Aptamers with fluorescence-signaling properties

Methods 37 (2005) 16–25

www.elsevier.com/locate/ymeth

Aptamers with Xuorescence-signaling properties

Razvan Nutiu, Yingfu Li ¤

Department of Biochemistry and Biological Sciences, McMaster University, Hamilton, Ont., Canada L8N 3Z5Department of Chemistry, McMaster University, Hamilton, Ont., Canada L8N 3Z5

Accepted 1 May 2005

Abstract

Aptamers are single-stranded DNA or RNA molecules with ligand-binding capabilities. Signaling aptamers refer to aptamers ormodiWed aptamers with recordable signal generation ability. Fluorescence-signaling aptamers, in particular, are valuable moleculartools that can be used to establish important techniques or assays for the interrogation of identities and concentrations of proteinsand metabolites. Since standard DNA and RNA aptamers themselves are not inherently Xuorescent, modiWcation methods arerequired for rationally converting non-Xuorescent aptamers into Xuorescent reporters or for selecting Xuorescent aptamers directlyfrom random-sequence DNA libraries by in vitro selection. This article will provide a brief review of various signaling aptamerdesign strategies as well as a detailed description of methods that can be used to generate, by both rational design and in vitro selec-tion, a special class of signaling aptamers dubbed “structure-switching signaling aptamers.” This class of signaling aptamers aredesigned to function by switching structures from a pre-formed, lowly Xuorescent duplex assembly to a ligand–aptamer complexhaving a higher level of Xuorescence. 2005 Elsevier Inc. All rights reserved.

Keywords: Aptamer; Fluorescence; Molecular design; In vitro selection; Biosensor; Molecular recognition element; DNA; RNA

1. Introduction

DNA, RNA, proteins, and metabolites are extremelyimportant biomolecules, and methods that can accuratelyreport their identities, concentrations or interactions,either in vivo or in vitro, are extremely useful in both basicscientiWc research and medical diagnosis. Because of thecomplex and dynamic nature of molecular composition ofa cell, a detection technique that can be used to simulta-neously interrogate a large number of gene products aswell as diVerent forms of gene products (RNA, proteins,and metabolites) is highly desirable. It would be ideal ifsuch a detection technique can be set up using a commonand easily obtainable material for the construction ofmolecular recognition elements (MREs), and a universaland convenient signal generation method that can report

* Corresponding author. Fax: +1 905 5229033.E-mail address: liying@mcmaster.ca (Y. Li).

1046-2023/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ymeth.2005.07.001

all probe–target interactions in question. Fluorescentreporters made of nucleic acids have the required charac-teristics to formulate such a technique [1].

Nucleic acids—DNA, RNA, and even various ver-sions of modiWed DNA and RNA [2–4]—are widelyknown for their ability to form Watson–Crick doublehelical structures. Because of the inherent duplex-form-ing capability, nucleic acids have been extensively uti-lized as MREs in the design of many important DNAand RNA detection techniques ranging from the simpleSouthern blotting procedure [5] to the complex DNAmicroarray technology [6,7]. Using nucleic acids asMREs has gained even more momentum in recent yearsdue to the discovery of a new class of nucleic acid mole-cules known as aptamers that have the ability to recog-nize a broad scope of non-nucleic acid ligands includingproteins and metabolites [1,8–10].

Aptamers refer to single-stranded DNA, RNA, andeven modiWed nucleic acid molecules that have the

R. Nutiu, Y. Li / Methods 37 (2005) 16–25 17

ability to form deWned tertiary structures to engage aspeciWc target for binding. Originally, aptamers wereknown exclusively as man-made molecules isolated fromsynthetic random-sequence DNA or RNA libraries by atechnique known as SELEX or “in vitro selection”[11,12]. However, recent studies have revealed thatMother Nature has been using many diVerent RNAaptamers (known as “riboswitches”) in cells to controlgene expression [13,14]. To date, a large number of artiW-cial aptamers have been reported as aYnity probes forproteins and small-molecule metabolites with high aYn-ity and speciWcity. The availability of diverse aptamershas laid solid foundation for exploring aptamers ashighly coveted molecular tools for both basic researchand medical diagnosis [15]. With their inherent ability torecognize DNA and RNA and their newfound capabil-ity to bind proteins and metabolites, nucleic acids havequickly become a popular building material for the con-struction of versatile MREs for biosensing applications.

Although aptamers have signiWcantly expanded theutility of nucleic acids as MREs, standard DNA andRNA aptamers do not have inherent properties withwhich a convenient method can be devised to report anaptamer–target interaction. Therefore, to take fulladvantage of aptamers as aYnity probes, it is necessaryto Wnd ways to link the molecular recognition capabilityof aptamers to a signal generation process that is simple(without the need of a complicated modiWcation schemeto alter an aptamer for signal generation), universal(regardless of what characteristics an aptamer and itstarget exhibits), and convenient to use (with minimalsample manipulation during the signal acquiring pro-cess). Considering all these, Xuorescence spectroscopybecomes a top-choice method.

