Bioorganic & Medicinal Chemistry Letters 24 (2014) 4678–4681

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Wavelength shifting oligonucleotide probe for the detectionof adenosine of a target DNA with enhanced fluorescence signal

http://dx.doi.org/10.1016/j.bmcl.2014.08.0310960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +91 361 258 2324; fax: +91 361 258 2349.E-mail address: ssbag75@iitg.ernet.in (S.S. Bag).

Subhendu Sekhar Bag ⇑, Manoj K. Pradhan, Suman K. Das, Subhashis Jana, Raghunath BagBioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology, Guwahati 781039, India

a r t i c l e i n f o

Article history:Received 13 May 2014Revised 23 July 2014Accepted 12 August 2014Available online 19 August 2014

Dedicated to Professor Isao Saito on theoccasion of his 75th birthday

Keywords:Fluorescent oligonucleotide probeDNA detectionFluorescenceWavelength shiftJust Mix & Read strategy

a b s t r a c t

The modulated photophysical property of strong electronically coupled naphthyl uridine linked via a sin-gle C–C bond was explored in DNA detection via wavelength shifting and enhanced fluorescence emissionby a simple ‘Just-Mix & Read’ strategy of homogeneous DNA detection.

� 2014 Elsevier Ltd. All rights reserved.

Hybridization sensitive fluorescent DNA labels have attractedimmense research interest in investigating interbiomolecularinteractions in DNA1 and nucleic acid analysis, such as the detec-tion of single nucleotide polymorphisms (SNPs).2 Creation of suchkind of hybridization sensitive probe nucleoside can be achievedby attaching an organic chromophore via a single C–C covalentbond to DNA bases.3 Such type of directly linked aromatic organicchromophores are expected to possess strong electronic couplingwith the p-electron cloud of the nucleobases which would installmodulated optical property into the nucleoside. Since long, organicchromophores via multiple C–C covalent bonds have been used tolink with DNA bases as label and utilized for genetic analysis.2h–l

However, there exist only two examples of organic chromophores(anthraquinone and pyrene) which are directly linked with20-deoxyuridine via a single C–C bond and have been utilized forDNA detection or studying DNA mediated charge transportprocesses.3e,f To the best of our knowledge, directly linked naph-thalene or its derivatives are not reported as a probe of DNA anal-ysis. Moreover, there is no report in the design of single C–C linkedfluorophore labeled solvatochromic nucleoside that discriminatesbetween the bases of a target DNA opposite of the labeled nucleo-side of the probe DNA via a shift in emission wavelength alongwith distinctly enhanced emission intensity.

Therefore, as a part of our ongoing research effort for the designof microenvironment sensitive fluorescent nucleoside,4 we reportherein the synthesis of naphthyl uridine (NaphU) labeled oligonu-cleotide probe for SNP detection (Fig. 1). We expected that strongelectronic coupling of naphthalene p-cloud with the uridinenucleus would yield modulated optical properties and thus it couldsense the opposite base of a target DNA via a change in absorptionand emission light. We also envisioned that upon binding with itsfully matched complementary sequence, the naphthyl chromo-phore (NaphU) would emit a strong fluorescence signal. On thecontrary, because of lack of Watson–Crick base pairing in themispaired position in a single base mismatched duplex, the fluoro-phore would preferably reside inside the duplex and exhibit eithervery weak or strong fluorescence depending on the nature of theflanking base pair. Thus, with this simple concept one can justmix the two complementary strands and read the fluorescencesignal. Following the ‘Just-Mix & Read strategy’ (Fig. 1A) we wereable to detect matched base adenosine of a target DNA oppositeto the labeled base (NaphU) of probe DNA via the enhanced inten-sity and wavelength shift of fluorescence when the NaphU is flankedbetween G:C base pair.

The possibility of electronic coupling between uridine nucleusand the aromatic p-cloud of naphthalene core in NaphU nucleosidewas first investigated by a theoretical calculation using Gaussian03 program package.5 Thus, based on time dependent density func-tional (TD-DFT) calculation at B3LYP/6-31G⁄ level of theory we

Figure 1. (A) Schematic presentation of the concept of DNA detection. (B) Chemicalstructure of the fluorescent nucleoside (NaphU) and its LUMO diagram.

