Aptamers

William James

inEncyclopedia of Analytical Chemistry

R.A. Meyers (Ed.)pp. 4848–4871

John Wiley & Sons Ltd, Chichester, 2000

APTAMERS 1

Aptamers

William JamesUniversity of Oxford, Oxford, UK

1 Introduction 1

2 In Vitro Evolution of Nucleic AcidLigands 12.1 Outline of Process 12.2 History of Discovery and

Development 32.3 Choice of Chemistry 32.4 Practical and Theoretical

Considerations 5

3 Properties of Published Aptamers 83.1 Structures 83.2 Aptamer Size 93.3 Aptamer Targets 93.4 Affinity 103.5 Specificity 11

4 Applications and DownstreamTechnologies 114.1 Conjugation to Detectable

Moieties 114.2 Aptamers in Biosensors and Other

Detectors 124.3 Aptamer Combination 124.4 Use of Aptamers In Vivo 134.5 Studies on Nucleic Acid

Structure 134.6 Isolation of New Catalysts 15

5 Perspectives and FutureDevelopments 15

Acknowledgments 15

Abbreviations and Acronyms 16

Related Articles 16

References 16

Aptamers are artificial nucleic acid ligands that canbe generated against amino acids, drugs, proteins andother molecules. They are isolated from complex librariesof synthetic nucleic acid by an iterative process ofadsorption, recovery and reamplification. They havepotential applications in analytical devices, includingbiosensors, and as therapeutic agents.

1 INTRODUCTION

A very neat job in a small space.1/

Aptamers range in size from approximately 6 to40 kDa and sometimes have complex three-dimensionalstructures, produced by a combination of Watson–Crickand non-canonical intramolecular interactions. They bindto their targets with KD typically in the low nanomolarrange and can distinguish enantiomers of small moleculesor minor sequence variants of macromolecules withfrequently several orders of magnitude KD ratio. Theyare typically composed of RNA, single-stranded DNA ora combination of these with non-natural nucleotides.

Aptamers are isolated from extremely complexlibraries of nucleic acids, generated by combinatorialchemistry, by an iterative process of adsorption, recoveryand reamplification. Additional sequence variation canbe introduced at each cycle and the process becomes anin vitro paradigm of Darwinian evolution. After suffi-cient enrichment, aptamers can be cloned and studied ashomogeneous sequence populations.

Aptamers can be used to analyze the natural pro-cesses of nucleic acid–protein recognition, to generateinhibitors of enzymes, hormones and toxins with poten-tially pharmacological uses, to detect the presence oftarget molecules in complex mixtures and to generatelead compounds for medicinal chemistry. Their advan-tages over alternative approaches include the relativelysimple techniques and apparatus required for their iso-lation, the number of alternative molecules that canbe screened (routinely of the order of 1015) and theirchemical simplicity. Disadvantages of aptamers includetheir pleiomorphism, their high molecular mass and therestricted range of target sites that appear to be suitable.

2 IN VITRO EVOLUTION OF NUCLEIC ACIDLIGANDS

2.1 Outline of Process

Aptamers are ligands derived by a process of combinato-rial chemistry, in which the desired property is identifiedby affinity chromatography and encoded genetically. Theprocess is illustrated in Figure 1 and described below.

2.1.1 Library Synthesis and Complexity

Combinatorial chemistry is the production of a verylarge number of different molecules by the repeated

Encyclopedia of Analytical ChemistryR.A. Meyers (Ed.) Copyright John Wiley & Sons Ltd

2 NUCLEIC ACIDS STRUCTURE AND MAPPING

T7 pro -> 5′ constant 3′ constant 36nt random

DNA library

RNA library

Target

In vitro transcription

RNA folding

Mixing Reverse transcription

PCR amplification

Enriched pool

Partition

Enriched pool

Discarded pool

Figure 1 Schematic representation of the in vitro selection of nucleic acid ligands (aptamers).

use of a limited range of synthetic steps. It takes manyforms, outside the scope of this article, but was inspiredby the sequential, solid-phase synthesis of oligopeptidesand oligonucleotides (oligos: Combinatorial ChemistryLibraries, Analysis of). An oligo of defined sequence iscommonly synthesized on a solid support by a cycle ofdeprotection of the acceptor end of the growing chain andits derivitization by using an excess of the next activatedmonomer in the mobile phase. By modifying this processso that the activated monomer is a mixture, rather thana single species, oligos of randomized sequence may bereadily obtained. For sequences of randomized length Nand y alternative monomers at each position, a library ofoligos of diversity yN can, in principle, be synthesized. Forexample, a nucleic acid library of randomized length 40has a maximum theoretical diversity of 440 D 1.2ð 1024.The theoretical diversity, otherwise known as sequencespace, of such a library will often exceed by many orders ofmagnitude the number of molecules that can be handledin practice. Convenience and expense generally restrictthe initial library to a sample of in the region of 1013 –1015

molecules taken, presumably randomly, from the largersequence space.