Fluorescence technique is highly compatible withnucleic acid aptamers. First, there are a large number ofwell-characterized Xuorophores and quenchers that canbe used to modify nucleic acids during the automatedsynthesis or using simple post-synthesis chemistries. Sec-ond, Xuorophore-dressing of nucleic acids eliminates theneed of target-labeling and therefore it can be univer-sally applied to any aptamer–target pair. Third, Xuores-cence oVers a very convenient way to report molecularinteractions because the detection can be carried out inreal time without the need for the separation of target–probe complexes from unbound probes. Furthermore,the availability of many diVerent Xuorophores withdiVerent excitation and emission wavelengths makes itfeasible to conduct multiplexed assays with which sev-eral targets within the same sample can be assessed atonce without cross interference. Because of these attrac-tive features associated with Xuorescence, signiWcantresearch activities have been directed at designing apta-mers with Xuorescence-signaling capabilities in recentyears [1,16]. Several rational design strategies have beenreported for the transformation of aptamers into

Xuorescent probes and they can be summarized into thefollowing seven categories:

1.1. Monochromophore approach (Fig. 1A)

This method involves covalent attachment of a singleXuorophore onto an aptamer at a position that can expe-rience signiWcant structural reorganization upon bindingof the target (grey star) [17,18]. If this change can

Fig. 1. Signaling aptamer rational design strategies. (A) Monochromo-phore approach. (B) Bischromophore approach. (C) Duplex-to-com-plex structure-switching approach. (D) In situ labeling approach. (E)Chimeric aptamer approach. (F) Dye-staining approach. (G) Apt-amer–polymer conjugate approach.

18 R. Nutiu, Y. Li / Methods 37 (2005) 16–25

substantially alter the electronic environment of theattached Xuorophore, an increase or decrease of Xuores-cence intensity will be produced.

1.2. Bischromophore approach (aptamer beacons) (Fig. 1B)

This strategy explores covalent attachment of a pair ofXuorophore and quencher [19–23], or two Xuorophores[24,25], onto an aptamer in such a way that binding of thetarget to the aptamer will either increase or decrease thedistance between the Xuorophore and the quencher (orthe second Xuorophore). The change of distance will alterthe eYciency of Xuorescence quenching between the Xuoro-phore and the quencher or that of Xuorescence resonanceenergy transfer (FRET) between the two Xuorophores,resulting in a change in Xuorescence intensity.

1.3. Duplex-to-complex structure-switching approach (Fig. 1C)

This method takes advantage of the fact that everyDNA or RNA aptamer has the ability to form a duplexstructure with a complementary sequence and complexstructure with its target [26]. The addition of the targetto a mixture containing the duplex of a Xuorophore-labeled aptamer and a quencher-modiWed antisensenucleic acid (QDNA) will force the release of the anti-sense from the aptamer, which is spontaneously accom-panied by the increase of Xuorescence intensity.

1.4. In situ labeling approach (Fig. 1D)

This technique uses in situ labeling of an aptamer with aXuorophore onto a reactive group placed on a nucleotide ofthe aptamer pre-dressed with another Xuorophore [27]. Iftarget-induced aptamer folding can alter the reactivity ofthe reactive group, a FRET signal (or loss of FRET) will beobserved as compared to the sample that lacks the target.

1.5. Chimeric aptamer approach (Fig. 1E)

This strategy is designed to link two aptamerdomains—one that engages a Xuorophore for signalingand the other that binds a non-Xuorescent target—intoone sequence in such a way that the binding of the non-Xuorescent target (the real target of interest) willstrengthen [28] or reduce [29] the aYnity of the chimericaptamer for the Xuorophore. The signal is producedbecause the aptamer–Xuorophore association or separa-tion is accompanied by a change in Xuorescence intensity.

1.6. Dye-staining approach (Fig. 1F)

This method utilizes a duplex-binding dye as anindicator to report the increase (or decrease) of helical

content within the folded structure of an aptamerinduced by target binding [30].

1.7. Aptamer–polymer conjugate approach (Fig. 1G)

This method is based on the Xuorescent (colorimetric)properties of water-soluble cationic polythiophene poly-mer that is conjugated to the anionic ssDNA [31–33].The formation of an aptamer–target complex preventsthe formation of the polymer–ssDNA conjugate, thusaltering the optical properties of the polymer [34].

2. Structure-switching signaling aptamers

Our group reported the duplex-to-complex structure-switching concept (Fig. 1C) [26]. This rational designapproach has the following key features [16].

2.1. Generality

The structure-switching approach is generally appli-cable to any aptamer regardless of its size and structuralproperties. This is because all aptamers share a commonability to form both a duplex structure with an antisenseDNA and a complex structure with their cognate target.

2.2. Simplicity

The structure-switching concept is easy to implement.The key step is to choose a suitable antisense sequence toa given aptamer for the formation of a duplex structurewith suitable strength. This task is easy to accomplishthrough experimenting diVerent number of base pairs[26] or introducing mismatch pairs [35].

2.3. Flexibility

It is Xexible to choose a Xuorophore-labeling site onthe aptamer (end-labeling vs. internal labeling) [26]. Inaddition, there is no restriction on the choice of a Xuoro-phore–quencher pair [16].