S. S. Bag et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4678–4681 4679

observed that the nucleoside NaphU has a large LUMO coefficient onthe carbon at the 2-position of naphthalene that connects to theC-5 of uridine moiety. Moreover, a significant amount of LUMOcoefficient was present on the �C@O and �C@C� functionalitiesof uridine nucleus (Fig. 1B). The significant overlap of the uridineacceptor and naphthalene donor orbitals supported the strongelectronic coupling between them. Therefore, the nucleoside NaphUexpectedly would show modulated optical properties which uponincorporation into short oligonucleotide sequence could sense DNAmicroenvironment via a change in absorption/emission intensityand wavelength.

With the above design concept and inspiring result from a the-oretical calculation we synthesized the naphthyl conjugated nucle-oside (NaphU) via a Pd(0)-mediated classical Suzuki3,6 couplingprotocol and characterized by NMR and mass spectrometry(Scheme 1). After having the pure nucleoside in hand, we firstexamined the UV–visible and fluorescence photophysical propertyof the nucleoside in various organic solvents. Thus, the UV–visiblespectra of NaphU (1) in all solvents exhibited structure-less bandswith a bathochromic shift of absorbance from that of pure naph-thalene (kmax (naphthalene) = 280–290 nm ? kmax (NaphU) = 302–312 nm) as the solvent polarity increases (see SI, Fig. S2). Uponexcitation of NaphU at the respective absorption wavelength max-ima of each solvent, broad, structureless and red shifted fluores-cence emission bands (386–404 nm) were observed as thesolvent polarity increases. All these spectroscopic propertiesclearly supported the strong electronic coupling between the twoaromatic parts of naphthalene and uridine in NaphU (1) that wereour expectation from the design and theoretical study.3f

The emissive state of the nucleoside NaphU was characterizedwith significant electron redistribution, that is, ICT feature as wasrevealed from the overlapping of HOMO–LUMO as well as the tran-sition oscillator strength. Thus, the calculated excitation energy (invacuum) for the transition of S0 ? S1 of NaphU was found to be

O

O

DMTrON

HNO

O

P NONC

Reagents and Conditions: (a) (i) n-BuLi, triispinacol, 1 : 1 Et2O-5% HCl, 40 oC, 2h; (b) 3, Pd((c) DMTrCl/Pyridine/DMAP, r.t.; (d) CH2Cl2, Et3N,

(b)

DNA

synthesis

ODN 1: 5'-CGCAX =

ODN 9: 5'-CGCA

6

O

OH

HON

HNO

O

I

4O

OH

HON

HNO

O

1 (65%

(

Scheme 1. Synthesis of fluorescent oligonucleotide

316 nm (with f = 0.17 and CI = 0.522) which was in close agree-ment with the experimental result of 312 nm (in toluene). Thesecalculations rationalized the explanation of ICT origin of the sol-vent polarity dependency of the fluorophores’ emission (see SI,Section 7).7

As the naphthalene is electronically coupled with the aromaticsystem of the uridine nucleus we expected that NaphU would beusable for sensing the dielectric property of the local microenviron-ment in DNA via the shift in wavelength of emission and enhancedintensity. Therefore, to establish our expectation the nucleosideNaphU was next incorporated into short 13-mer oligonucleotidesequence containing cytosine as the flanking base for DNA detec-tion via standard DNA synthesis protocol in an automated DNA/RNA synthesizer (Scheme 1, Table 1). The ODN was purified andcharacterized by MALDI-TOF mass spectrometry (see SI, Section 3).