2.1.2 Selection of the Desired Property

The desired property is, typically, the ability to bind toa molecule of interest. Depending on the anticipatedapplication, the desired binding properties may be a fastassociation rate, slow dissociation rate, high affinity, low

affinity to closely related molecules, or a combinationof these. This property will be a function of the three-dimensional structure of the folded nucleic acid and willbe a combination of its van der Waals surface contacts,hydrogen bonds, stacking interactions and other non-covalent bonds that can form between the aptamer andits target. It is a necessary assumption that, to a reason-able approximation, the three-dimensional structure ofan aptamer is uniquely determined by the sequence of itsbases. These assumptions will be explored below.

By mixing the solution-phase library with the targetmolecule and subsequently retrieving the target andremoving unbound or loosely bound nucleic acids, thefew nucleic acids that have the desired property can berecovered from the library. This partitioning step is a formof affinity chromatography and the methodology can bevaried depending on the nature of the target and the exactproperty desired. This will be discussed in detail below.The selection step differs from screening methods usedin other branches of combinatorial chemistry because ofthe numbers of candidate molecules that can be reviewedat one time. Conventional libraries of potential drugsmay consist of the order of 106 molecules, which canbe screened for activity by robotic means in a fewmonths. Combinatorial libraries of greater than about105 are impractical to screen by conventional, iterativemethods, as vanishingly few molecules of each specieswould be present in each assay. Perhaps the most sensitivemethods involve affinity partitioning followed by massspectrometry (MS)..2/

APTAMERS 3

2.1.3 Genetic Encoding

In contrast to other forms of combinatorial chemistry,where a variety of methods have been devised to encodethe structure of each agent, or at least its synthetic recipe,in the solid-phase support, aptamers contain withinthemselves the genetic code for their own amplificationand synthesis. Nucleic acid polymerase enzymes areused to convert the target-bound aptamer, if it wasbased on RNA, into DNA, to amplify the copy numberexponentially (by a factor of at least 1010) and, again ifthe aptamer is RNA, to transcribe the amplified DNAtemplate back into RNA.

2.1.4 Enrichment and Evolution

Because the affinity-based partitioning methods areimperfect, the cycle of partitioning and amplification isnormally repeated 6–12 times, sometimes more, beforemolecules of the desired property predominate in thepopulation of nucleic acids. The process, as describedabove, represents a reduction in sequence diversity fromthe initial sample of sequence space (e.g. 1015 out of aspace of 1024) to a population of the order of typically10 distinct sequences that show appreciable binding tothe target. In addition, however, the error-prone natureof the enzymes used to amplify the selected sequencesleads to the introduction of mutations that effectivelyallow the procedure to sample a greater proportion ofthe sequence space than was initially sampled. This leadsto the appearance of clearly related but evolutionarydivergent sequences within the final aptamer population,in a process akin to Darwinian evolution by naturalselection.

2.2 History of Discovery and Development

While experiments involving the isolation of nucleic acidsfrom artificial libraries on the basis of their biochemicalproperties were being widely discussed during 1988 and1989, three groups independently published their resultsin 1990. First, the Joyce group reported the use of invitro mutation, selection and amplification to isolateRNAs that were able to cleave DNA..3/ They beganwith a natural RNA-cleaving ribozyme, rather than apurely random library, and were looking for a novelenzymic activity rather than a selective ligand. However,their experiment had most of the essential featuresdescribed in this article, including the repeated cyclesof reaction performed in a single vessel. Second, theGold group described experiments designed to identifythe sequence requirements of T4 DNA polymerase,in which the library was based on the natural targethairpin structure but with the eight loop nucleotidesrandomized..4/ The process of in vitro selection, for which

they coined the term ‘SELEX’ (selective expansion ofligands by exponential enrichment), was able to identifythe natural target of the enzyme as the predominant,high-affinity ligand, with one major variant emergingwith similar affinity. The authors patented their process.Less than a month later, the Szostak group reportedthe use of in vitro selection to isolate ‘molecules withspecific ligand-binding activities’..5/ More radically thanthe previous reports, they began with a library thatwas structurally unrelated to any known nucleic acid,having 100 nucleotides of randomized sequence, andchose targets that had no previously identified nucleicacid ligands. They used affinity chromatography to isolateRNAs with specific and selective binding characteristicsfor a number of organic dyes (chosen because of theirpotential as H-bond partners and their planar structuresthat might be expected to form stacking interactions withthe nitrogenous bases of RNA). It was this group thatcoined the term ‘aptamer’ for such nucleic acid ligands.

2.3 Choice of Chemistry

Aptamers are nucleic acids that can be composed ofnaturally occurring monomers or chemically synthe-sized derivatives. For a review of this field, see Eatonand Pieken..6/ As outlined above, the nucleotides mustbe compatible with RNA-dependent DNA polymerases(reverse transcriptase) and with DNA-dependent RNApolymerases (such as T7 RNA polymerase) for the enzy-matic steps required in the production of RNA-basedaptamers, or with thermostable DNA-dependent DNApolymerases (such as Taq polymerase) for the productionof DNA-based aptamers..7,8/ This restricts the availablechemistry more strictly than is the case with purely syn-thetic oligonucleotides. Nevertheless, researchers haveadopted many modifications of natural nucleotides inorder to overcome two substantial problems posed bynatural nucleic acids.