2.4. Cost-eVectiveness

When working with two or more aptamers, the struc-ture-switching approach is highly cost-eVective becausea common Xuorophore-labeled DNA molecule can beused to tag any plain aptamer sequence in question [26].

2.5. Performance of signaling aptamers

Signaling aptamers designed by structure-switchingmethod come with a large signaling magnitude and a fastand real-time reporting capability [26]. Signaling apta-mers exhibiting large Xuorescence enhancements upon

R. Nutiu, Y. Li / Methods 37 (2005) 16–25 19

target binding increase the sensitivity and accuracy ofdetection assays. The real-time reporting capabilityallows rapid sample measurements and permits demand-ing applications such as high throughput screening. Thesensitivity of structure-switching aptamers has recentlybeen illustrated by us using a signaling DNA aptamerthat can distinguish the subtle diVerence between adeno-sine 5�-monophosphate (AMP) and adenosine. Thus, thisaptamer can monitor the enzymatic reaction of AMP-to-adenosine conversion by alkaline phosphatase [36].

Further added to the above list of attractiveness is thepossibility to combine structure-switching concept within vitro selection practice, which allows the isolation ofstructure-switching aptamers directly from a random-sequence DNA or RNA library [37]. Through this inte-gration, the aptamers to be isolated are inherentlyencoded with a duplex-to-complex switching capabilityand can be instantly converted into signaling aptamersupon their isolation [37].

Taking together, structure-switching aptamers areparticularly useful as reagents to build solution-based,real-time Xuorescence assays for various in vitro applica-tions. In this paper, we will use speciWc examples to dis-cuss detailed methods and protocols for the rationaldesign of structure-switching signaling aptamers fromplain aptamers, for the creation of structure-switchingsignaling aptamers by in vitro selection, and for theexploration of signaling aptamers as reporter moleculesto monitor enzymatic reactions for various applications.

3. Description of methods

3.1. Rational design of structure-switching signaling aptamers

Structure-switching aptamers are designed to func-tion by switching structures from a pre-formed duplexassembly between a Xuorophore-labeled aptamer and aquencher-labeled DNA sequence (denoted QDNA) to atarget-induced complex structure. The displacement ofQDNA by the aptamer target will result in thedequenching of the Xuorophore and an increase of Xuo-rescence intensity.

Two essential steps have to be followed when design-ing a structure-switching aptamer from a standard DNAaptamer. First, the aptamer needs to be labeled with aXuorophore (F) at a location that will not inactivate theaptamer. Second, one or several QDNA molecules needto be produced and examined for target-induced struc-ture-switching ability. A key factor for considerationwhen choosing a suitable QDNA is the stability of theaptamer–QDNA duplex. QDNA must be long enoughto form a suYciently stable duplex with the aptamer sothat the Xuorescence intensity of the aptamer solution islow in the absence of the target. However, QDNA can-

not be too long; otherwise the duplex will be too stableto allow the release of the QDNA by the target. A goodapproach is to test a few QDNAs with variable lengthsor targeting diVerent stretches of the aptamer sequencefor binding. We have observed that short DNAsequences of 11–13 nucleotides oVers both a low Xuores-cence background and high structure-switching ability[26]. Longer QDNAs may also be used if one or two mis-match base pairs are included in the design [35]. Theswitching ability of a duplex assembly can also be exam-ined as a function of temperature—a duplex that doesnot switch at a given temperature may switch easily at anelevated temperature [26]. Furthermore, we have foundthat a ‘nonsense’ sequence of 4–6 nucleotides can beadded onto one end of the aptamer as part of QDNAbinding sequence. With this arrangement, fewer nucleo-tides of target-binding domain in the aptamer are occu-pied by the QDNA, which makes the displacement ofQDNA considerably easier [26].

A previously isolated ATP-binding aptamer [38] isused here as an example to illustrate how to design a suc-cessful structure-switching signaling aptamer design(Fig. 2). Three separate DNA molecules are used:FDNA1 (a 15-nt DNA molecule with a Xuoresceinattached to its 5�-end), QDNA1 (12-nt DNA moleculewith a DABCYL attached to its 3�-end), and MAP (themodiWed aptamer). MAP contains the sequence of theoriginal ATP-binding DNA aptamer (underlined nucle-otides, 27 nt) and an additional 15-nt element that cananneal to FDNA1. In addition, six nucleotides (italicizedletters) are inserted between the aptamer domain and theFDNA1-binding domain. The 12-nt QDNA1 is designedto bind the last Wve nucleotides of this nonsense regionas well as the seven adjacent nucleotides on the aptamerelement. This arrangement makes the structure-switch-ing process considerably easier. This is because the target(ATP) only needs to compete with seven nucleotides inQDNA1 that block the target-binding site in the apt-amer while overall stability of QDNA1–MAP duplex isstill high. After the formation of ATP–aptamer complex,the remaining 5-bp duplex between QDNA and Wveinserted nucleotides in MAP is not strong enough tohold QDNA onto MAP. The 12-nt QDNA1 oVers botha low melting point (desirable for quick target-inducedstructure switching) and a low level of background Xuo-rescence (indicative of a stable QDNA–MAP duplex).The choice of the tripartite duplex assembly, in particu-

Fig. 2. A structure-switching signaling aptamer for ATP reporting. It isa duplex assembly made of FDNA1, QDNA1, and MAP. There arethree segments within the sequence of MAP: ATP binding domain(underlined letters), FDNA1-binding domain (grey letters), and non-sense domain (italicized letters).

