approach is particularly useful for the synthesis of

either highly polar nucleotides (e.g. tetraphosphate analogs)

or those bearing a modified oligophosphate bridge (Fig. 3C).

In this case a “reverse” strategy has been employed, in

which the fluorophosphate unit is activated as an electro-

phile and an appropriate nucleotide is used as an nucleo-

philic agent. In each case the pyrophosphate bond formation

is highly accelerated by the presence of excess divalent met-

al chloride, either ZnCl2 or MgCl2.

Based on the presented methodology we have synthe-

sized, with good to excellent yields, over 20 different nucle-

oside (oligo)phosphates bearing terminal fluorophosphate

moiety. Some of these nucleotides were additionally modi-

fied by the phosphorothioate, boranophosphate, meth-

ylenebis(phosphonate) or imidodiphosphate groups. Alt-

hough, syntheses of some fluorophosphate nucleotides have

been reported earlier,1,2

this, to our knowledge, is the first

attempt at a comprehensive synthetic method that would

provide straightforward access to a variety of modified

fluorophosphate nucleotides. Several of the synthesized

compounds are reported for the first time, including a new

class of nucleoside diphosphate analogs, namely fluoro-

[methylenebis(phosphonate)] nucleosides, and yields for the

known compounds have been notably improved. The syn-

thesized compounds have the potential to become useful

probes for studying enzymatic phosphoryl transfer processes,

spy molecules for pyrophosphatase inhibitor screening and

many others by means of 19

F NMR. Some initial results on

their application for studying protein nucleotide interactions

and following pyrophosphatase mediated reactions will also

be presented.

REFERENCES

1. Bollmark, M., Stawinski, J., Nucleos Nucleot Nucleic

Acids, 2006, 663-680

2. Stumber, M., et al. FEBS J., 2002, 3270-3278

n=0, 1, 2 or 3X = O, CH2 or NH Y = O, S, BH3

Method I

Method II

Method III

TBAFZnCl2

DMSO

A

MCl2

C

B

DMF

MCl2

DMF

Figure 1. Overview on the synthesis of nucleoside oligophos-phate analogs bearing fluorophosphate moiety.

129

2'-BISPYRENE-LABELED OLIGO(2'-O-METHYLRIBONUCLEOTIDE) PROBES AS USEFUL

TOOLS FOR RNA DETECTION

Krasheninina Olga,1,2,*

Novopashina Darya,1 Venyaminova Alya

1

1 Institute of Chemical Biology and Fundamental Medicine SB RAS, acad. Lavrentiev ave. 8, 630090, Novosibirsk, Russia and

2Novosibirsk State University, Pirogova str. 2, 630090, Novosibirsk, Russia * Correspondence to: okrash-

[email protected]

ABSTRACT

Series of novel 2'-bispyrene-labeled oligo(2'-O-

methylribonucleotides) (19 nt) which contained one in-

sertion of 2'-bispyrenylmethylphosphorodiamidate de-

rivative of ribonucleoside (A, C, G or U) and “inverted”

thymidine at the 3'-end were synthesized. The properties

of conjugates as fluorescent probes for RNA detection

were investigated.

INTRODUCTION, RESULTS AND DISCUSSION

At present, the most appropriate techniques for identifica-

tion of sequence, structure, quantity and function of NAs are

various fluorescent assays. An application of these tech-

niques implies the use of fluorescent-labeled probes which

exhibit hybridization-induced changes in fluorescence spec-

tra, possess high affinity to target NAs and nuclease re-

sistance. Here we present a new type of 2'-bispyrene-labeled

oligo(2'-O-methylribonucleotides) as useful tools for fluo-

rescent detection of RNA.

Series of novel 2'-bispyrene-labeled oligo(2'-O-

methylribonucleotides) (19 nt) were synthesized as de-

scribed in [1]. The conjugates contained one insertion of 2'-

bispyrenylmethylphosphorodiamidate derivative of ribonu-

cleoside (A, C, G or U) within the chain and “inverted”

thymidine at the 3'-end (Fig 1). The sequences of probes

were complementary to two accessible regions of MDR1

mRNA. The structures of 2'-bispyrene-modified probes

were confirmed by MALDI TOF mass-spectrometry, UV-

and fluorescent spectroscopy. Thermal stability of duplexes

of probes with model NA targets corresponding to frag-

ments 113-137 (ON1-ON8) and 315-336 (ON9-ON16)

MDR1 mRNA was investigated by thermal denaturation

method. Steady state fluorescence emission spectra of the

2'-bispyrene-modified probes and their duplexes with corre-

sponding NA targets were recorded and analyzed.

Fluorescence emission quantum yields of probes and their

duplexes with model short RNAs were determined. The

hybridization of the most sensitive probes to model short

RNA/DNA targets and 5'-terminal fragment of MDR1

mRNA (nucleotides 1-678) was investigated by fluores-

cence-monitored titration with an increasing target concen-

tration.

We found that the properties of the new probes varied

depending on position of 2'-bispyrene-modified nucleoside

within the chain. The UV melting studies indicate that the

conjugates obtained form stable complexes with the model

short RNA/DNA. The fluorescence spectra of the probes

displayed significant increase of eximer fluorescence inten-

sity (480 nm) upon binding with both complementary short

and extended (678-mer fragment of MDR1 mRNA) RNA

targets (Fig 2).

The results obtained in this study indicate that the 2'-

bispyrene-labeled oligo(2'-O-methylribonucleotide) probes

can be useful tools for detection of specific RNA.

REFERENCES 1. Krasheninina, O.A., Novopashina, D.S. Venyaminova,

A.G. Russ. J. Bioorg. Chem., 2011, 37, 273-277.

Fig. 2 (a) Schematic representation of the 2’-bispyrene-labeled

probes principle of action. (b) Fluorescence emission spectra

of single stranded ON2 (curve 1) and the duplexes with com-

plementary short DNA (curve 2) and RNA targets (curve 3)

and extended 678 nt RNA target (curve 4).

Fig. 1 Structure and sequences of 2’-bispyrene-labeled probes.

(a)

(b)

130

TRITYL RADICALS AS SPIN LABELS FOR NANOMETER DISTANCE MEASUREMENTS

Nitin C. Kunjir1*, Gunnar W. Reginsson

2, Olav Schiemann

2 and Snorri Th. Sigurdsson

1

1Science Institute, University of Iceland, Dunhagi-3, 107-Reykjavik, Iceland.

2Centre of Magnetic Resonance, University of St Andrews, St Andrews, KY169ST, UK.

ABSTRACT

We report the syntheses of trityl biradicals and trityl-

nitroxide biradicals for distance measurement by pulsed

EPR spectroscopy. Electron Paramagnetic Resonance

(EPR) studies show that accurate distances can be meas-

ured between these radicals, which bodes well for their

use in structural studies of nucleic acids.


CONCLUSION

Structure and dynamics of a biomolecule on the

molecular level is directly related to the function of the

biomolecule. Therefore, to understand the mechanism, one

needs information about the structure of the biomolecule.

Various biochemical and biophysical methods (e.g. NMR

and X-ray) have been used to study biomolecules in their

native conditions, but their use can be hampered by the size

and physical state of the biopolymers [1].

Electron paramagnetic resonance (EPR) spectroscopy is

increasingly being used to study the structure and motion of

large biomolecules. In this method, paramagnetic species

(spin labels) are incorporated into the biomolecule at

specific sites, referred to as site-directed spin-labelling

(SDSL), without perturbing the strurcture of biomolecule.

EPR can be used to get information about secondary

structure and interspin distances between two paramagnetic

centers attached to the large biopolymers [2]. EPR analyses

of biopolymers can give distances up to 25 Å using CW-

EPR and up to 80 Å by pulsed EPR, specifically by pulsed

electron-electron double resonance (PELDOR) [3].

Nitroxides are commonly used for SDSL studies of nu-

cleic acids [4]. However nitroxides have a very short relaxa-

tion time and inhomogeneously broaden spectra. Therefore,

distance measurements with PELDOR are carried out at

cryogenic temperatures, which is not pertinent to the bio-

molecules. Nitroxides also suffer low stability in the reduc-

ing environment present within cells. In contrast, triaryl

methyl (trityl) radicals have a long relaxation time and nar-

row linewidths in both solid and liquid states at ambient

temperatures [5]. They also show good biostability within

cells [6, 7]. Therefore, trityl radicals are candidates for in-

cell distance measurements of biopolymers by EPR spec-

troscopy.

We have prepared a series of trityl-nitroxides as well as

trityl-trityl biradicals using trityls, nitroxides and polyaro-

matic linkers of different sizes (Figure 1). PELDOR meas-

urements of trityl-nitroxide biradicals and double quantum

coherence (DQC) measurements of trityl biradicals, carried

out at Prof. Olav Schiemann’s laboratory, yielded the ex-

pected distances. Experiments are under way to incorporate

trityl radicals into nucleic acids.

REFERENCES

1. Schiemann, O., Prisner, T.F., Q Rev Biophys 2007, 40,

1-53.

2. Steinhoff, H.J., Biol Chem 2004, 385, 913-920.

3. Sale, K., Faulon, J.L., Protein Science 2004, 13, 2613-

2617.

4. Shelke, S. A.; Sigurdsson, S. T., Eur. J. Org. Chem.

2012, 12, 2291-2301.

5. Liu, Y. P., Villamena, F. A., Rockenbauer, A., et al.,

Chem Comm 2010, 46, 628-630.

6. A. Bobko, I. Dhimitruka, J. Zweier, J. Am. Chem. Soc

2007, 129, 7240-7241.

7. Y. Liu, F. Villamena, J. Sun, Y. Xu, I. Dhimitruka, J.

Org Chem 2008, 73, 1490-1497.

S

S

S

S

S

S

S

S

S S

S S

OO

OO

O

OO

HN N Olinker

S

S

S

S

S

S

S

S

S S

S S

OO

OO

O

O

S

S

S

S

S

SS

S

SS

SS

OO

O

O

O

O

linker

Figure 1. Structures of trityl-nitroxide biradicals and trityl-trityl

biradicals prepared in this study. The atomic structures of the link-

ers are not shown.

Figure 1.. Structure of trityl biradical.

131

CHROMOPHORE LABELLED NUCLEOBASES DERIVED FROM 5-AMINOURACIL

Augusto Matarazzo1* and Robert H. E. Hudson

1


*Correspondence to: Email address [email protected]

ABSTRACT

5-aminouracil (5-AU) is readily available, under uti-

lized, starting material for the synthesis of labelled nu-

cleobase analogues. We have investigated the ability to

derivatize 5-AU with amine-reactive chromophores for

the purpose of producing new base discriminating fluor-

ophores. For example, 9-chloroacridine adds to 5-AU to

produce a fluorescent nucleobase that is suitable for in-

corporation into peptide nucleic acid (PNA). The spec-

troscopic properties of this compound and others will be

reported.

INTRODUCTION

Natural nucleic acids are essentially nonfluorescent, so

engendering them with fluorescence dramatically expands

their range of applications. Nucleobases that are also lumi-

nophores may exhibit fluorescence that is dependent on

their environment, thus being able to report the difference

between the single-stranded state and duplex as well as the

difference between a complementary base and a mismatch.

Nucleobases that possess this ability have been termed

“base discriminating fluorophores” (BDFs).1 BDFs have

uses in fluorimetric sequence detection, detection of single-

nucleotide polymorphorisms and the ability to visualize oli-

gonucleotide trafficking in biological systems which can

shed light on important biological processes such as RNAi

and microRNA functions.2

Although the development of fluorescent uridine ana-

logues has been extensively investigated, the derivatization

of 5-AU with chromophoric molecules remains relatively

unexplored.3


For some time, we have been interested in a minimalist

approach to the transformation of essentially nonfluorescent

natural nucleobases into base-pairing competent intrinsic

fluorophores. Approaches for the conversion of uracil to a

luminophore have focused predominantly on derivatization

at C5. In this vein, 5-aminouracil represents a convenient

starting material for derivatization with small, amine-

reactive fluorophores. For example, the acridine-labelled

base can be accessed via reaction with 9-chloroacridine.

Preparation of 9-chloroacridine is readily achieved from N-

phenylanthranilic acid (figure 1). Further standard trans-

formations yield a monomer suitable for peptide nucleic

acid oligomer synthesis. The spectroscopic properties of

this compound as a monomeric unit and once incorporated

into oligomeric PNA will be reported.

Figure 1. Synthesis of a fluorescent 5-aminouracil-derived PNA

monomer.