We next studied the UV–visible absorption and fluorescenceemission properties of the single strand probe ODN 1 as well asits various duplex ODNs. Thus, from the UV–visible spectra, itwas observed that various single base mismatched duplexes ofODN 1 exhibited a bathochromic shift along with a hypochromiceffect in comparison to the corresponding single strand ODN 1(Fig. 2A, Table 1). This indicated that in the mismatched duplexesa rotation of NaphU of probe ODN 1 might lead to pairing betweenthe mismatched base of the target ODN 3–7 and naphthyl unit ofprobe ODN 1 resulting in an intercalation of naphthyl moietybetween the base stack. This intercalation reasonably was moreprominent when the opposite base of target ODN is an abasic site(U) wherein the bathochromic shift and hypochromism was max-imum. On the other hand, the fully matched duplex ODN 1�2 con-taining adenosine natural base in target ODN 2 opposite to theNaphU nucleoside of probe ODN 1 showed minimal shift in absorp-tion band and a small hyperchromism (Fig. 2A). This clearly sup-ported that NaphU of probe ODN 1 involved in Watson–Crick basepairing with the opposite matched base adenosine of target ODN2. Thus, rigid naphthyl unit involved in a partial intercalation typeof stacking interaction with the bases pointing along the majorgroove of compact hydrophobic environment.8 The comparablethermal melting stability of the fully matched duplex ODN 1�2(Tm = 59.3 �C) to that of an unmodified duplex ODN 8�2(Tm = 58.8 �C) also supported our explanation (Table 1).3e,3f

From the fluorescence spectra (Fig. 2B, Table 1) it was observedthat the probe ODN 1 upon hybridization with any of the single basemismatched ODNs (ODN 3–6) or with abasic site containing ODN 7showed drastically quenched fluorescence emission. This might bebecause of the intercalation of naphthyl moiety between the flank-ing G:C base pair which was also supported from the shift in UV–visible spectra. Interestingly, perfectly matched duplex ODN 1�2

Br

BOO

opropylborate, THF, -78 oC, 15 min, (ii)PPh3)4, 2:1 toluene-1N NaOH, reflux, 12 h;Diisopropylchlorophosphoramidite, 3-4 h.

AC X CAACGC-3'NaphUAT X TAACGC-3'

)O

OH

DMTrON

HNO

O

5 (65%)

(d)c)

(a)2

3 (90%)

probe containing fluorescent nucleoside, NaphU.

Figure 3. (A) Deconvoluted CD spectra (inset: induced CD) of all the duplexes used.(B) Amber⁄ optimized geometry of the fully matched duplex ODN 1�2.

Table 1Summary of photophysical properties of ODN 1 and its various duplexes

ODNs Sequence

ODN 1 50-CGCAAC NaphU CAACGC-30

ODN 2–7 30-GCGTTG N GTTGCG-50 [N = A, G, C, T, MeC, U]ODN 8 50-CGCAAC T CAACGC-30

ODNs kabs (nm) emax � 103 Dkabs kfl (nm) Dkfl Uf Tm (�C)

1 256, 313 10.4 — 434 — 0.05 —1�2 255, 313 10.9 — 408 26 0.09 59.31�3 256, 318 10.1 5 432 02 0.02 53.11�4 256, 318 10.5 5 424 10 0.02 53.51�5 256, 317 9.9 4 421 13 0.03 53.41�6 257, 317 9.7 4 427 7 0.03 51.31�7 257, 319 8.4 6 429 5 0.01 54.2

Tm of unmodified duplex (ODN 8�2) in G–C flanking sequence = 58.8 �C. Error in Tm

is estimated at ±0.3 �C.

Figure 2. UV–visible (A) and fluorescence emission (kex = 310 nm) (B) spectra ofODN 1 and its various duplexes with ODN 2–7. Final duplex concentration was2.5 lM in 50 mM sodium phosphate, 0.1 M NaCl, pH 7.0, rt.

4680 S. S. Bag et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4678–4681

showed a large enhancement of emission at 434 nm upon excita-tion at 310 nm. Moreover, all the duplexes exhibited blue shiftedemission compared to their corresponding single stranded ODN 1.Most notably, the blue shift in emission wavelength was maximum(26 nm) in case of the fully matched duplex ODN 1�2. Therefore, theprobe ODN 1 having G:C flanking pair was able to detect matchedadenosine base of the target ODN 2 opposite of labeled base NaphUof probe ODN 1 via the generation of a distinct, drastically enhancedand maximally blue shifted fluorescence signal. The drastic changein fluorescence emission intensity along with a maximum blue shiftof wavelength for perfectly matched duplex ODN 1�2 indicated thatthe naphthyl moiety located in the compact hydrophobic microen-vironment pointing toward the major groove of the duplex whichwas also supported from UV–visible spectroscopy (Fig 2A). Similarresult of blue shifting (27 nm) was also observed with other fluores-cent probe ODN 9 (ODN 9: 50-CGCAAT NaphU TAACGC-30) containing–T– flanking base which showed reasonably non-discriminatingfluorescence enhancement upon pairing with any of the comple-mentary target ODNs (see SI, Fig. S5). In case of all mismatchedduplexes the naphthyl moiety, similar to the case of C:G flankingpair sequence, also involved in intercalation between the flankingnon-quenching T:A base pair. However, in contrast to the C:G flank-ing base pair (Fig. 2B), the non-quenching nature of A:T flankingpair resulted in enhanced fluorescence signals from all the mis-matched duplexes comparable to the intensity of the signal gener-ated from fully matched duplex (see SI, Fig. S5).