20 R. Nutiu, Y. Li / Methods 37 (2005) 16–25

lar, is due to the consideration of cost-eVectivenessbecause this same FDNA1 can be used to assemblediVerent Xuorescence reporters from diVerent aptamers.The sequence of FDNA1 is arbitrarily chosen. A short,GC-rich FDNA1 is used to insure that the FDNA1–MAP duplex has a high stability. Longer FDNAs withor without high GC content can also be used.

3.2. In vitro selection of structure-switching signaling aptamers

As discussed above, rational design approaches canbe applied to generate Xuorescent reporters from exist-ing aptamers. However, when no aptamer is available fora target of interest, it becomes fairly tedious andresource-consuming to Wrst create a non-Xuorescent apt-amer by in vitro selection and then to convert it into aXuorescent reporter by a rational design method. Analternate approach is to integrate in vitro selection andrational design into one method so that aptamers to beisolated will have an encoded Xuorescence-signalingcapability. With this idea in mind, we have developed anovel in vitro selection technique that incorporates thestructure-switching concept (Fig. 3) [37].

A DNA library used for a typical SELEX contains acentral random-sequence domain Xanked by two primer-binding domains (PBDs); the DNA library used for ourstructure-switching based SELEX also contains twoPDBs, but it has two short random-sequence domains(segments in light blue) sandwiching a central Wxed-sequence motif (red). The central Wxed-sequence domainis design to form a duplex structure with a complementary

Fig. 3. In vitro selection of structure-switching signaling aptamers. (I)L1 (DNA library), BDNA (biotin-containing DNA), P1, and P2 arehybridized and immobilized on avidin-coated beads. (II) Aptamerscapable of switch structures from the duplex with BDNA to the com-plex with the target are isolated by target elution. (III) The selectedsequences are ampliWed by PCR using P2 and P3 as primers. (IV) Thesense strand is isolated PAGE puriWcation following a treatment withNaOH. The resulted DNA molecules are again annealed to BDNA,P1, and P2 for a new round of selection.

DNA biotinylated at the 5�-end (denoted BDNA, shownin pink), which permits the immobilization of the DNAlibrary onto avidin-coated beads. With this arrangement,the potential aptamers in the DNA library will adopt aduplex structure and stay in column-bound state until thetarget is used to release them from the duplex assembly.Therefore, the aptamers obtained by this approach willhave an encoded structure-switching capability.

Two short DNA molecules, named P1 and P2 inFig. 3, that are complementary to each of PDBs are alsoincluded in the duplex assembly for two important pur-poses. First, they will block the physical involvement ofPDBs in the tertiary folding because single-strandedDNA has an enhanced chance to participate in struc-tural folding than a duplex DNA. More importantly,one of these two DNA molecules can be chosen asFDNA while BDNA can be redesigned into QDNA. Inother words, an aptamer can be instantly formulatedinto a Xuorescence reporter upon its isolation by attach-ing a Xuorophore on P1 (or P2) and a quencher toBDNA—no optimization steps are needed.

We have demonstrated the above method by con-ducting an in vitro selection using four standard nucleo-tide 5�-triphosphates (ATP, GTP, CTP, and UTP) as amixture of targets. Structure-switching aptamers that areresponsive to ATP or GTP have been isolated (forunknown reasons, we have failed to isolate CTP andUTP binding aptamers). The results from this study havebeen published elsewhere [37] and the detailed protocolfor in vitro selection is given in Section 5.

3.3. Application of structure-switching signaling aptamers

The straightforward application of a structure-switch-ing signaling aptamer is the detection of the cognate targetof the aptamer. For example, the FDNA1–QDNA1–MAP signaling aptamer duplex can function as a sensitivereal-time reporter for ATP. When this reporter is exposedto ATP, the structure-switching process is completed inless then two minutes and more than 10-fold Xuorescenceenhancement can be observed (Fig. 4).

Fig. 4. An ATP reporting assay. The Xuorescence intensity of MAP–FDNA1–QDNA1 duplex solution is continuously monitored. At the6th min, ATP is added.

R. Nutiu, Y. Li / Methods 37 (2005) 16–25 21

Two or more structure-switching aptamers labeledwith diVerent Xuorophores can be used to report multi-ple targets simultaneously. Fig. 5 is an illustration of atwo-color assay that can be used to report the presenceof both ATP and GTP in the same solutions. The ATP-binding aptamer, ATP1.1, can form a signaling duplexwith FDNA1 (carrying Xuorescein) and QDNA2 whilethe GTP-binding aptamer, GTP1.2 can form a signalingduplex with FDNA2 (carrying Cy3) and QDNA2. Whenboth reporters are mixed into a single solution, theresulting mixture can generate a real-time signal for bothATP and GTP.