In addition, other structurally small, environmentally re-

sponsive fluorophores are being evaluated. In a second ap-

proach, we are investigating the preparation of the N1-

alkylated 5-AU to afford an intermediate that may be deri-

vatized with a variety of labels, such as 7-nitrobenzo-2-oxa-

1,3-diazole (NBD), (figure 2).

Figure 2. Synthesis towards 5-aminouracil derivatives via the N1-

alkylated nucleobase.

CONCLUSION

5-Aminouracil represents a convenient material for sim-

ple access to new fluorescent uracil derivatives. These nu-

cleobase analogues are suitable for incorporation into PNA

or nucleic acids,3

and may be exploited for probe develop-

ment.

REFERENCES

1. Okamoto, A., Saito, Y., Saito, I. J. Photochem. Photo-

biol., 2005, 6, 108-122.

2. Dodd, W.D., Hudson, R.H.E. Mini-Reviews in Organic

Chemistry. 2009, 6, 378-391.

3. (a) Barawkar, D.A., Ganesh, K.N. Nucleic Acids

Res.1995, 23, 159-164. (b) Gondela, A.; Kumar, T.S.;

Walczak, K.; Wengel, J. Chem. Biodivers. 2010, 7, 350-

362.

132

INVESTIGATION OF THE REPAIR OF O6-ALKYLGUANINE AND O

4-ALKYLTHYMINE

INTERSTRAND CROSS-LINKED DNA BY O6-ALKYLGUANINE DNA

ALKYLTRANSFERASES

Francis P. McManus,1 Derek K. O’Flaherty,

1 Jordan Vergara,

1 Anne M. Noronha,

1

and Christopher J. Wilds1,*

1Concordia University, Department of Chemistry and Biochemistry, 7141 Sherbrooke St. West, Montréal, Canada


ABSTRACT

DNA duplexes containing O6-2’-deoxyguanosine-alkyl-

O6-2’-deoxyguanosine (O

6-dG-alkyl-O

6-dG) and O

4-2’-

deoxythymidine-alkyl-O4-2’-deoxythymidine (O

4-dT-

alkyl-O4-dT) interstrand cross-links (ICL), where the

linked atoms are joined by a tetra- and heptamethylene

linker, have been prepared to investigate whether the

alkyl lesions can be removed by the action of O6-

alkylguanine-DNA alkyltransferase (AGT) proteins

from human (hAGT) and E. coli (Ada-C and OGT). It

was shown that the O6-dG-alkyl-O

6-dG ICL were re-

moved by the hAGT, however the O4-dT-alkyl-O

4-dT

ICL evaded repair by the human and E. coli AGTs.


CONCLUSION

The introduction of ICLs and their impact on events in

the cell involving DNA unwinding is exploited in cancer

chemotherapic regimens employing bis-alkylating agents.[1]

The potency of these agents may be reduced by the repair of

the lesions which these therapeutic drugs induce. Various

DNA repair pathways including direct, base- and nucleo-

tide-excision repair (NER), homologous recombination

(HR), non-homologous end joining and DNA-mismatch

repair have been shown to remove several DNA lesions.

AGT proteins, found in numerous organisms, are respon-

sible predominantly for the repair of O6-methyl-2’-

deoxyguanosine, and to an extent O4-methyl-2'-

deoxythymidine, playing a role in maintaining genomic in-

tegrity.[2] hAGT, the most thoroughly characterized AGT

protein, repairs alkylated DNA by flipping the alkylated

base from the helix and into its active site where transfer of

the alkyl group from the base to the C145 residue in the

active site occurs. Once alkylated, this protein is degraded

by the ubiquitin pathway.

It was shown that hAGT can remove an O6-dG-heptyl-

O6dG ICL (Figure 1a) in mismatched and 5’-GXC se-

quence motif DNA, the latter designed to mimic the lesion

formed by hepsulfam.[3,4] However, neither of the E.coli

AGT homologues (Ada-C or OGT) were capable of repair-

ing these ICL. To enhance our understanding of the range of

repairable substrates of AGT, we prepared O4-dT-alkyl-O

4-

dT ICL (Figure 1b) by adapting methods involving convert-

ible nucleosides described by Swann.[5,6] These ICL were

prepared by solid-phase oligonucleotide synthesis and the

influence of various alkyl linker lengths on duplex stability

and structure as well as repair by AGTs from human and E.

coli were explored. The oligonucleotides were purified by

polyacrylamide gel electrophoresis and ESI mass spectrom-

etry analysis revealed they had molecular weights consistent

with the expected values. UV thermal denaturation experi-

ments demonstrated that directly mismatched O4-dT-alkyl-

O4-dT ICL containing a tetra- or heptamethylene linker in

an 11-bp duplex exhibited an increased Tm relative to a con-

trol duplex (not cross-linked). Circular dichroism experi-

ments on these ICL duplexes revealed minimal difference

from B-form DNA structure. The O4-dT-alkyl-O

4-dT ICL

prepared in this study were found to evade repair by hAGT

and the E. coli OGT and Ada-C.

REFERENCES

1. Dronkert, M.L., Kanaar, R. Mutat. Res.2001, 486, 217-

247

2. Pegg, A.E. Mutat. Res. 2000, 462, 83-100.

3. Fang, Q., Noronha, A.M., Murphy, S.P., Wilds, C.J.,

Tubbs, J.L., Tainer, J.A., Chowdhury, G., Guengerich,

F.P., Pegg, A.E. Biochemistry, 2008, 47, 10892-10903.

4. McManus, F.P., Fang, Q., Booth, J.D., Noronha, A.M.,

Pegg, A.E., Wilds, C.J. Org. Biomol. Chem., 2010, 8,

4414-4426.

5. Xu, Y.Z., Swann, P.F. Nucleic Acids Res., 1990, 18,

4061-4065.

6. McManus, F.P., O'Flaherty, D.K., Noronha, A.M,

Wilds, C.J., Org. Biomol. Chem., 2012, submitted.

Figure 1.Chemical structures of the (a) O6-dG-alkyl-O6-dG and (b) O4-dT-alkyl-O4-dT ICLs, where n=1 or 4.

133

CLICK, SUBSTITUTE AND FLUORESCE: SYNTHESIS AND APPLICATIONS OF 2,6-DI-(1,2,3-TRIAZOLYL)-PURINE NUCLEOSIDES

Irina Novosjolova*, Armands Kovaļovs, Inga Bižāne, Ērika Bizdēna and Māris Turks

Faculty of Material Science and Applied Chemistry, Riga Technical University, 14/24 Azenes str., Riga LV1007, Latvia. *Correspondence to: [email protected]

ABSTRACT

A novel class of ditriazolylpurine nucleosides were ob-

tained from 2,6-diazido precursors via copper catalyzed

azide-alkyne cycloaddition. These intermediates ap-

peared to be very reactive towards N- and S-

nucleophiles and thus selectively gave C(6)-substituted

analogs with triazolyl moiety at C(2)-position. The latter

products exhibit interesting fluorescence properties.


CONCLUSION

Application of copper catalyzed azide-alkyne 1,3-dipolar

cycloaddition [1] in nucleoside, nucleotide and oligonucleo-

tide chemistry was recently reviewed [2]. Since 2002 many

different nucleoside and nucleotide derivatives containing

1,2,3-triazolyl moiety were synthesized and investigated.

Nevertheless, only few literature reports deal with either 2-

or 6-(1,2,3-triazol-1-yl)-purine nucleosides [3].

Here we report a straightforward way to 2,6-di-(1,2,3-

triazol-1-yl)-purine nucleosides that represent a novel struc-

tural entity in nucleoside chemistry. Their synthesis from

corresponding 2,6-diazidopurine nucleosides will be dis-

cussed. It was discovered that triazolyl moiety at C(6) of the

purine base undergoes a facile nucleophilic aromatic substi-

tution with amines, hydrazines and thiols (Fig. 1). This ap-

proach provides a versatile and user friendly method for the

synthesis of various 6-amino-2-(1,2,3-triazol-1-yl)-purine

nucleosides, including those that have been described earli-

er, albeit prepared by a longer synthetic sequence [3].

Figure 1. Synthesis of 2,6-di-(1,2,3-triazol-1-yl)-purine nucle-osides 2 and their transformation into fluorescent derivatives 3.

We have demonstrated that the aforementioned structures

with general formula 3 posses fluorescent properties. 2,6-

Ditriazolyl purine nucleosides 2a-d were prepared in four

distinct series. These include ribo-furanosyl- (2a), deoxyri-

bo-furanosyl- (2b), arabino-furanosyl (2c) and arabino-

pyranosyl nucleosides (2d) (Fig. 2.). Aromatic nucleophilic

substitution proceeds equally well in all four series 2a-d,

giving a broad range of products 3.

Figure 2. Four series of 2,6-di-(1,2,3-triazol-1-yl)-purine nu-cleosides.

All products with general formula 3 possess similar fluo-

rescence properties with emission maxima between 410 and

450 nm (Fig. 3). The fluorescence is observed in various

solvents (THF, MeCN, DMSO, H2O). Emission properties

of compounds 3 such as quantum yields and decay times are

influenced by substitution pattern.

Potential applications of the discovered transformation

2→3 will be discussed.

441,96; 891,85

0,0

0,5

1,0

1,5

2,0

0

200

400

600

800

1000

210 320 430 540 650

Ab

so

rpti

on

Flu

ore

sc

en

ce

In

ten

sit

y (

a.u

.)

Wavelength, nm

Figure 3. Absorption (red) and emission (blue) spectrum of 9-

α-D-arabinopyranosyl-2-(4-(2-hydroxypropan-2-yl)-1H-1,2,3-

trizol-1-yl)-6-pyrrolidin-1-yl-9H-purine (7.7•10-7 M) in water.

REFERENCES 1. a) Tornoe, C.W., Christensen, C., Meldal, M. J. Org.

Chem. 2002, 67, 3057-3064; b) Rostovtsev, V.V.,

Green, L.G., Fokin, V.V., Sharpless, K.B. Angew.

Chem. Int. Ed. 2002, 41, 2596-2599.

2. Amblard, F., Cho, J.H., Schinazi, R.F. Chem. Rev. 2009,

109, 4207-4220.

3. a) Cosyn, L., Palaniappan, K.K., Kim, S-K., Duong,

H.T., Gao, Z-G., Jacobson, K.A., Van Calenbergh, S. J.

Med. Chem. 2006, 49, 7373-7383; b) Lakshman, M.K.,

Singh, M.K., Parrish, D., Balachandran, R., Day, B.W.

J. Org. Chem., 2010, 75, 2461-2473.

134

PREPARATION OF A NOVEL RNA DETECTING PROBE WHICH INCREASE FLUORESCENT

INTENSITY BY BINDING TO THE 3’-END OF A TARGET RNA.

Kentaro Ohno, Akira Ono and Itaru Okamoto*

Kanagawa Univesity, 3-27-1 Rokkakubashi, Kanagawa-ku Yokohama, Kanagawa, 221-8686 Japan.


ABSTRACT

The aim of this study is to develop new RNA detecting

probe which consisting of a DNA strand, a fluorescing

group and a phenyl boronic acid residue. The DNA

strand of the probe binds to a 3’-end sequence of a target

RNA to form a double helical structure, then the boronic

acid residue of the probe and a diol group at the 3’- end

of RNA form boronate esters which results increasment

of fluorescence intensity by cancelling photo-induced

electron transfer (PET) between the fluorescent group

and an amino group to which the phenyl boronic acid

group attached.


CONCLUSION

Recently, functions of RNA molecules in the cell have

been of prime interest. For investigation of RNA functions,

efficient methods for selectively detecting a target RNA

molecule in a mixture of numerous numbers of RNA

molecules in a cell have been desired.

Figure 1. Schematic representation of the RNA detecting probe

In this report, we propose a new method for sequence

specifically detecting a RNA molecule by using a novel

synthetic DNA strand carrying a boronic acid group

attached to a fluorescent group (Figure 1) [1]. A

fluorescence intensity of the fluorescent group is decreased

by photo-induced electron transfer (PET) between the

fluorescent group and an amino group to which the phenyl

boronic acid attached [2]. The DNA strand of the probe

binds to a 3’-end sequence of a target RNA to form a double

helical structure, then, the boronic acid of the probe and a

diol at the 3’-end of the RNA, those placed close to each

other, form a cyclic boronate ester structure [3-5]. The

formation of the cyclic boronate esters changes the electron

density of the phenyl group, which may some degree cancel

PET between the fluorescent group and the amino group.

Consequently, the fluorescence intensity increases.