To know about the spatial disposition of the naphthyl chromo-phoric moiety inside the duplex, we examined the global propertyof all possible duplexes using circular dichroism (CD) spectroscopy.Thus, all spectra showed a positive band at around k = 281–285 nmand a negative band at around k = 250–252 nm of varying magni-tude with intersection at around k = 263–265 nm (Fig. 3A and seeFig. S6). Thus, CD spectra reflected that the naphthyl unit of NaphUstabilized B-form conformation of the duplexes. The fully matchedduplex ODN 1�2 showed a distinct CD signal with a 4 nm blue shift

of positive band of highest intensity indicating a strong stackinginteraction between naphthyl unit of NaphU and the base pairs ofduplex. The induced CD signal between 300–350 nm correspond-ing to the chromophores’ absorption band supported that the con-formational freedom of naphthyl moiety was restricted in thegroove in case of fully matched duplex (ODN 1�2, NaphU:A) andinside the duplex upon intercalation in case of single base mis-matched duplexes (ODN 1�3–6) and abasic duplex (ODN 1�7)(Fig. 3A).9 This observation was also supported by the increase influorescence anisotropy of the duplexes (see SI, Table S4) in com-parison to the single strand probe ODN 1 and a macromodel study(Fig. 3B and see SI, Fig. S7).

It is worth noting that the nucleoside labels attached via multipleC–C covalent bonds and flanked between G:C base pair were foundto be unable to generate discriminating fluorescence signal in sens-ing of opposite base of target DNAs which might be because ofquenching of fluorescence by G:C flanking base pair.2h,i,l,4b On thecontrary and very interestingly, inspite of flanked between G:C basepair, our present probe ODN 1 is capable of sensing oppositematched base –A– of target ODN 2 with a clear discriminating signalto noise ratio (S/N = 2.0). This might be because of more rigid dispo-sition of the labeled nucleoside NaphU compared to the nucleosidelabeled with fluorophore via multiple C–C bonds within the duplex.Though, the present probe relies on the light of excitation in theUV–region, the concept of wavelength shift-guided DNA detectionand our inspiring result would shed light for the design of more ofsuch long wave length emissive probe which could be utilizedfor DNA detection even under the quenching environment of G:Cflanking base pair.

In conclusion, we established that the strong electronic cou-pling between naphthalene p-cloud and uridine nucleus linkedvia a single C–C bond led to a modulated photophysical propertyin NaphU which was explored, for the first time, in DNA detection.The oligonucleotide probe containing NaphU was found to be highlyspecific for the detection of matched base adenosine of a targetDNA opposite to the labeled base (NaphU) of probe DNA via maxi-mum shift in emission wavelength. Interestingly, the probe nucle-oside flanked between G:C base pair could sense efficiently thepresence of opposite matched base –A– of target DNA both viathe maximum shift in wavelength and enhancement of intensityof fluorescence signal which is a first report to the best of ourknowledge. The concept of wavelength shift-guided DNA detectionis very important and would shed light in the design of long wave-length emissive fluorescent nucleoside which could be applied incellular DNA detection. Designing more of such probes for DNAdetection is our current research focus.

Acknowledgments

Author S.S.B. is thankful to Department of Biotechnology, Govt. ofIndia for a financial support [DBT: BT/PR5169/BRB/10/1065/2012].

S. S. Bag et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4678–4681 4681

M.K.P., S.K.D. and S.J. are thankful to UGC and CSIR, respectively, fortheir fellowships.

Supplementary data

Supplementary data (synthesis, spectroscopic data, macromod-el study) associated with this article can be found, in the onlineversion, at http://dx.doi.org/10.1016/j.bmcl.2014.08.031.

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