If a signaling aptamer can be designed to produce adiVerential signal for the starting material and the prod-uct of a given enzymatic reaction, the aptamer can thenbe used to monitor the activity of the concerned enzyme.For example, we have found that the FDNA1–QDNA1–MAP structure-switching signaling aptamer exhibitsdiVerent Xuorescence intensities in the presence of anequal amount of adenosine and AMP; thus this aptamercan be used as a reporter for nucleotide-dephosphoryl-ating enzymes such as alkaline phosphatase (ALP),which is known to remove the 5�-phosphate groups fromAMP and convert it into adenosine (Fig. 6). Althoughthe signaling aptamer may not have a highly practicalvalue for use as an ALP reporter as many convenientmethods are already available for ALP detection [39–41], it is conceivable that the structure-switching mecha-nism can be generalized for the future design of newaptamers for monitoring the activity of any enzyme thatdoes not have an easy reporting method.

Fig. 5. Two-color detection of ATP and GTP in a single solution.ATP1.1 (an ATP-binding DNA aptamer) is labeled with Xuorescein,GTP1.2 (an GTP-binding aptamer) is labeled with Cy3. QDNA2(DNA sequences are given in Section 4) is used as a common quencherfor both aptamers. Simultaneous addition of ATP and GTP results inthe increase of Xuorescence intensity at both 520 and 563 nm, indica-tive of ATP and GTP binding, respectively.

It is also conceivable that several structure-switchingsignaling aptamers carrying diVerent Xuorophores orquenchers can be generated through the combination ofin vitro selection and signaling aptamer engineering.These aptamers could be used to establish various formsof multiplexed assays for real-time monitoring of eithermulti-step enzymatic reactions or diVerent enzymaticactivities occurring in the same solution. The capabilityof signaling aptamers to monitor enzymatic reactionswill certainly expand the utility of signaling aptamers asXuorescent reagents to quantitate enzyme concentra-tions and to conduct high throughput screening ofsmall-molecule libraries for enzyme inhibitors.

4. Reagents

4.1. DNA oligonucleotides

DNA oligonucleotides are purchased from KeckFoundation Facility Center, Yale University. They arepuriWed and quantitated using the procedures describedin Section 5. The stock concentration of each DNA oli-gonucleotide is set at 100�M. The sequences of all oligo-nucleotides are given below.

ATP1.1: 5�-CCTGCCACGCTCCGCACTTCGGAGGAGTTCTGCAGCGATCTTGATCGGGGACGGGGGAGAAAGGTTTTAAGCTTGGCACCCGCATCGT-3�BDNA: 5�-Biotin-TACCGCAAAAAAAAACAAGAATCGCTGCAG-3�

Fig. 6. Monitoring the activity of ALP in real time using the signalingaptamer that reports AMP-to-adenosine transition. Two levels of sig-nals are observed with MAP–FDNA1–QDNA1 signaling aptamer,Wrst upon addition of AMP at the 11th min, and then upon addition ofcalf intestine ALP at 21st min. The reaction of dephosphorylation ofAMP by ALP is shown at the top.

22 R. Nutiu, Y. Li / Methods 37 (2005) 16–25

FDNA1: 5�-Fluorescein-GCGGAGCGTGGCAGG-3�FDNA2: 5�-Cy3-CTCTCCTCTTACCAGATTCG-3�GTP1.2: 5�-CGAATCTGGTAAGAGGAGAGGGGTGGTTTCCGCAGCGATTCTTGATCGCGGAAGTCGGTGGGGAGGGTTAAGCTTGGCACCCGCATCGT-3�L1: 5�-CCTGCCACGCTCCGCAAGCTTN10CTGCAGCGATTCTTGATCGN20TAAGCTTGGCACCCGCATCGT-3�MAP: 5�-CCTGCCACGCTCCGCTCACTGACCTGGGGGAGTATTGCGGAGGAA GGT-3�P1: 5�-GCGGAGCGTGGCAGG-3�P2: 5�-ACGATGCGGGTGCCAAGCTTAr-3�P: 5�-GCCTCGCACCGTCC-3�QDNA1: 5�-CCCAGGTCAGTG-3�-DABCYLQDNA2: 5�-TACCGCAAAAAAAAACAAGAATCGCTGCAG-3�-DABCYL

4.2. Chemicals and enzymes

Radioactive nucleotides are purchased from Amer-sham Biotechnologies. Enzymes and non-radioactivenucleotides are bought from MBI Fermentas. Otherchemicals are obtained from Sigma–Aldrich. Multi-com-ponent chemical solutions are home-made using high-concentration stocks of individual gradients. pH valuesof the concerned solutions are measured at 23 °C.

• ATP: 100 and 75 mM in water.• GTP: 100 mM in water.• AMP: 75 mM in water.• Adenosine: 10 mM in water and 75 mM in DMSO.

100£ NTP mixture: 10 mM ATP, 10 mM GTP,10 mM CTP, and 10 mM UTP.

• 10£ dNTP mixure: 2 mM dATP, 2 mM dGTP, 2 mMdCTP, and 2 mM dUTP.