In Figure 2, fluorescence spectra of a probe in the

presence of complementally strands are shown. By adding

an oligonucleotide having a ribonucleoside residue at the

3’-end, the fluorescence intensity of the probe increased

(solid line). Contrarily, the fluorescence intensity did not

change at all by addition of a deoxyribo-strand (dotted line).

Figure 2. Fluorescence spectra of solutions containing an

appropreate duplex (2 μM) in 100 mM Phosphate buffer (pH 8), 1

M NaClO4, λex 379 nm.

REFERENCES

1. Fujii, S. et al., 89th Annual Spring Meeting of the

Chemical Society of Japan, 2009, Japan.

2. James, T. D. et al., J. Am. Chem. Soc., 1995, 117,

8982-8987.

3. Cai, S. X., Keana, J. F. W. Bioconjugate Chem., 1991,

2, 317-322.

4. Martin, A. R. et al., Angew. Chem. Int. Ed., 2011, 50,

4193-4196.

5. Luvino, D. et al., Chem. Comm., 2008, 2352-2354.

135

HETEROARYL-LINKED C8-DEOXYGUANOSINE NUCLEOSIDES ACT AS FLUORESCENT

REPORTERS OF DNA STRUCTURE AND BASE PAIRING IN THE NAR1 SEQUENCE

Katherine M. Rankin (née Schlitt), Michael Sproviero and Richard A. Manderville*

University of Guelph, 50 Stone Road East, Guelph, Canada N1G 2W1 * Correspondence to: [email protected]

ABSTRACT

Heteroaryl-linked C8-deoxyguanosine adducts were

incorporated into the Nar1 restriction sequence to act as

fluorescent reporters of the DNA environment. These

modified oligonucleotides possess the ability to both sta-

bilize mismatches, and distinguish from syn versus anti

glycosidic bond conformation, in the duplex.


CONCLUSION

An intriguing property of modified DNA nucleosides is

their ability to exhibit strong fluorescence, as the intrinsic

fluorescence of DNA is very weak. Fluorescent modified

bases can closely resemble the structure of the natural nu-

cleosides, with minimal disruption to the DNA structure, or

can be fluorophores-linked nucleosides, in which aromatic

moieties are attached to the natural nucleoside.1 Of particu-

lar interest are C8-aryl-purine adducts that display useful

fluorescent properties, examples of which include the

strongly emissive 8-(2"-furyl)-G,2 and 1-propyl-8-(2"-

pyrrolyl)-G, a synthetic nucleoside that can stabilize a 3-

point Hoogsteen interaction with G.3 Herein, we demon-

strate the functionality of a series of C8-heteroaryl-2'-

deoxyguanosine (dG) derivatives as fluorescent sensitive

probes of the DNA environment.

The series of C8-heteroaryl-dG derivatives shown in Fig-

ure 1 were synthesized by a Suzuki-Miyaura cross-coupling

reaction with 8-Br-dG and the appropriate boronic acid.4

All C8-heteroaryl-dG derivatives exhibit red-shifted absorb-

ance compared to that of the natural nucleoside dG, indicat-

ing these derivatives could be selectively excited in the

presence of DNA, and possess a considerably higher quan-

tum yield than that of dG, with a quantum yield of fl = 0.78

for Ind-dG. In addition, these adducts possess interesting

solvatochromic properties; for example, the fluorescence of

Ind-dG is quenched in chloroform compared to that in wa-

ter, and thus could act as an ‘on/off’ switch in DNA.

Each of the C8-heteroaryl-dG derivatives were incorpo-

rated into the Nar1 restriction sequence, a known ‘hotspot’

for arylamine modification.5 Modification at position X of

5'-CTCGGCXCCATC was accomplished by one of two

methods; phosphoramidite chemistry, or postsynthetic gua-

nine arylation by Suzuki-Miyaura cross-coupling.6 The

effect of this modification on duplex stability was deter-

mined. In each instance when the modified oligonucleotide

was hybridized to a complementary strand in which ‘C’ was

opposite X, a destabilization, compared to the unmodified

duplex, was observed. Interestingly, when the modified

oligonucleotide was hybridized to a complementary strand

in which ‘G’ was opposite X, stabilization was observed.

Finally, excitation and emission spectra of the hybridized

modified oligonucleotides was obtained. Measurements

were first carried out at 10 oC with the desired duplex, fol-

lowed by heating to 80 oC to induce denaturation. For all

duplexes with ‘C’ opposite X = Ind-dG, Bfur-dG or Bth-

dG, the fluorescence intensity decreased as the duplex dena-

tured, while when ‘G’ was opposing, the intensity was ob-

served to increase. This suggests that upon mismatch for-

mation, the C1'-N glycosidic bond of the C8-heteroaryl-G

derivative adopts the syn conformation, resulting in an in-

crease in stacking interactions.

In conclusion, incorporation of C8-heteroaryl-dG deriva-

tives into the Nar1 sequence has resulted in modified oligo-

nucleotides with fluorescent sensitivity to base-pairing

properties and glycosidic bond conformation.

REFERENCES

1. Millar, D.P. Nucleic Acids 1996, 6, 322-326.

2. Sinkeldam, R.W., Greco, N.J., Tor, Y. Chem. Rev. 2010,

110, 2579-2619.

3. Sessler, J.L., Jayawickramarajah, J., Sherman, C.L.,

Brodbelt, J.S. J, Am, Chem. Soc. 2004, 126, 11460-

11461.

4. Western, E.C., Daft, J.R., Johnson, E.M., Gannett, P.M.,

Shaughnessy, K.H. J. Org. Chem. 2003, 68, 6767-6774.

5. Elmquist, C.E., Stover, J.S., Wang, Z., Rizzo, C.J. J.

Am. Chem. Soc. 2004, 126, 11189-11201.

6. Omumi, A., Beach, D.G., Baker, M., Gabryelski, W.,

Manderville, R.A. J. Am. Chem. Soc. 2011, 133, 42-50.

Figure 1. C8-heteroaryl-dG derivatives.

136

OXIDATION OF H-PHOSPHONATES WITH IODINE BY INTRAMOLECULAR SUPPORT

OF A 2-PYRIDYL THERMOLABILE PROTECTING GROUP

Tomasz Ratajczak and Marcin K. Chmielewski*

Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14, Poznań, Poland * Correspondence to: [email protected]

ABSTRACT

Acceleration of H-phosphonate diester oxidation with

iodine accompanied by a thermolabile protecting group

(TPG) is presented. It is shown that the intermediate

product of this reaction is an oxazaphospholidine oxide

which forms a phosphate diester only when a 2-pyridyl

TPG is applied. 31

P NMR spectroscopy was used to eval-

uate the relationship between chemical shift and abso-

lute configuration at the phosphorus center of H-

phosphonate diesters, oxazaphospholidine oxides and

phosphorothioate diesters.

INTRODUCTION

Thermolabile protecting groups (TPGs) have been

introduced to enhance effective protection of a phosphate,

hydroxyl or amine center. Removal of these groups is based

on intramolecular cyclization depending on temperature [1].

However, phosphate triesters with some TPGs are very

unstable [2] and to enhance their stability a “click-clack”

approach has been applied [3]. This approach increases 2-

pyridyl TPG stability by forming a five-membered

oxazaphospholidine ring. A linear form of TPG may be

easily recovered by acid hydrolysis, during which also an H-

phosphonate diester is formed. H-phosphonate diesters also

get oxidized to the corresponding phosphates by iodine in a

basic environment like pyridine. In this case oxidation of an

H-phosphonate diester involves a multi-step mechanism

where an phosphoroiodidate derivative and further

pyridinium cation is formed [4,5]. We discovered that H-

phosphonate diesters which contain a 2-pyridyl moiety get

easily oxidized to phosphate by using the iodine only.


Our study shows a possibility of using N-(2-pyridyl)ethyl

moiety as a thermolabile protecting group as well as

intramolecular catalyst of the oxidation reaction. We take

advantage of the nucleophilic and basic properties of exo-

cyclic nitrogen atom (3, 5), which is involved in a five-

membered ring with a phosphate center after substituting the

hydrogen atom by iodine (Fig. 1). The oxazaphospholidine

oxide 9 formed from the 2-pyridyl TPG (3) opens very fast

(less then 1 min) in the presence of water giving 11 while 10

is very stable under these conditions. While studying the

mechanisms of phosphate cyclization it is important to

assign absolute configuration to H-phosphonate center. We

have enzymatically determined the absolute configuration of

thiophosphate 12 while configurations of 3, 5, 9, 10 were

assigned using correlation methods (Fig. 2).

CONCLUSION

Our work presents the application of a 2-pyridyl TPG which

intramolecularly catalyzes oxidation of H-phosphonate

diesters with iodine only. However, forming a phosphate

diester in this reaction is possible only when a 2-pyridyl

TPG is applied. Moreover, the process is very fast and quan-

titatively completed within 15 minutes.

REFERENCES

1. Grajkowski, A., Wilk, A., Chmielewski, M.K., Phillips,

L.R., Beaucage, S.L. Org. Lett. 2001, 3, 1287-1290.

2. Cieślak, J., Beaucage, S.L. J. Org. Chem. 2003, 68,

10123-10129.

3. Chmielewski, M.K. Org. Lett. 2009, 11, 3742-3745.

4. Stawiński, J., Stromberg, R., Zain, R. Tetrahedron Lett.

1992, 33, 3185-88.

5. Garegg, P.J., Regberg, T., Stawiński, J., Stromberg, R. J.

Chem. Soc. Perkin Trans. I 1987, 6, 1269-1274.

NH

O

ON

O

OAc

OPH

OON

HX

NH

O

ON

O

OAc

OPN

O

X

NH

O

ON

O

OAc

OPN

O

X

O

NH

O

ON

O

OAc

OPOH

OON

HN

NH

O

ON

O

OAc

OPS-

OON

H

S8/Py

BTT

MeCN Iodine/MeCN

H2O 11

9 X=N10 X=CH2

X=N

X=CH2

3 X=N5 X=CH2

12

1 X=N2 X=CH2

PyH+

Figure 1. Oxidation of H-phosphonate diesters to phosphate by

the intramolecular oxidative coupling.

Figure 2. Correlation between chemical shifts and absolute con-figuration of 5, 10 and 12.

137

SOLID PHASE SYNTHESIS OF NUCLEOSIDE- AND OLIGONUCLEOTIDE 5’-TRIPHOSPHATES WITH CYCLOSALIGENYL PHOSPHITYLATING REAGENTS

Ivo Sarac1* and Chris Meier1

1Organic Chemistry, Department of Chemistry, Faculty of Sciences, University of Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany. * Correspondence to: [email protected]

ABSTRACT

Starting from immobilized nucleosides or oligo-nucleotides the corresponding 5’-cycloSal-phosphate triesters were directly synthesized with cycloSaligenyl phosphitylating reagents. These compounds were re-acted with pyrophosphate yielding nucleoside- and oligo-nucleotide 5’-triphosphates after cleavage from various solid supports.

INTRODUCTION

2’-Deoxyribo- and ribonucleoside 5’-triphosphates are the building blocks for enzymatic synthesis of DNA and RNA in vivo and in vitro.[1] While DNA 5’-triphosphates are mostly used in the biotechnology industry to obtain syn-thetic genes, RNA 5’-triphosphates have a broader spectrum of applications. For example, RNA 5’-triphosphates are used in the induction of antiviral immunity and for the ligation of RNA fragments.[2]

Nucleoside- as well as oligonucleotide 5’-triphosphates are very important compounds in biological systems with therapeutic applications. Although there have been a num-ber of different approaches reported, the general access to these important classes of compounds is still a challenge.

Therefore, we attempted to develop a generally applic-able route to both nucleoside- and oligonucleotide 5’-tri-phosphates.


In an earlier approach 5’-cycloSal-nucleotides were con-verted to a wide range of different biomolecules such as nucleoside 5’-triphosphates using an in-solution method.[3]

Later we reported on a solid-phase synthesis route to nucleoside 5’-triphosphates by first synthesizing 5’-cycloSal-nucleotides along with the linker unit and attaching these building blocks to polystyrene. The sub-sequent reaction with pyrophosphate led to nucleoside 5’-triphosphates in high yields after cleavage from the resin, which facilitated the purification.[4]

With regard to a more versatile and modular approach not only for immobilized nucleotides but also oligo-nucleotides these compounds were directly converted to 5’-cycloSal-phosphate triesters using cycloSaligenyl phosphitylating reagents and oxidation. The advantage of this new approach is a faster, more flexible and more con-venient way (Scheme 1) to nucleoside- and oligonucleotide

5’-triphosphates compared to our first published method in which protection as well as purification steps of the 5’-cycloSal-nucleotides were involved.