• [�-32P]ATP: 1.66 �M (6000 Ci/mmol).• [�-32P]dGTP: 3.33 �M (3000 Ci/mmol).• Sodium acetate (pH 7.0): 3 M.• Sodium acetate (pH 5.2): 3 M.• Triethylammonium acetate (TEAA; pH 7.0): 0.1 M.• Acetonitrile: 100%; HPLC grade.• NaOH: 0.25 M.• Urea: 8 M.• Ethanol: both 70 and 100%, stored at ¡20 °C.• Water (ddH2O): deionized and nuclease-free.• Avidin-coated agarose beads (Sigma–Aldrich): 2–

4 mg avidin mL¡1 beads suspension.• 2£ assaying buVer (2£ LB, pH 8.3): 600 mM NaCl,

10 mM MgCl2, and 40 mM Tris–HCl.• 2£ PAGE loading buVer (2£ LB, pH 8.3): 16 M urea,

100 mM Tris–borate, 0.6 M sucrose, 0.1% (w/v) SDS,1 mM EDTA, 0.025% (w/v) xylene cyanol, and 0.025%(w/v) bromphenol blue.

• 1£ elution buVer (1£ EB, pH 7.5): 10 mM Tris–HCl,200 mM NaCl, and 1 mM EDTA.

• 2£ selection buVer (2£ SB, pH 8.3): 600 mM NaCl,100 mM KCl, 20 mM MgCl2, and 100 mM Tris–HCl.

• 10£ PCR buVer (pH 9.0): 750 mM Tris–HCl, 20 mMMgCl2, 500 mM KCl, and 200 mM (NH4)2SO4.

• 10£ PNK buVer (pH 7.6): 500 mM Tris–HCl, 100 mMMgCl2, 50 mM DTT, 1 mM spermidine, and 1 mMEDTA.

• 50£ SybrGreen (Invitrogen): Diluted from10,000£ stock in DMSO.

• T4 Polynucleotide kinase (PNK): 10 U �L¡1.• Taq DNA polymerase (Biotools Labs): 5 U �L¡1.• Calf intestine alkaline phosphatase (calf intestine

ALP): 10 U �L¡1.

5. Protocols

5.1. Oligonucleotide synthesis

Both standard and modiWed DNA oligonucleotidesare prepared by automated DNA synthesis usingcyanoethyl-phosphoramidite chemistry. 5�-Fluores-cein and 3�-DABCYL (4-(4-dimethylamino-pheny-lazo)benzoic acid) moieties (in FDNA and QDNA,respectively) are introduced using 5�-Xuorescein phos-phoramidite and 3�-DABCYL-derivatized controlledpore glass (CPG).

5.2. PuriWcation of modiWed oligonucleotides

FDNA and QDNA are puriWed by reverse-phaseHPLC on a Waters XTERRA C18 HPLC column withdimensions of 4.6 mm £ 50 mm and a 2.5-�m beaddiameter. A two-solvent system is used for the puriWca-tion of all DNA species, with solvent A being 0.1 Mtriethylammonium acetate (TEAA, pH 7.0) and sol-vent B being 100% acetonitrile. The best separationresults are achieved by a nonlinear elution gradient ata Xow rate of 1 mL/min. The solvent gradient forFDNA puriWcation is: 7% B for 2 min, 7% B to 20% Bover 20 min (for FDNA separation), 20% B to 100% Bover 2 min (for column cleanup), 100% B to 7% B in2 min, followed by 7% B for 4 more minutes (forcolumn re-equilibration); the solvent gradient forQDNA puriWcation is: 10% B for 2 min, 10% B to 40%B in 20 min (for QDNA separation), 40% B to 100% Bin 2 min (for column cleanup), 100% B to 10% B in2 min, followed by 10% for 4 more minutes (for columnre-equilibration). The main peak shows strong absorp-tion at both 260 nm (for any DNA) and at the wave-length were the modiWcation moiety shows maximumabsorption. For the highest purity, only the fractionwithin 2/3 of the peak-width should be collected. Thus,obtained DNA-containing solutions are dried undervacuum.

R. Nutiu, Y. Li / Methods 37 (2005) 16–25 23

5.3. PuriWcation of standard DNA oligonucleotides

UnmodiWed DNA oligonucleotides are puriWed by10% preparative denaturing (8 M urea) polyacrylamidegel electrophoresis (PAGE). The DNA can be visualizedwith a handheld UV lamp when the gel wrapped withthin transparent plastic Wlm (such as SaranWrap Wlm) isplaced directly on top of a Xuorescent TLC (thin-layerchromatography) plate. The dark DNA bands are cut,crushed and eluted overnight at 37 °C in 1£ elutionbuVer. The DNA is recovered by ethanol precipitation(see the next protocol).

5.4. Recovery of DNA by ethanol precipitation

Adjust the sodium concentration of a DNA contain-ing solution to approximately 0.3 M using 3 M sodiumacetate, pH 7.0 (for example, 40 �L of 3 M NaOAc isadded to 400 �L of DNA in water), followed by the addi-tion of approximately 2.5 volume of cold 100% ethanol(1000 �L of ethanol to be added to the solution in theabove example). If the amount of DNA is extremely low(such as 0.1 pmol or lower), 1–20 �g of glycogen can beadded to facilitate the formation of the precipitant. Toprevent the non-speciWc binding of DNA to the tubewall, a small amount (such 20 pmol) of a short oligonu-cleotide (such as a PCR primer) that will not interferewith subsequent experiments can be added. The resul-tant solution is thoroughly mixed and then centrifugedat 15,000 rpm at 4 °C for 15 min. After decanting thesupernatant, 100 �L of 70% cold ethanol is carefullyadded to the wall of the microcentrifuge tube in a tiltedposition. The tube with the cap closed is turned slowlyallowing the cold ethanol to rinse the entire tube wall.The ethanol is then carefully removed using an Eppen-dorf pipette. Residue ethanol can be removed by vacuumdrying (a few minutes in a speedvac).