YOP

OX

2. oxidation

1.

phosphorylation

B

O

O

PO

HO

O B

O

O

ONC

n

O

NH

O

OPG/H

OPG/H

B

O

O

PO

O

O B

O

O

ONC

n

O

NH

O

OPG/H

OPG/H

OP

OX

O

B

O

O

PO

O

O B

O

O

ONC

n

O

NH

O

OPG/H

OPG/H

POPOPO

O O O

O O O

B

O

O

PO

O

O B

O

OH

O

n

OH/H

OH/H

POPOPO

O O O

O O O

deprotectionand cleavage

PG: protecting group

--B: nucleobasex.X: Cl, NO2, Ac

x.Y: Cl, phosphoramidite

x.--: for n=0 aminomethyl polystyrene, for n>0 controlled pore glass

2

Scheme 1. General synthesis of immobilized nucleoside and oligo-nucleotide 5’-triphosphates

CONCLUSION

With our new method both nucleoside- and oligo-nucleotide 5’-triphosphates were synthesized in a more convenient way. This approach opens a general way to a variety of complex biomolecules.

REFERENCES

1. Burgess, K., Cook, D. Chem Rev. 2000, 100, 2047-2059.

2. Zlatev, I., Lavergne, T., Debart, F., Vasseur, J-J., Manoharan, M., Morvan, F. Org. Lett. 2010, 12, 2190-2193.

3. Warnecke, S., Meier, C. Nucleic Acids Symp. 2008, 52, 583-584.

4. Tonn, V. C., Meier, C. Chem. Eur. J. 2011, 17, 9832-9842.

138

AN APPLICATION OF THE FUNCTIONALITY TRANSFER REACTION FOR THE SELECTIVE

AND SENSITIVE DETECTION OF O6-METHYL-2’-DEOXYGUANOSINE IN DNA

Kazumitsu Onizuka,1,2

Takamasa Nishioka,1 Zhichun Li,

1,2 Daichi Jitsuzaki,

1,2 Yosuke Taniguchi,

1,2

and Shigeki Sasaki1,2*

1Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582,

Japan,and 2CREST, Japan Science and Technology Agency, 4-1-8 Motomachi, Kawaguchi, Saitama 332-0012, Japan.

* Correspondence to: [email protected].

ABSTRACT

The hybridization-induced transfer of the functional

group has enabled the selective modification on the 4-

amino group of the cytosine base or the 2-amino group

of the guanine base of RNA depending on the buffer

conditions. This paper describes a new application of

this method to selective and sensitive detection of O6-

methyl-2’-deoxyguanosine (O6-Me-dG) in DNA.


CONCLUSION

O6-Methyl-2’-deoxyguanosine (O

6-Me-dG) is formed by

the reaction with alkylating agents, and induces GC to AT

transition mutations during DNA replication both in vitro

and in vivo. The genotoxicity of O6-Me-dG is thought to be

involved in carcinogenesis of a variety of tissues. In the cur-

rently used methods, O6-Me-dG is analysed after enzymatic

hydrolysis of DNA samples. In this study, we have estab-

lished a simple procedure to achieve the highly specific de-

tection of O6-Me-dG in DNA in a sequence specific manner

based on the selective functionality-transfer reaction within

the duplex DNA developed by our group.1

We have developed a unique method for the site-

specific, internal modification of RNA based on the

functionality transfer strategy.2 The ODN probe incorpo-

rating 2’-deoxy-6-thioguanosine (GS) functionalized with

the 2-methyliden-1,3-diketone group, the functionality-

transfer ODN probe, is hybridized with the target RNA to

allow the transfer reaction of the functional group to the

amino group of the target nucleobase. At neutral pH, the

4-amino group of the cytosine base is selectively

modified,3 whereas the transfer reaction to the 2-amino

group of the guanine base is selectively accelerated at

alkaline pH,4 or in the preence of NiCl2 at neutral pH.

5

Subsequently, the transfer group reaction was applied to

an internal labelling of RNA with a variety of functional

groups via the copper-catalyzed azide-alkyne

cycloaddition reaction.6 In this study, we hypothiesized

that the 2-amino group of the O6-alkylated dG would be

more reactive than that of the non-alkylated dG at neutral

pH (Figure 1).

During the first step, the alkyne-containing transfer

group of FT-ODN is transfered to O6-Me-dG in DNA,

followed by the second step for the click reaction with

the azide derivative of fluorescent FAM. The remaining

acetylene groups of the probe ODN was hydrolized in in

NH4OH solutions to produce the 2-NH2-modified dG as a

sole FAM-labelled DNA species. As a result, the FAM-

modified DNA was easily obtained without any

purification procedure such as by HPLC. The bitoin-

azide derivative was similarly applied to the click reac-

tion.

Thus, this study has established a simple method for

the specific detection of O6-Me-dG in DNA based on a

functionality-transfer strategy. We expect that this meth-

od may be useful to analyse O6-Me-dG in the pathogenic

DNA regions.

REFERENCES

1. K. Onizuka, T. Nishioka, Z. Li, D. Jitsuzaki, Y. Tanigu-chi, S. Sasaki,,Chem. Commun., 2012, 48, 3969-3971.

2. S. Sasaki, K. Onizuka, Y. Taniguchi, Chem. Soc. Rev., 2011, 40, 5698 - 5706

3. K. Onizuka, Y. Taniguchi, S. Sasaki, Bioconjugate Chem. 2009, 20, 799-803.

4. K. Onizuka, Y. Taniguchi, S. Sasaki, Nucleic Acids Res. 2010, 38, 1760-1766.

5. K. Onizuka, Y. Taniguchi, S. Sasaki, Bioconjugate Chem. 2010, 21, 1508-1512.

6. K. Onizuka, A. Shibata, Y. Taniguchi, S. Sasaki, Chem. Comm., 2011, 47, 5004-5006.

Figure 1. Discrimination of O6-Me-dG from dG by the Func-tionality Transfer reaction

139

SELECTIVE DETECTION METHOD for 5-FORMYL-2'-DEOXYURIDINE in DNA USING a FLUOROGENIC REAGENT

Kousuke Sato, Wataru Hirose and Akira Matsuda*

Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Kita-12, Nishi-6, Sapporo, Japan. * Correspondence to: Email address [email protected]

ABSTRACT

We report here the selective detection method for 5-

formyl-2'-deoxyuridine (dfo

U) by derivatization to 5-(5,6-

dimethoxybenzothiazole-2-yl)-2'-deoxyuridine (dbt

U),

which has a strong fluorescent in aqueous 100 mM

NaOH. This fluorogenic derivatization has also been

successful in oligodeoxyribonucleotides (ODNs) contain-

ing dfo

U. Finally, 8.9 dfo

U/106 bases/Gy was detected in -

irradiated calf thymus DNA by our detection method.


CONCLUSION

DNA in living cell is damaged by reactive oxygen spe-

cies derived from UV light, ionizing radiation, and cellular

respiration. 5-Formyl-2'-deoxyuridine (dfo

U), an oxidized

thymidine lesion generated in yields comparable to that of

2’-deoxy-8-oxoguanine,[1]

induces mutation in DNA

through the mispairing of dfo

U with other nucleobases in-

stead of adenine during replication.[2]

It appears that the

formation of dfo

U may cause carcinogenisity and/or aging of

cells. A selective and more convenient method for detecting

dfo

U would be highly desirable, since the existing methods

are complicated, involve time-consuming analysis by HPLC

and/or mass spectrometry following complete enzymatic

hydrolysis of the target DNA, and require isotope-labeled

dfo

U (13

C and/or 15

N) as an internal standard.[3]

Herein, we report a new concept for the simple detection

of dfo

U in damaged DNA with a fluorogenic reagent. The

reagent, substituted 2-aminothiophenol, shows no fluores-

cent before reaction with the target dfo

U in DNA. However,

upon the reaction of substituted 2-aminothiophenol with

dfo

U, the formyl group at the 5-position of dfo

U is converted

into a benzothiazol-2-yl group, which is directly conjugated

with the uracil group. This ring system is similar to luciferin,

which undergoes the luciferase reaction to produce lumines-

cence. Thus, dfo

U in DNA could be detected directly by

fluorescence measurement without enzymatic hydrolysis of

the target DNA to its corresponding nucleosides, HPLC

separation, and analysis. We first synthesized some 5-(benzothiazol-2-yl)-2'-

deoxyuridine derivatives via the reaction between dfo

U and

corresponding substituted 2-aminothiophenols. The effects

of substituent of benzothiazole and UV absorbance and fluo-

rescence properties of 5-(benzothiazol-2-yl)-2'-deoxyuridine

derivatives under various pH conditions were investigated.[4]

2-Amino-4,5-dimethoxythiophenol (1) was chosen as the

specific fluorogenic reagent for a selective and convenient

method for its detection.[5]

5-(5,6-Dimethoxybenzothiazole-

2-yl)-2'-deoxyuridine (dbt

U), which has a strong fluores-

cence at 458 nm in aqueous 100 mM NaOH. This fluoro-

genic derivatization has also been successful in oligodeoxy-

ribnucleotides (ODNs) containing dfo

U in >95% yield. In

control experiments, the fluorescence intensity of the ODN-

T, which contains thymidine (Thy), and ODN-AP, which

contains one abasic site, were measured, after their fluoro-

genic reaction with 1 under the same conditions. Although

abasic sites are generated as a major aldehyde source in

DNA, there was no fluorescence of ODN containing thymi-

dine and abasic site. Finally, 8.9 dfo

U/106 bases/Gy was de-

tected in -irradiated calf thymus DNA. Our detection

method does not need long reaction times, any enzymatic

digestion, HPLC separation, or mass spectrometric analysis.

REFERENCES

1. Kasai, H., Iida, A., Yamaizumi, Z., Nishimura, S., Ta-

nooka, H. Mutat. Res., 1990, 243, 249-253.

2. Masaoka, A., Terato, H., Kobayashi, M., Ohyama, Y.,

Ide, H. J. Biol. Chem., 2001, 276, 16501-16510.

3. Hong, H., Wang, Y. Anal. Chem., 2007, 79, 322-326.

4. Hirose, W., Sato, K., Matsuda, A. Eur. J. Org. Chem.,

2011, 2011, 6206-6217.

5. Hirose, W., Sato, K., Matsuda, A. Angew. Chem. Int.

Ed., 2010, 49, 8392-8394.

Figure 1. Fluorogenic detection method for dfoU in DNA.

140

DEVELOPMENT OF BACKBONE FIXED AND TERMINALLY MODIFIED OLIGONUCLEOTIDES

CAPABLE OF SHORT RNA SELECTIVE BINDING

Yoshihiro Iijima, Sayako Kurohagi, Takashi Kanamori, Hirosuke Tsunoda, Akihiro Ohkugo, Mitsuo Sekine* and Kohji Seio*

Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta,Midori-ku, Yokohama, Japan.*Kohji Seio: Email address for [email protected]; Mitsuo Sekine: Email address for [email protected]

ABSTRACT

We have reported the oligodeoxynucleotide containing

bulky and anionic modification (dAChcmp

) at its 5´-

terminus which selectively hybridized with the short

complementary RNA. The position of dAChcmp

was fixed

by incorporating a propylene bridge between the 5´-

phosphate the 5-position of the 3´-downstream uridine

residue. Short-RNA selective binding abilities of the

modified oligodeoxynucleotides were assessed by the Tm

measurements.


CONCLUSION

There are a lot of studies to introduce conformational re-straint in the nucleoside moiety and the phosphodiester backbones in order to modify the structures and the hybridi-zation properties of oligonucleotides.

1 For example, the

fixation of the sugar puckering by the introduction of addi-tional ring structure has been proved to be effective to in-crease the hybridization stability of oligonucleotides. In addition, there are several examples of the conformational fixation of the sugar-phosphate backbones. We have also reported the incorporation of the cyclic nucleoside residue having a propylene linkage between the uracil moiety and the phosphorous atom forming the phosphotriester structure, and their hybridization properties.

2

This time, we studied the synthesis of oligonucleotides hav-

ing conformationally fixed phosphotriester backbone previ-

ously reported2 and the dA

ChcmP residue

3 at the 5'-end of the

oligonucleotide aiming the development of oligonucleotide

probes capable of short RNA selective binding.3

The Rp and Sp isomers of the dAChcmP

-c3T dimer unit (Figure

1) was synthesized from deoxyadenosine and deoxyuridine

and purified by silica gel column chromatography. The oli-

godeoxynucleotides incorporating the dimer unit at the 5’-

termini was performed by the conventional solid-phase syn-

thesis. The Tm analyses revealed the short RNA selective

binding properties of the conformation fixed probes which

are useful for the development of miRNA detection methods.

REFERENCES

1. a) Veedu, R. N., Wengel, J. Chem. Biodivers. 2010, 7,

536-542. b) Leumann, C. J. Bioorg. Med. Chem. 2002,

10, 841-854. c) Dupouy, C., Iché-Tarrat, N., Durrieu, M.

P., Vigroux, A., Escudier, J. M. Org. Biomol. Chem.

2008, 6, 2849-2851.

2. Sekine, M., Kurasawa, O., Shohda, K., Seio, K., Wada,

T. J. Org. Chem. 2000, 65, 6515-6524.

3. Seio, K., Kurohagi, S., Kodama, E., Masaki, Y., Tsu-

noda, H., Ohkubo, A., Sekine, M. Org. Biomol. Chem.

2012, 10, 994-1006.

Figure 1. Structure of backbone fixed and terminally modified oli-gonucleotides.

141

mailto:for%[email protected]

http://www.ncbi.nlm.nih.gov/pubmed?term=Leumann%20CJ%5BAuthor%5D&cauthor=true&cauthor_uid=11836090

http://www.ncbi.nlm.nih.gov/pubmed/11836090

ENZYME-LINKED SMALL-MOLECULE DETECTION USING SPLIT APTAMER LIGATION

Ashwani K. Sharma*, Alexandra D. Kent, and Jennifer M. Heemstra

Department of Chemistry and the Center for Cell and Genome Science, University of Utah, Salt Lake City, Utah 84112, United States. * Correspondence: [email protected]

ABSTRACT

A split aptamer based analogue of the widely-used

ELISA diagnostic assay is described for the detection of

cocaine. This assay utilizes the ligation of two split ap-

tamer fragments in the presence of cocaine, providing a

colorimetric output that can be quantified using an ab-

sorbance plate reader.


CONCLUSION

The detection of small molecules is an active area of re-

search as these molecules play a critical role in disease pro-

gression, environmental health, and clinical diagnostics.

Antibodies have long been the first choice for use in molec-

ular recognition due to their high affinity and broad sub-

strate scope. However, nucleic acid aptamers have emerged

as a promising alternative to antibodies, as they are easily

obtained through in vitro selection and benefit from higher

chemical stability relative to antibodies. Moreover, aptamers

can be generated for a broad range of targets ranging from

small molecules to proteins and enzymes.

Figure 1. Enzyme-linked cocaine detection using split aptamer ligation.

We have recently demonstrated novel Split Aptamer

Proximity Ligation (StAPL) technology1 in which attach-

ment of reactive groups to the termini of split aptamer frag-

ments2 enables translation of a small molecule signal into

the output of DNA ligation. Herein, we describe the use of

this technology for the development of an enzyme-linked

assay for detecting cocaine. The capture and detection anti-

bodies used in the standard ELISA assay were replaced by

chemically modified fragments of the cocaine split aptamer.

In brief, a capture strand having an azide at one terminus

and an amine at the opposite terminus was attached to an N-

hydroxysuccinimide (NHS)-functionalized microplate via

amide bond formation (Figure 1). The detection strand, hav-

ing a cyclooctyne at one terminus and a biotin at the oppo-

site terminus, is added to the DNA functionalized micro-

plate well along with a test sample containing varying con-

centrations of cocaine. If present, cocaine directs assembly

of the aptamer fragments, bringing the azide and cy-

clooctyne groups into close proximity and thus promoting a

cycloaddition to ligate the two fragments3. The resulting

ligation yield is dependent upon cocaine concentration, and

is measured as a colorimetric signal by adding streptavidin-

horseradish peroxidase (SA-HRP) conjugate that binds to

biotin and converts a colorless tetramethylbenzidine (TMB)

substrate into an optically observable blue product. This

assay is capable of detecting cocaine at concentrations of

100 nM-100 µM in buffer and 1-100 µM in human blood

serum. The detection limit of 1 µM in serum is two orders

of magnitude better than previously reported split aptamer-

based sensors.

In conclusion, we demonstrate use of the cocaine split ap-

tamer to construct the first DNA-based analogue of sand-

wich ELISA capable of small molecule detection. The total

assay time is less than two hours. Moreover, the enzyme-

linked format provides a convenient colorimetric output and

functions analogously to antibody-based ELISA, the current

standard in clinical diagnostic laboratories.

REFERENCES

1. Sharma, A. K.; Heemstra, J. M. J. Am. Chem. Soc. 2011,

133, 12426-12429.

2. (a) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J.

Am. Chem. Soc. 2000, 122, 11547–11548. (b) Zhang, J.;

Wang, L.; Pan, D.; Song, S.; Boey, F. Y. C.; Zhang, H.;

Fan, C. Small 2008, 4, 1196–1200.

3. Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.;

Bertozzi, C. R. ACS Chem. Biol. 2006, 1, 644-648.

.

142

NONCOVALENT SITE-DIRECTED SPIN-LABELING OF ABASIC SITES IN DUPLEX DNA

Sandip A. Shelke1* and Snorri Th. Sigurdsson

1

1Science Institute, University of Iceland, Dunhagi 3, 107 Reykjavik, Iceland. * Correspondence to: [email protected]

ABSTRACT

A noncovalent and site-directed spin-labeling ap-

proach was developed for nucleic acids using the rigid

spin label ç. EPR spectroscopic studies revealed that the

spin label ç fully and specifically bound to abasic sites in

duplex DNA at low temperatures. Factors that influence

the binding of ç to the abasic site, such as the structure

of the abasic site, flanking nucleotide sequence and func-

tional groups at the N3 positions of the ç, were optimized.


CONCLUSION

Nucleic acids play vital roles in biological systems. In-

formation about their structure and dynamics (motion) is the

key to understanding their functions. Among the techniques

that are available for structure determination of nucleic ac-

ids, EPR spectroscopy has become a prominent technique

due to its high sensitivity, no molecular size limit and ability

to study biopolymers under bioorthogonal experimental

conditions. EPR detects free radicals or paramagnetic cen-

tres associated with biopolymers. Since nucleic acids are

diamagnetic, EPR studies require incorporation of persistent

free radicals (spin labels) at specific site(s), termed site-

directed spin-labeling (SDSL). SDSL of nucleic acids gen-

erally achieved by covalent attachment of organic molecules

that bears stable nitroxide radicals. However, such spin-

labeling usually time consuming and requires extensive syn-

thetic efforts.

Here we report a general and straight-forward approach

for SDSL of nucleic acids using noncovalent interactions [1].

We have designed and synthesized the spin label ç, which

binds to a nucleic acid that contains an abasic site utilizing

hydrogen bonding and -stacking interactions (Figure 1).

The binding of ç to an abasic site in duplex DNA was stud-

ied by EPR spectroscopy and revealed that the spin label is

fully and specifically bound to the abasic site at low temper-

atures. This technique provides an easy access to spin-

labeled nucleic acids for structural studies using pulsed EPR

techniques, such as pulsed electron-electron double reso-

nance (PELDOR) or DEER, which are generally carried out

in frozen solutions. The spin-labeled nucleic acids were pre-

pared simply by mixing the spin label with the DNA con-

taining the abasic site(s) and lowering the temperature.

Factors that influence the noncovalent binding of the spin

label ç, such as effect of the hydrogen bonding and effect of

the structure of the abasic site, were studied and shown that

the spin label binds most efficiently when it forms three

hydrogen bonds with guanine (G). Furthermore, we found

that the binding of ç is highly flanking-sequence dependent

[2]. Generally, 5’-dG nucleotide favors the binding whereas,

5’-dC disfavors the binding of the spin label. To improve

the binding affinity of the spin label, several N3-derivatives

were prepared. Derivatives containing basic functional

groups, such as ethyl amino and ethyl guanidino showed

higher binding affinities and increased solubility in aqueous

solution, which makes them promising candidates for non-

covalent SDSL of nucleic acids for distance measurements

using PELDOR [3].

REFERENCES

1. Shelke, S.A., Sigurdsson, S.T. Angew. Chem. Int. Ed.,

2010, 49, 7984-7986.

2. Shelke, S.A., Sigurdsson, S.T. Nucleic Acids Res., 2012,

40, 3732-3740

3. Shelke, S.A., Sigurdsson, S.T. ChemBioChem., 2012,

13, 684-690.

Figure 1. Noncovalent and site-directed spin-labeling. A. Structure of the spin label ç and its base-pairing scheme with G. B. A model of a duplex DNA containing an abasic site (grey) and spin label ç (black), EPR spectra of ç in free state and in DNA containing an abasic site.

143

SYNTHESIS AND INCORPORATION OF DIAZIRINE-MODIFIED URIDINE PHOSPHORAMIDITE

Christine Smith1* and Christian J Leumann

1

1Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3008 Bern, Switzerland

* Correspondence to: Email address: [email protected]

ABSTRACT

A diazirine-modified uracil analogue was synthesised

and incorporated into three RNA 21-mers for use in de-

tecting RNA –RBP interactions.

INTRODUCTION

The role of many RNA binding proteins

(RBPs), particularly in the context of non-coding RNAs,

remains widely unknown. There is a lack of methodology

available to determine the specific interactions between

RNA and RBPs via covalently linking the two biomolecules.

Current methods in this context include CLIP1, where RNA

is cross-linked to the RBP by short wavelength UV radiation

followed by immunoprecipitation. However, this process is

both inefficient and will lead to damage in living cells. 4-

thiouridine has also been used2, which requires milder

(>300nm) cross-linking conditions, but the radical mecha-

nism leads to lower efficiencies, typically <40% and the

base-pairing properties of the 4-thiouridine-containing RNA

are compromised.

More recently, chemistry has been developed to crosslink

DNA to DNA binding proteins3. This involved the modifi-

cation of the thymidine base with a diazirine moiety, which

upon irradiation with UV in the range of 360nm forms a

carbene. The carbene inserts into the C-H bonds of adjacent

proteins via a non-radical mechanism.


We have used this chemistry to modify the RNA base ur-

idine via the synthetic route shown in Figure 1. Then, using

phosphoramidite chemistry, we have incorporated the modi-

fied uridine into a 21-mer RNA hairpin structure. 4 The fluo-

rescein-tagged sequences that were synthesised and the cor-

responding mass analyses are reported in Table 1.

Sequence Expected

Mass

Found

Mass

1 6-FAM-AAU CCA UUG CAC UCC GGA UUU 7131.5 7131.0

2 6-FAM-AAU CCA XUG CAC UCC GGA UUU 7411.8 7412.0

3 6-FAM-AAU CCA UXG CAC UCC GGA UUU 7411.8 7410.0

4 6-FAM-AAU CCA UUG CAC XCC GGA UUU 7411.8 7410.2

Table 1. Unmodified and modified RNA sequences synthesised. X represents the diazirine modified uridine incorporation.

Figure 1. Synthesis of phosphoramidite X. a) DMTCl, DMAP, pyri-dine; b) TBSCl, DMF, 62% over 2 steps; c) 4-pentyn-1-ol, CuI, Pd(PPh3)2Cl2, Et3N, DMF, 71%; d) 3-(α-iodo-p-tolyl)-3-(trifluoromethyl)-3H-diazirine5, NaH, THF, 51%; e) TBAF, THF, 73%; f) TBSCl, AgNO3, pyridine, THF, 72%; g) CEPCl, DIPEA, THF, 75%.

An irradiation assay using the modified oligonucleotide

sequences is being developed for use with SDS-PAGE, us-

ing a combination of fluorescence imaging and staining to

show the migration of the peptide, RNA and the RNA-

peptide conjugates.

CONCLUSION

A diazirine modified uridine (X) has been synthesised

and successfully incorporated into three RNA 21-mers via

phosphoramidite chemistry. It’s crosslinking potential to

single stranded binding proteins (SSBs) has been evaluated.

REFERENCES

1. Ule, J.; Jensen, K. B.; Ruggiu, M.; Mele, A.; Ule,

A.; Darnell, R. B. Science 2003, 302, 1212-1215.

2. Favre, A.; Moreno, G.; Blondel, M. O.; Kliber, J.;

Vinzens, F.; Salet, C. Biochem. Biophys. Res.

Commun. 1986, 141, 847-854.

3. Winnacker, M.; Breeger, S.; Strasser, R.; Carell, T.

Chembiochem 2009, 10, 109-118.

4. Oubridge, C.; Ito, N.; Evans, P. R.; Teo, C. H.;

Nagai, K. Nature 1994, 372, 432-438.

5. Shih, L. B.; Bayley, H. Analytical Biochemistry

1985, 144, 132-141.