5.5. Determination of DNA oligonucleotide concentrations

PuriWed oligonucleotides are re-dissolved in ddH2Oand the absorbance of each DNA solution at 260 nm isdetermined on any UV–vis absorption spectrophotome-ter. The concentration of each DNA sample is calculatedusing the Biopolymer Calculator program (it can beaccessed at: http://paris.chem.yale.edu/extinct.frames.html).

5.6. Radioactive labeling of DNA oligonucleotide by T4 PNK (T4 polynucleotide kinase)

Twenty picomole of a concerned oligonucleotide ismixed with 1£ PNK buVer, 3�L [�-32P]ATP, and 1 �L ofPNK in a reaction volume of 10 �L. After incubation at37 °C for 1 h, the radioactive DNA is recovered by

ethanol precipitation, and if desirable, further puriWedon 10% PAGE.

5.7. Real-time polymerase chain reaction

A 100 �L mixture for a standard real-time polymerasechain reaction (real-time PCR) contains a desirableamount of a given template DNA (such as the DNAfraction selected by the in vitro selection techniquebelow), 50 pmol of forward primer, 50 pmol of reverseprimer, 1£ PCR buVer, 1£ dNTP mix, 1 �L of DNApolymerase, and 1£ SybrGreen. The real-time PCR canbe performed in a real-time PCR machine (such asSmartCycler from Cepheid). The reaction should bestopped 3–4 cycles after the Xuorescence intensity of thesolution plateaus.

5.8. PCR for DNA labeling

This protocol is designed to obtain highly radioactiveDNAs that contain an internal 32P-labeled phosphodies-ter bond. One microliter of DNA solution from a stan-dard PCR is used to set up a 25 �L mixture that contains12.5 pmol of each primer, 1£ PCR buVer, 0.1£ dNTP,1£ SybrGreen, 3 �L of [�-32P]dGTP, and 2.5 �L of TaqDNA polymerase. The amount of dNTPs is reduced 10times to allow a high yield of incorporation of radioac-tive deoxyguanosine into the ampliWed DNA. The reac-tion is performed and monitored in the same way asdescribed for the standard PCR.

5.9. Alkaline hydrolysis of ribonucleotide linkage

The PCR-ampliWed double-stranded DNA contain-ing a ribonucleotide embedded in one of the two DNAchains is Wrst recovered by ethanol precipitation. At theend of ethanol wash step, 90�L of 0.25 M NaOH is usedto dissolve the DNA pellet. The resultant DNA–NaOHmixture is incubated at 90 °C for 10 min. This step willresult in the cleavage of the ribonucleotide linkage in theantisense strand and produce three single-strandedDNA molecules with diVerent sizes. Ten microliter of3 M NaOAC (pH 5.2) is then added to neutralize NaOH.After DNA recovery by ethanol precipitation, the single-stranded DNAs can be separated by 10% denaturingPAGE.

5.10. Cloning and sequencing

DNA sequences in the Wnal pool by in vitro selectionare cloned into a vector by the TA cloning method. Plas-mids containing individual aptamer sequences are pre-pared using the Qiagen Mini-Prep Kit. DNA sequencingis performed on a CEQ 2000XL capillary DNAsequencer (Beckman-Coulter) following the manufac-turer’s recommended procedures.

24 R. Nutiu, Y. Li / Methods 37 (2005) 16–25

5.11. In vitro selection of ATP and GTP responsive signaling aptamers

Two thousand picomole of the DNA library L1,together with 20 pmol of the 32P-labeled L1, is mixedwith 10,000 pmol of BDNA, 2500 pmol each of P1 andP2, and adequate amount of 2£ SB and the resultantmixture was incubated at 4 °C overnight. The long incu-bation at low temperature is carried out to insure thehigh-percentage hybridization of all the DNA oligonu-cleotides. The DNA mixture is then incubated with200�L of avidin-coated agarose beads for 45 min atroom temperature to allow the binding of BDNA to theavidin beads (the beads should be pre-washed severaltimes and equilibrated with 1£ SB). The supernatantcontaining the unhybridized oligonucleotides is dis-carded and the beads are quickly washed Wve more timeswith 1£ SB to remove the weak-binding sequenceswithin the DNA library. The washes can be simply doneby gently shaking the tubes containing the beads in500�L of 1£ SB, followed by a short low-speed centrifu-gation on a mini-centrifuge to facilitate the precipitationof the beads. The beads are Wnally incubated with 500�Lof 1£ SB as the last wash and the radioactivity of thiswash solution is recorded as the background radioactiv-ity. The last wash is followed by the incubation with a500�L target-containing solution (0.1 mM each ATP,GTP, CTP, and UTP). After incubation for 1 h, thesupernatant is separated from the beads and its radioac-tivity is counted. The switching activity is taken as theratio between the radioactivity of the target-containingsolution and that of the last wash.