144

DEVELOPMENT OF A GENERAL APPROACH FOR SOLID-PHASE SYNTHESIS OF

OLIGONUCLEOTIDES BEARING C8-ARYL GUANINE ADDUCTS

Michael Sproviero, Katherine M. Rankin, and Richard A. Manderville*

University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada * Correspondence to: [email protected]

ABSTRACT

Through the use of modified pixyl protection (DMPx), a

general means for incorporating multiple acid sensitive

C8-modified dG adducts into longer oligonucleotides has

been established. The procedure employs slightly modi-

fied phosphoramidite synthesis protocols and allows for

incorporation of many biologically relevant nucleotide

adducts.


CONCLUSION

Past research in the Manderville laboratory has

demonstrated the ability to generate oligonucleotides con-

taining C8-modified deoxyguanosine (dG) adducts using an

on-strand Suzuki-Miyaura coupling reaction.1 This ap-

proach to generating modified oligonucleotides is ideal for

incorporating one bulky adduct into relatively short oligo-

nucleotides as it requires the separation of unreacted oligo-

nucleotides from reacted ones via HPLC. Typical phospho-

ramidite synthesis has been shown to be inappropriate for

C8-phenol modified dG adducts due to problems encoun-

tered with the phenolic hydroxyl protection and their in-

creased acid sensitivity (up to 200 times more sensitive than

dG).2

Herein we report the synthesis and stability of a se-

ries of C8-modified deoxyguanosine adducts as well as their

incorporation into 12mer oligonucleotides containing the

Nar1 recognition sequence (5'-G1G2CG3CC-3'). Typical

phosphoramidite synthesis was utilized to generate the

strands of interest, albeit with poor yields. The presence of

large quantities of failed sequences with no fluorescence in

their corresponding HPLC traces was indicative of deglyco-

sylation occurring due to acid exposure (Figure 1).

Figure 1: Crude HPLC trace of Phenyl-dG adduct incorpo-

rated into the Nar1 12-mer oligonucleotide.

It was thus necessary to minimize acid exposure

for each Deblock cycle during automated DNA synthesis.

This was achieved by replacing the 5′ DMT protecting

group with a 5′ DMPx protecting group which lowered the

effective Deblock concentration required for complete re-

moval of the 5′ protecting group from 3% DCA to 0.5%

DCA.3 This modification allowed for successful incorpora-

tion of C8-modified dG adducts and resulted in no acid cata-

lysed deglycosylation (Figure 2).

Figure 2: Crude HPLC trace of Furan-dG adduct incorpora-

tion into the Nar1 12mer oligonucleotide using DMPx pro-

tection.

In summary, we show that the ability to generate

such oligonucleotide strands, especially when the modifica-

tion is located closer to the 3' end, is dependent on replacing

the Dimethoxytrityl (DMT) 5'OH protection with a more

acid labile protecting group. This is achieved through the

use of Dimethyl Pixyl (DMPx) protection for use in DNA

synthesis which can be removed under much milder condi-

tions. These adaptations provide a general means for incor-

porating multiple acid sensitive C8-modified dG adducts

into longer oligonucleotides using slightly modified phos-

phoramidite synthesis protocols.

REFERENCES

1. Omumi, A; Beach, G.D; Baker, M; Manderville, R.A;

Gabryelski, W. JACS 2011,133, 42-50.

2. Schlitt, M.K.; Sun, K.M.; Paugh, R.J.; Millen, A.L.;

Navarro-Whyte, L.; Wetmore, S.D.; Manderville, R.A.

J. Org. Chem. 2009, 74, 5793–5802.

3. Tram, K; Sanghvi, Y.S; Yan, H. Nucleosides, Nucleo-

tides and Nucleic Acids, 2011, 30, 12-19.

145

STRETEGIES TO PREPARE N3-THYMIDINE-ALKYL-N

3-THYMIDINE

INTERSTRAND CROSS-LINKED DNA

Gang Sun,1 Anne M. Noronha,

1 and Christopher J. Wilds

1,*

1Concordia University, Department of Chemistry and Biochemistry, 7141 Sherbrooke St. West, Montréal, Canada.


ABSTRACT

DNA duplexes containing an N3-thymidine-butyl-N

3-

thymidine interstrand cross-link (ICL) were prepared

using an on-column orthogonal deprotection strategy to

permit different nucleotide sequence composition

around the cross-linked site. Efficient removal of 5’-O-

allyloxycarbonyl and 3’-O-tert-butyldimethylsilyl protec-

tive groups enabled successful coupling of 2’-

deoxyphosphoramidites to produce the desired duplex

with a 31 % yield after deprotection and purification.


CONCLUSION

Cellular DNA, on exposure to various agents, can under-

go damage with numerous modifications identified includ-

ing ICLs.[1,2] ICLs block DNA strand separation interfer-

ing with replication and transcription. ICL-inducing agents

are employed in chemotherapeutic treatments for many can-

cers. However, resistance to ICL-inducing agents may be

due in part to efficient DNA repair. One approach to inves-

tigate the role that repair pathways play in removing ICL is

to use chemically synthesized oligonucleotides which con-

tain adducts that represent the lesions introduced by ICL-

inducing agents. Differing strategies have been developed to

produce ICL-containing DNA. One avenue to ICL DNA

synthesis involves the generation of reactive intermediates

which cross-links to the opposing DNA strand.[3] Another

approach involves the synthesis of cross-linked nucleosides

incorporated into a DNA duplex by solid-phase synthesis to

directly introduce the ICL, which enables exact placement

of the desired ICL.

We have previously reported the synthesis of N3-

thymidine-butyl-N3-thymidine (N

3T-butyl-N

3T, Figure 1)

ICL duplexes with either partial or complete symmetry

around the site of the cross-link using nucleoside dimers

containing DMT and TBS protecting groups on the 5’ and

3’-O functionalities using mono- and bis-phosphoramidite

approaches.[4,5] In order to accomplish the synthesis of

asymmetric sequences it is imperative to design the dimer

phosphoramidite to contain three different protective groups

around the 5’- and 3’-O functionalities that are compatible

with each other for selective removal without compromising

the ICL lesion in order to introduce the cross-link into the

DNA duplex.

An 11-bp ICL duplex containing a N3T-butyl-N

3T ICL in

a 1,2 staggered motif has been successfully synthesized us-

ing a phosphoramidite dimer containing 5’-O-

allyloxycarbonyl and 3’-O-tert-butyldimethylsilyl protective

groups. Selective removal of these groups and chain exten-

sion around the ICL site by solid-phase synthesis produced

the desired duplex in a 31% yield from a 1 µmol scale syn-

thesis. The orthogonal deprotection strategy may be valua-

ble for the synthesis of other ICL duplexes to produce sub-

strates for various DNA repair studies.

REFERENCES

1. Dronkert, M.L., Kanaar, R. Mutat. Res.2001, 486, 217-

247

2. Noll, D.M., Mason, T.M., Miller, P.S. Chem. Rev. 2005,

106, 277–301.

3. Hentschel, S., Alzeer, J., Angelov, T., Schärer, O.D.,

Luedtke, N.W. Angew. Chem., Int. Ed. Engl., 2012, 51,

3466-3469.

4. Wilds, C.J., Noronha, A.M, Robidoux, S, Miller, P.S. J.

Am. Chem. Soc. 2004, 126, 9257-9265.

5. Wilds, C.J., Palus, E., Noronha, A.M. Can. J. Chem.

2007, 85, 249-256.

Figure 1.Chemical structure of the N3T-butyl-N3T (n=2).

146

SYNTHESIS AND PHOTOPHYSICAL PROPERTIES OF NOVEL PUSH-PULL-TYPE FLUORESCENT PURINE- AND 7-DEAZAPURINE 2'-DEOXYNUCLEOSIDES

Azusa Suzuki1, Isao Saito2*, Nobukatsu Nemoto1 and Yoshio Saito1*

1Department of Chemical Biology and Applied Chemistry, School of Engineering, Nihon University, Koriyama, Fuku-

shima 963-8642, Japan and 2NEWCAT Institute, School of Engineering, Nihon University. * Correspondence to: Email

address [email protected] (Y. Saito)

ABSTRACT

We have synthesized novel push-pull-type fluorescent

punine- and 7-deazapurine 2'-deoxynucleosides, contain-

ing a 1,6-disubstituted pyrene chromophores. Among

them, 7-deazaadenosine derivatives, CNZ

A, was found to

exhibit a remarkable solvatofluorochromicity.


CONCLUSION

Environmentally sensitive fluorescence nucleosides in which emission spectra and quantum yields change sensi-tively according to solvent polarity are of greatest interest owing to their wide range of applications [1,2]. They are used as fluorescence sensors in various fields, as for exam-ple, incorporation of such solvatofluorochromic nucleosides into oligonucleotides provides powerful tools for the detec-tion of target DNA and SNP genotyping [1].

Recently, we reported C8-substituted push–pull-type flu-orescent guanosines, Ac

G and CNG, which contain a cova-

lently linked electron donor–acceptor system consisting of guanosine as electron donor and pyrene fluorophore as ac-ceptor (Figure 1a)[3]. Although these guanosine derivatives exhibited interesting solvatofluorochromic properties, the steric bulk of the C8-substituents in both Ac

G and CNG caus-

es considerable destabilization of the DNA duplex structure due to their syn-conformation, and thus, these molecules may not be suitable for the use as fluorescent DNA probes. Thus, we have designed novel push-pull-type 7-deazapurine 2'-deoxynucleosides. An electron-withdrawing 4-cyano-phenyl group (acceptor) and 7-deazapurine nucleoside (do-nor) are directly attached to pyrene chromophore via triple bonds in order to construct an intramolecular donor–acceptor system. We report herein the synthesis and photo-

physical properties of novel push–pull-type 7-deaza-2'-deoxyadenosine (CNZ

A) and 7-deaza-2'-deoxyguanosine (CNZ

G) containing a 1,6-disubstituted pyrene chromophore (Figure 1b).

CNZA was synthesized from 7-iodo-7-deaza-2'-deoxy-

adenosine prepared according to protocol Seela et al. [4]. CNZ

G was also synthesized through a similar reaction route. The photophysical properties of newly synthesized push-

pull-type 7-deazapurine derivatives, CNZA and CNZ

G, were examined. Initially, we measured the fluorescence spectra of CNZ

A in various solvents of different polarity. Upon excita-tion of CNZ

A at 416 nm in chloroform, strong fluorescence emission was observed at 470 nm (Φfl = 0.448). Upon exci-tation of CNZ

A in THF, we observed moderate emission at 510 nm (Φfl = 0.305). In contrast, very weak fluorescence emission was observed at 530 nm in a polar solvent such as DMF (Φfl = 0.089). As expected, push–pull-type 7-deaza-2'-deoxyadenosine derivative CNZ

A exhibited a considerable solvatofluorochromicity (Δλfl.max = 60 nm).

The photophysical properties of 7-deaza-2'-deoxy-guanosine derivative CNZ

G were also examined. While the fluorescence intensity of CNZ

G is strong in low-polarity sol-vents such as chloroform, weak fluorescence was observed in polar solvents. In the case of CNZ

G, no remarkable red-shift of fluorescence emission was observed by changing solvent polarity, unlike 7-deazapurine derivative, CNZ

A

which exhibited a redshift with increasing solvent polarity. In summary, we have synthesized novel push-pull-type fluorescent 7-deazapurine nucleosides CNZ

A and CNZG, the

first highly fluorescent pyrene containing 7-deaza-2'-deoxypurine nucleosides. Among them, CNZ

A exhibited strong fluorescence at long wavelength and a remarkable solvatofluorochromicity (Δλfl.max = 60 nm). Such environ-mentally sensitive and strongly fluorescent nucleosides may be used as a fluorescence sensor for structural studies of nucleic acids and as a building block for constructing a fluo-rescent DNA nanostructure. REFERENCES 1. Saito, Y., Miyauchi, Y., Okamoto, A., Saito, I. Tetra-

hedron Lett. 2004, 45, 7827-7831. 2. Tainaka, K., Tanaka, K., Ikeda, S., Nishiza, K., Unzai,

T., Fujiwara, Y., Saito, I., Okamoto, A. J. Am. Chem.

Soc. 2007, 129, 4776-4784. 3. Saito, Y., Suzuki, A., Imai, K., Nemoto, N., Saito, I.

Tetrahedron Lett. 2010, 51, 2606-2609. 4. Seela, F., Zulauf, M. synthesis, 1996, 726-730.

Figure 1. Strucure of push-pull-type fluorescent nucleosides containing a disubstituted pyrene chromophore.

147

RECOGNITION AND FLUORESCENCE DETECTION OF 8-OXO-2’-DEOXYGUANOSINE IN DNA BY ADENOSINE-1,3-DIAZAPHENOXAZINE DERIVATIVE

Yosuke Taniguchi, Yohei Koga, Keitaro Fukabori, Ryota Kawaguchi and Shigeki Sasaki*

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 Ja-pan, * Correspondence to: Email address for. [email protected]

ABSTRACT

The sequence specific detection of 8-oxo-2’-deoxyguanosine (8-oxodG) in DNA without chemical or enzymatic treatment is an attractive tool for genomic research. We designed and synthesized the non-natural nucleoside analogue, the adenosine-1,3-diazaphenoxazine derivative (Adap), for selective recog-nition of 8-oxodG in DNA. This study has clearly shown that Adap has a highly selective stabilizing effect of the duplex having the Adap-8-oxodG base pair and an abil-ity to detect the 8-oxodG in DNA.

INTRODUCTION, RESULTS AND DISCUSSION, CONCLUSION

Cellular DNA is continuously exposed to a variety of chemically reactive species such as alkylating agents, reac-tive oxygen species (ROS), etc., resulting in DNA damage that may increase the risk of developing diseases. 8-Hydroxy-2’-deoxy-guanosine (or 8-oxo-2’-deoxyguanosine, 8-oxodG) is the representative damaged nucleoside, which is generated by the oxidation of 2’-deoxyguanosine triphos-phate (dGTP) in the triphosphate nucleotide pool or 2’-deoxyguanosine (dG) in DNA [1]. There are several meth-ods to detect the 8-oxodG nucleoside using the degradation products of DNA. Therefore, the selective detection of 8-oxo-2’-deoxyguanosine (8-oxodG) in DNA without chemi-cal or enzymatic treatment is an attractive tool for genomic research.

We designed and synthesized the non-natural nucleoside analogue, the adenosine-1,3-diazaphenoxazine derivative

(Adap), for selective recognition of 8-oxodG in DNA with a focus on the glycocil conformation of it. We have success-fully synthesized the Adap phosphoramidite, and it was in-corporated into oligodeoxynucleotide (ODN). From the re-sults of Tm analysis, it is clearly shown that Adap has a high-ly selective stabilizing effect of the duplex having the Adap-8-oxodG base pair. Moreover, the fluorescent property of Adap has been shown to be useful for the selective detection of 8-oxodG in the duplex DNA. To the best of our knowledge, this is the first successful demonstration of the non-natural nucleoside with a high selectivity for 8-oxodG in DNA [2].

Furthermore, we developed a OFF-to-ON type FRET probe, in which one strand contains Adap and another con-tains natural nucleoside for the formation of a less stale double strand. Each strand was labeled with Cy3 or BHQ2 at the 5’-end or 3’-end, respectively. It was expected in this system that fluorescence of the duplex probe was first quenched by FRET, but the target DNA strand containing 8-oxodG at the complementary site of Adap would enhance the displacement reaction of the less stable duplex probe that results in the fluorescence recovery. The results showed that the duplex probe containing the Adap-T base pair ex-hibited a complete discrimination between 8-oxodG and dG in DNA by fluorescence enhancement [3].

In conclusion, we have shown that Adap has a highly se-lective stabilizing effect on the duplex containing the Adap-8-oxodG base pair. And the fluorescent property of Adap has been shown to be useful for the selective detection of 8-oxodG in DNA. Furthermore, we have designed the off-to-on type fluorescent probe for the detection of 8-oxodG. The Adap-T pair masks the base recognition ability of Adap and shows that the effective strand displacement reaction only occurs in the presence of 8-oxodG in the target DNA.

REFERENCES

1. (a) Risom, L.; Moller, P.; Loft, S. Mutat. Res. 2005, 592, 119–137. (b) Knaapen, A. M.; Güngör, N.; Schins, R. P. F.; Borm, P. J. A,; van Schooten, F. J. Mutagenesis 2006, 21, 225–236. (c) Valavanidis, A.; Vlahogianni, T.; Dassenakis, M.; Scoullos, M. Ecotoxicol. Environ. Saf. 2006, 64, 178–189. (d) Halliwell, B. Biochem. J. 2007, 401, 1–11.

2. Taniguchi, Y., Kawaguchi, R., Sasaki, S. J. Am. Chem.Soc., 2011, 133, 7272-7275.

3. Taniguchi, Y., Koga, Y., Fukabori, K., Kawaguchi, R., Sasaki, S., Bioorg. Med. Chem. Lett., 2012, 22, 543-546.

Figure 1. Speculated recognition structure of Adap-oxodG pair.

Adenosine 1,3-diazaphenoxadine

(Adap)

8-oxo-2’-deoxyguanosine(8-oxodG)

148

INVESTIGATION OF RNA APTAMERS TARGETING MITOCHONDRIA BY MITOCHONDRIA-BASED SELEX

Yuri Tawaraya,1* Mamoru Hyodo,

2 Yuma Yamada,

1 and Hideyoshi Harashima

1,2

1 Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-Ku, Sapporo, Japan.

2Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hok-

kaido University, Kita 12, Nishi 6, Kita-Ku, Sapporo, Japan * Correspondence to: [email protected]

ABSTRACT

We have investigated two RNA aptamers (mitomer1,

2) which bind isolated mitochondria identified via mito-

chondoria based SELEX method. We confirmed that

mitomer2 binds to rat liver mitochondria well and short-

mitomer2, which is truncated form of mitomer2, binds

stronger than mitomer2.


CONCLUSION

Mitochondria are essential organelle that produce ATP

and metabolize lipids. It has been reported that functional

abnormalities of mitochondria is related to various diseases

such as diabetes, obesity and so on (1). Exploring drug de-

livery system to mitochondria can open the gate to cure such

diseases. In our research, we chose RNA aptamers as a lig-

and for mitochondria. We selected aptamers by mitochon-

dria-based SELEX (Systematic Evolution of Ligands by

Exponential enrichment) method which is modified from

cell-SELEX (2).

First, we established mitochondria-based SELEX method

(Figure 1). In this method, we applied RNA library that con-

tains random sequences to mitochondria isolated from rat

liver, collected bound RNAs, reverse-transcribed, amplified

and recovered library sequences. At 6th

round, we intro-

duced counter selection using nuclei and collected unbound

sequences. After 7th

round, we cloned and sequenced our

library and obtained two candidate sequences which were

named mitomer1, mitomer2, respectively.

Next, we checked each binding affinity toward isolated

mitochondria. FAM-modified mitomers were incubated

with isolated mitochondria, and bound RNAs were collected

and measured their fluorescence intensity. As the result,

mitomer2 showed higher binding affinity compared to mi-

tomer1. We analysed the secondary structure of mitomer2

and it contains one stem-loop structure with flanking single

strand region. The secondary structure of RNA contributes

the binding capacity to the target and the removal of non-

essential nucleotides increases the binding affinity of ap-

tamers (3). We designed short-mitomer2 which contains

only stem-loop structure of mitomer2 and checked their

affinity. Then, short-mitomer2 showed significantly higher

affinity than mitomer2 and random sequence. It was close to

the same level as D-arm which is the special sequence taken

from Leishmania tRNA which is already known as the tar-

geting device to mitochondria (4). We proved that the stem-

loop structure of mitomer2 is essential to bind mitochondria.

We have established mitochondria-based SELEX method

and obtained two candidates for mitochondria-binding ap-

tamers. We truncated the better candidate, mitomer2 and

short-mitomer2 showed higher binding affinity to isolated

mitochondria.

REFERENCES

1. Johannsen, D. L., Ravussin, E. Curr. Opin. Pharmacol.,

2009, 9, 780-786.

2. Sefah, K., Shangguan, D., Xiong, X. et al., Nat. Protoc.,

2010, 5, 1169-1185.

3. Zhou, J., Battig, M. R., Wang, Y. Anal. Bioanal. Chem.,

2010, 398, 2471-2480.

4. Mahapatra, S., Ghosh, S., Bera, S. K., Ghosh, T., Das,

A., Adhya, S. Nucleic Acids Res., 1998, 26, 2037-2041.

Figure 1. Schematic representation of mitochondria-based SELEX.

Figure 2. Binding assay of mitomer2 and short-mitomer2.

149

HYPOXANTHINE-CONTAINING PNA PROBES FOR IMAGING THE MUTATIONS OF KRAS2 ONCOGENE MRNA

Chang-Po Chen1, Dalip Sethi1, Mathew E. Wampole1, Jeffrey M. Sanders1, Yuan-Yuan Jin1, Mathew L. Thakur2, 3, and Eric Wickstrom1,3*

1Department of Biochemistry & Molecular Biology, 2Radiology; 3Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107 *Correspondence to: Eric Wickstrom. email: [email protected]

ABSTRACT

We hypothesize that determining KRAS2 cancer gene mutation status by external PET imaging of mutant KRAS2 mRNA overexpression will inform decisions on EGFR-targeted cancer therapy. We synthesized a wobble base hypoxanthine PNA monomer to incorporate into radionuclide-chelator-spacer-peptide nucleic acid (PNA)-spacer-insulin-like growth factor 1 (IGF1) analogs for radioimaging of mutant KRAS2 mRNA in suspect masses.

INTRODUCTION

Cancer cell growth depends on high expression of cancer genes. Overexpression of epithelial growth factor receptor (EGFR) stimulates lung cancer cell growth. Anti-EGFR antibodies or tyrosine kinase inhibitors (TKIs) fail in most lung cancer patients. KRAS2 activation makes cancer cell proliferation independent of EGFR activity. As a result, treating lung cancer with anti-EGFR antibodies or TKIs fails when KRAS2 has been mutated. We hypothesize that determining KRAS2 cancer gene mutation status by external genetic PET imaging of mutant KRAS2 mRNA overexpression, as an adjunct to biopsy, will enable physicians to decide on alternatives to EGFR-directed therapies. We designed radionuclide-chelator-spacer-peptide nucleic acid (PNA)-spacer-insulin-like growth factor 1 (IGF1) analogs to enable IGF1R-mediated cellular uptake and radioimaging of mutant KRAS2 mRNA in suspect masses. We demonstrated sequence-specific radioimaging and quantitation of high levels of mutant KRAS2 mRNA in pancreas cancer xenografts after tail vein injection of 99mTc (SPECT) [1] or 64Cu (PET) [2] PNA-spacer-IGF1 probes. However, imaging several specific 12th codon mutants of KRAS2 mRNA would require administration of a cocktail of specific agents. As an alternative, we synthesized a hypoxanthine PNA monomer to enable wobble basepairing to the most frequent base mutations. The monomer was inserted at one or two positions in a chelator-spacer-PNA-spacer-peptide.


In order to develop a general probe for imaging of multi-mutations in KRAS2 mRNA, hypoxanthine-containing PNA monomer was prepared (Scheme 1). Chelator-PNA-peptide sequences were designed (Table 1), assembled, purified, and

characterized. PNA/RNA duplex stabilities were predicted by molecular dynamics. The melting temperatures of the PNA/RNA duplexes were determined.

Scheme 1

OHN

NH

O

O O

N

N N

N

O

COOCH2CH3

N

NH N

N

ON

N N

N

O

COOH

ONH

NO

OO

N

N N

N

OPhe

O

ONH

N

OO

N

N N

N

OH

O

HO

Phe PhePhe

CONCLUSION

In vivo imaging of KRAS2 mRNA mutants in lung cancer is possible. Supported by NIH CA148565; IP owned by EW/MLT, licensed to MTTI.

REFERENCES

1. Amirkhanov, N.V., Zhang, K., Aruva, M.R., Thakur, M.L., and Wickstrom, E. (2010) Bioconj. Chem. 21(4):731-740. PMID: 20232877.

2. Chakrabarti, A., Zhang, K., Aruva, M.R., Cardi, C.A., Opitz, A.W., Wagner, N.J., Thakur, M.L., and Wickstrom, E. (2007) Cancer Biol. Ther. 6(6):948-956. PMID: 17611392.

Table 1. Chelator-AEEA-PNA-AEEA-peptide sequences KRAS2 G12CDV complement SBTG2-DAP-AEEA-GCCAHHAGCTCC-AEEA-D(Cys-Ser-Lys-Cys) KRAS2 G12DV complement SBTG2-DAP-AEEA-GCCAHCAGCTCC-AEEA-D(Cys-Ser-Lys-Cys) KRAS2 G12 wild type SBTG2-DAP-AEEA-GCCACCAGCTCC-AEEA-D(Cys-Ser-Lys-Cys) KRAS2 G12CDV peptide mismatch SBTG2-DAP-AEEA-GCCAHHAGCTCC-AEEA-D(Cys-Ser-Ala-Cys) AEEA: aminoethoxyethoxyacetyl spacer; DAP: diaminopropanoyl; SBTG: S-benzyl thioglycolyl chelator.