The DNA from the target-containing solution isrecovered by ethanol precipitation and dissolved in50 �L of water. Four polymerase chain reactions (100 �Leach) are performed using the real-time PCR protocoldescribed above. For each reaction, 12.5�L of the DNAis used as the DNA template, P2 and P3, are used as theprimers. Upon the completion of the reactions, DNAsamples are combined and 1 �L of the combined mixtureis used as the template to generate highly radioactivedouble-stranded DNA using the labeling PCR protocolgiven above.

All PCR solutions are combined and the double-stranded DNA is recovered by ethanol precipitation.The ribonucleotide linkage within the antisense DNAstrand is cleaved by the alkaline hydrolysis protocol dis-cussed above. After DNA recovery by ethanol precipita-tion, the single-stranded DNA corresponding to the sizeof L1 is isolated by 10% denaturing PAGE. The recov-ered DNA was subjected to additional rounds of selec-tion and ampliWcation.

Starting from the second round, 100�L of avidinbeads are used. The ratio of L1, P1, P2, and BDNA is setto be the same as that in the Wrst round. The beads arewashed more stringently (at least 10 times) following

each annealing step to eliminate the sequences from eachpopulation that only weakly bind to BDNA. Moreover,only 2 �L (out of 500 �L) of the target containing solu-tion is directly used as the template (without an ethanolprecipitation step) to set up a single PCR (100 �L). Theabove selection eVort has eventually led to the isolationof many ATP and GTP binding structure-switchingaptamers and the results have been published elsewhere[37].

5.12. ATP detection assay (Fig. 4)

A signaling mixture made of FDNA1–QDNA1–MAP duplex in 1£ assaying buVer is prepared. Twomeasures can be taken to insure that the signaling willhave a low background Xuorescence reading. First, theratio of FDNA1–MAP–QDNA1 is set to be 1:2:3(achieved by mixing 40 nM FDNA1, 80 nM MAP, and120 nM QDNA1) so that every FDNA1-containingduplex will also have a QDNA1. Second, the DNA mix-ture is incubated for a considerable length of time (suchas making the mixture the day before the experiment) toallow the maximal duplex formation.

Four hundred and ninety-Wve microliter of the aboveDNA mixture is transferred to a cuvette and its Xuores-cence is recorded for 5 min at the room temperature.Next, 5 �L of 100 mM ATP is added and the Xuorescenceintensity of the DNA–ATP mixture is immediately mon-itored and recorded every minute for 30 min. A concen-trated stock of ATP is used to minimize the dilution ofthe DNA concentrations. The raw Xuorescence data aregiven in Fig. 4. However, the raw data can also be nor-malized using the formula F/F0, where F is the Xuores-cence intensity at every time point and F0 represents thebackground Xuorescence right before target addition.The overall performance of the system can be judgedusing two parameters: the Xuorescence intensityenhancement and the response time. The Xuorescenceintensity enhancement is given by the formula F

Wnal/F0.We deWne the response time as the time needed for thesystem to reach 50% of the maximal Xuorescence aftertarget addition. A short response time (in the order ofminutes or seconds) is a good indicator of excellent real-time signaling capability of a signaling aptamer. Thisprotocol can also be used to verify the performance ofany rationally designed or in vitro selected structure-switching signaling aptamer.

5.13. A two-color assay that simultaneously report ATP and GTP (Fig. 5)

A DNA solution is prepared that contains 62.5 nM ofFDNA1, 62.5 nM FDNA2, 125 nM each of ATP1.1 andGTP1.2, 375 nM of P2, and 1000 nM of QDNA2 in1£ SB. Fifty microliter of the DNA solution was placedin a cuvette and the Xuorescence intensities at 520 nm

R. Nutiu, Y. Li / Methods 37 (2005) 16–25 25

(emission of Xuorescein) and 563 nm (emission of Cy3)are recorded every minute for 30 min when the sample isexcited at 490 nm (for excitation of Xuorescein) and547 nm (for excitation of Cy3). 0.5�L of 100 mM ATPand GTP are added to the DNA solution right after thereading made at the third minute.

5.14. Monitoring ALP-catalyzed AMP-to-adenosine conversion by a signaling aptamer (Fig. 6)

A signaling mixture made of FDNA1–QDNA1–MAPduplex in 1£assaying buVer is prepared. Four hunderdand ninety-Wve microliter of the signaling mixture is incu-bated in the absence of any target for 10min at 22°C, fol-lowed by the addition of 75 mM AMP. The resultantaptamer–target mixture is incubated at the same tempera-ture for 10 more minutes. At this point, 0.5U of calf intes-tine ALP are introduced, and the resultant solution isfurther incubated for 50 additional minutes. A Xuores-cence reading at the emission wavelength of 520 nm (exci-tation wavelength is set at 490nm) is recorded everyminute. The raw Xuorescence data are shown in Fig. 6.

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