150

PCR-FREE TELOMERASE ASSAY WITH CYCLING PROBE TECHNOLOGY TOWARD VALID EVALUATION OF TELOMERASE INHIBITOR

Hidenobu Yaku,1,2,3

Takashi Murashima,1,2

Daisuke Miyoshi 1,2*

and Naoki Sugimoto 1,2*

1Faculty of Frontiers of Innovative Research in Science and Technology (FIRST) and

2Frontier Institute for Biomolecu-

lar Engineering Research (FIBER), Konan Univ., 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan, 3Advanced Technology Research Laboratories, Panasonic Corp., 3-4 Hikaridai, Seika-cho, Soraku-gun, Kyoto 619-0237, Japan. * Correspondence to: (D. M.) [email protected] and (N. S.) [email protected]

ABSTRACT

Novel PCR-free assay for telomerase activity was de-

veloped. The assay is based on a catalytic hydrolysis by

RNase H. Detection limit of this assay was 50 HeLa

cells/L.

INTRODUCTION

Telomerase is responsible for an elongation of telomere

DNA, which is composed of a repeated sequence of 5’-

TTAGGG-3’, at chromosome ends (1). In 85-90% human

tumor cells, telomerase is highly activated, which plays a

role in carcinogenesis (2). Since it was found that G-

quadruplex of telomere DNA inhibits telomerase activity,

development of G-quadruplex ligands as a telomerase inhib-

itor has been attracting attention. For the development, ap-

propriate method for evaluation of telomerase activity is

essential. Telomeric repeat amplification protocol (TRAP)

assay, which utilizes polymerase chain reaction (PCR), is

the most widely-used method because of its high sensitivity

(2). However, many G-quadruplex ligands have been mise-

valuated with TRAP assay because of their PCR inhibitory

effect. Thus, there has been a compelling need for a PCR-

free assay for telomerase activity. Here, we describe a novel

PCR-free assay for telomerase activity based on a catalytic

signal amplification with RNase H toward the valid evalua-

tion of telomerase inhibitory effect of G-quadrulex ligands.


A new telomerase assay proposed here is composed of

four steps (Figure 1): (1) Telomerase elongates the telomere

DNA sequence. (2) Probe RNA hybridizes with the elongat-

ed telomere DNA sequences. The probe RNA has a fluoro-

phore, which is quenched by a quencher. (3) Catalytic

cleavage of the hybridized probe RNA occurs by RNase H.

This catalytic reaction results in the fluorescence enhance-

ment because of release of the fluorophore from the quench-

er. (4) The cycling of the reactions leads to an amplification

of the fluorescence signal reflecting to telomerase activity.

A FRET-based probe RNA, 5’-CCCUAACCC-3’, modi-

fied with FITC (fluorophore) and Dabcyl (quencher) at the

5′ and the 3′ ends, respectively, was synthesized. Firstly, to

proof of the concept, a model sequence of telomerase prod-

uct (MSTP), 5′-(TTAGGG)16-3′, was utilized. After anneal-

ing the sample including 100 nM probe RNA and various

concentrations of MSTP in a buffer containing 50 mM Tris-

HCl (pH8.0), 4 mM MgCl2, the hydrolysis reaction was

carried out with 0.1 U/L RNase H at 30 °C for 30 min. The

fluorescence intensity of FITC at 512 nm with excitation

wavelength of 482 nm increased as a function of the con-

centration of MSTP. The result indicates that the assay de-

veloped here can be used for the detection of telomerase

activity. We then attempted for the detection of telomerase

in HeLa cells with the assay. Various concentrations of

lysed HeLa cells, 100 nM probe RNA, and 0.1 U/L RNase

H were incubated in the reaction buffer at 30 °C for 1 hour

to carry out the simultaneous reactions of telomerase elon-

gation and hydrolysis by RNase H. The fluorescent intensity

increased as a function of HeLa cell concentration and the

detection limit of HeLa cell was 50 cells/L. These results

demonstrate that telomerase activity in HeLa cells is detect-

able without PCR amplification.

CONCLUSION

A novel assay for telomerase activity based on catalytic

signal amplification with RNase H was developed. This

assay can be applied for the valid evaluation of telomerase

inhibitory effect of G-quadruplex ligands without PCR am-

plification.

REFERENCES 1. Morin, G. B. Cell 1989, 59, 521-529.

2. Kim, N. W., et al. Science 1994, 266, 2011-2015.

Figure 1. Novel PCR-free assay for telomerase activity.

RNase H

TS primer

Telomerase

Probe RNA

FluorophoreQuencher

1. Telomerase reaction

3. RNase H reaction

2. Hybridization

4. Signal amplificationFluorescence

RNase HRNase H

TS primer

Telomerase

Probe RNA

FluorophoreQuencher

1. Telomerase reaction

3. RNase H reaction

2. Hybridization

4. Signal amplificationFluorescence

151

ANTHRACENE TAGGED DNA AS FLUORESCENCE SENSORS

Zhengy-yun Zhao,* Jean-Louis Duprey, Jack Manchester and James H. R. Tucker

School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. *e-mail: [email protected]

ABSTRACT

Anthracene with flexible linker as well as a defined thre-

oninol moiety either D or L configuration can be incor-

porated into DNA sequence as a probe. This probe dur-

ing the hybridisation process shows a fluorescence

changes hence could be used to sense base variations as

well as base modifications.


CONCLUSION

The association of single nucleotide polymorphisms (SNPs)

with a number of diseases, for example Alzheimers and

cancer,1,2

has led to the need for direct, rapid, and inexpen-

sive processes to detect specific polymorphisms. One new

approach for establishing base identity in a sequence is to

use modified DNA probes containing base-discriminating

fluorophores, and measure the output upon duplex for-

mation, as depicted in Figure 1. Pyrene-tagged fluorescent

probes and fluorescent nucleobase analogues were reported

in the literature for this purpose.

The current method utilising 1L anthracene, Figure 2, has

been successfully employed to detect a SNP in sequences

associated with prostate cancer.3 It can also be used employ-

ing 4D anthracene to detect epigenetic sequences resulting

from the enzymatic methylation of cytosine.4,5

Recently it

has demonstrated that this probe can differentiate the gua-

nine from 8-oxoguanine due to oxidations in a cellular situa-

tion. The current probes demonstrate versatile uses to detect

various modifications associated with medical importance.

The mechanisms depend on the interaction of fluorophore

with the modification in a local environment through inter-

calation, electron transfer or both to alter the fluorescence

properties.

Host Guest H-bonded duplex

Figure 1 Principle of fluorescence detection

3’ end

Figure 2, L-Threoninol (R,R) linked anthracene moiety

REFERENCES

1. P. Hollingworth, D. Harold, L. Jones, M. J. Owen, J.

Williams, Int. J. Geriatr. Psych., 2011, 26, 793-802.

2. L. J. Engle, C. L. Simpson, J. E. Landers, Oncogene,

2006, 25, 1594-1601.

3. Z. Zhao, M. San, J-L, H. A. Duprey, J. R. Arrand, J. S.

Vyle and J. H. R. Tucker, Bioorg. Med. Chem. Lett.,

2012, 22, 129-132.

4. J-L H. A. Duprey, Z. Zhao, D. M. Bassani, J. Manches-

ter, J. S. Vyle and J. H. R. Tucker. Chem. Commun.,

2011, 47, 6629-6631.

5. J-L. H. A. Duprey, D. M. Bassani, E. I. Hyde, C. Lud-

wig, A. Rodger, J. S. Vyle, J. Wilkie, Z. Zhao and J. H.

R. Tucker, Supramol. Chem., 2011, 23(3-4), 273-277.

152

mailto:[email protected]

FLUORESCENT CAP ANALOGUES FOR MRNA LABELLING

M. Ziemniak1*

, M. Szabelski2, M. Lukaszewicz

1, E. Darzynkiewicz

1, Z. Wieczorek

2 and J. Jemielity

1

1 Division of Biophysics, Faculty of Physics, Warsaw University, Zwirki i Wigury 93, 02-089 Warsaw, Poland. 2

Department of Physics and Biophysics, University of Warmia and Mazury, Oczapowskiego 4, 10-719 Olsztyn, Poland *Correspondence to: [email protected]

ABSTRACT

Five fluorescently labelled triphosphate cap analogues

incorporable into mRNA via in vitro trancription were

designed and synthesised. These compound possess ei-

ther antranoyl (Ant) or mantranoyl (Mant) moiety with-

in the ribose of 7-methylguanosine. The fluorescent

properties of these novel analogues make them valuable

tolls for biophysical studies on cap-binding proteins and

mRNA turnover.

INTRODUCTION

A characteristic feature of almost all eukaryotic mRNA

molecules is the presence of a cap moiety at their 5' termi-

nus. The cap is a distinctive residue composed of 7-

methylguanosine connected to mRNA body via triphosphate

linkage. This unusual chemical structure of the cap deter-

mines its interesting physicochemical properties and it is

crucial for various stages of mRNA metabolism such as

mRNA splicing, intercellular transport, translation and turn-

over. Owing to the paramount importance of that structure

synthetic cap analogues appears to be versatile tools not

only in fundamental research but also in biotechnology and

medicine [1].

Figure 1. Structures of cap analogues 1-5: 1: (R1=H, R2=H, Z=O, X=O) 2: (R1=CH3, R

2=H, Z=O, X=O);

3: (R1=CH3, R2=H, Z=O, X=CH2); 4: (R1=CH3, R

2=H, Z=CH2,

X=O); 5: (R1=H, R2=CH3, Z=O, X=O)

RESULTS AND DISCUSSION,

Five triphosphate cap analogues bearing either Ant or

Mant fluorescent tag were designed and prepared (Fig 1).

Two of these compounds (1-2) are derivatives of a canonical

cap structure m7GpppG without any modification of phos-

phate chain whereas two other compounds (3-4) have one of

the bridging oxygen atoms substituted by a methylene

bridge in order to increase their resistance to enzymatic deg-

radation. All these cap analogues exists as a mixture of 2'

and 3' (M)Ant regioisomers which are in a dynamic equilib-

rium. However, compound 5 bear 2'-O-methyl group in the

m7Guo residue blocking the isomerisation, which results in

a defined position for Ant moiety.

In the first step of the synthesis of compounds 1-5 Ant

and Mant-labelled nucleotides were prepared. It was

achieved by following the synthesis procedure for Ant-GTP

reported by Hiratsuka [2] with modifications. The second

step in preparation of compound 1-5 was the ZnCl2-

mediated coupling of two nucleotide subunits to form a di-

nucleotide 5',5'-triphosphate. For this purpose one subunit is

converted to active P-imidazolide and a second reactant act

as a nucleophilic agent, which allows formation of the pyro-

phosphate bond.

Both absorption and emission spectra of cap analogues 1-

5 are insensitive to pH changes in the range from 5 to 9. The

absorption spectra of cap analogues measured in PBS dis-

play two maxima at 254 nm and 337 nm (Ant) or 349.5 nm

(Mant) which are associated with 7-methylguanosine resi-

due and Ant (Mant) group, respectively. The emission spec-

tra of attached fluorophores measured in PBS display one

maximum at 422.5 nm (Ant) or 446 nm (Mant)

CONCLUSION

Compact size of Ant and Mant residues as well as their high

quantum yields and photostability ensure that fluorescent

cap analogues 1-5 can be used as effective tools in research

on cap-binding proteins and mRNA turnover. Due to re-

placement of one bridging oxygen by methylene moiety cap

analogues 3 and 4 are stable to degradation by Dcp1/2 com-

plex and DcpS enzyme, respectively [3]. It creates an inter-

esting opportunity to research on decapping enzymes in vivo.

On the other hand compound 5 has a defined position of

fluorescent tag, hence it may be used to examine interac-

tions with eIF4E and other cap binding proteins without

reducing fluorescence signal and hindering interpretation of

experimental data.

REFERENCES

1. Jemielity J., Kowalska J., Rydzik A.M., Darzynkiewicz

E. New J. Chem., 2010, 34, 829-844

2. Hiratsuka,T. J. Biol. Chem., 1985, 260, 4784–4790.

3. Grudzien E., Kalek M.,Jemielity J., Darzynkiewicz E.,

Rhoads R. J. Biol. Chem., 2006, 280, 1857-1867

153

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