2.3.1 Phosphodiester Bond Hydrolysis

This is a particular problem in natural RNA, as thehydroxyl at the 20-position is reactive, particularly athigher than neutral pH, and will attack the neigh-boring phosphodiester bond to produce a cyclic 20,30-phosphate, thereby breaking the nucleic acid backbone(see Figure 2). This reaction is catalyzed by many tran-sition metals ions, particularly lead and iron, and by arange of ribonucleases found ubiquitously in biologicalsamples. To overcome this problem, one has turned tomodifications of either the 20-position of the ribose moi-ety or modifications of the phosphodiester backbone. Byfar the most common approach is the use of nucleotidessubstituted at the 20-position with either an amino groupor fluorine atom. These modifications were pioneered

4 NUCLEIC ACIDS STRUCTURE AND MAPPING

N

NH2

O

O−

O

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PO O−

O

P O−

OH

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NHN

N

O

NH2

OH OH

N

cytosine

guanosine

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OH

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PO O−

OH

OP

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NHN

N

O

NH2

OH OH

N

(c)

O

Figure 2 Representative RNA components. (a) Diagram ofthe structure of a CG dinucleotide, showing the numberingconvention for carbons in the ribose moiety. (b) The 20,30-cyclicphosphate that forms as the result of nucleolysis. (c) A CGdinucleotide containing 20-fluorine.

by the Eckstein group.9,10/ and are compatible with T7RNA polymerase for efficient in vitro transcription..11/

Where they have been compared, 20-deoxy and 20-amino chemistries have been found to produce aptamerswith similar affinity, although different structure (foran example, against immunoglobulin E (IgE), see Wie-gand et al..12/). However, 20-fluoro chemistry producesaptamers with greater thermal stability and probablyhigher affinity than 20-deoxy or 20-amino chemistry..13/

Although one cannot generally change the chemistryof the nucleotides after selection without changing thestructure of the aptamer and frequently abolishing itsproperties as a ligand, this can often be done in a moreselective manner (A. Tahiri-Alaoui, unpublished work).For example, further modifications at the 20-position thatgive additional stability can be incorporated syntheticallyinto aptamers with 20-OH, -H, -NH2 or -F that havebeen identified through in vitro evolution. For example,20-O-methyl has been introduced at certain purines in20-F-pyrimidine aptamers against the vascular endothe-lial growth factor (VEGF), with beneficial effects..14/

The alternative approach, to modify the phosphodiesterbackbone, is more challenging to the enzymology of invitro evolution. Nevertheless, by using a-thio-substituteddeoxynucleoside triphosphates (dNTPs), a phosphoroth-ioate DNA library was successfully screened for aptamersagainst the transcription factor NF-IL6,.15/ and someprogress has been made toward the use of analogous,‘thio-RNA’ aptamers..16,17/

Another way to produce nuclease-resistant aptamersis to select an aptamer that binds the enantiomer ofthe eventual target, then synthesize the enantiomer of theaptamer as a nuclease-insensitive ligand of the normaltarget. Such ‘spiegelmers’ have been made in L-DNAagainst the peptide hormone, vasopressin.18/ and in L-RNA against L-adenosine and L-arginine..19,20/

2.3.2 Absence of Hydrophobic or Basic Residues

In contrast to proteins, nucleic acids are strikingly uniformin their hydrophilicity and low pI. In spite of theselimitations, a surprising range of enzymatic activities arepossible for nucleic acids and the hydrogen bond andstacking interactions of their component bases provide adiverse toolbox of structural motifs..6/

Nevertheless, this uniformity almost certainly limitsthe ease with which aptamers can be made to certaintargets and a greater range of side chains, includingsome with basic or hydrophobic character would bewelcome additions to the armamentarium. The challenge,as with substitutions giving nuclease resistance, is todiscover approaches that are consistent with the enzymesused during aptamer isolation. One approach, using5-(1-pentynyl)-20-deoxyuridine, succeeded in producingadditional aptamers to thrombin,.21/ though no reports

APTAMERS 5

have emerged showing that this approach has producedaptamers against more refractory targets.

It has long been speculated that the precursor toour DNA-genome, protein-catalyst world of organismswas a world in which RNA functioned for genomes,ligands and enzymes.22/ and this was consolidated bythe discovery of ribozymes..23,24/ (In his paper onthe ‘Evolution of the Genetic Apparatus’, Orgel.22/

speculated on ‘life based on nucleic acids without agenetic code’ and said that ‘it seems to me quite possiblethat polynucleotide chains could make primitive selectionamong organic molecules such as amino acids by formingstereospecific complexes stabilized by hydrogen-bondingand hydrophobic interactions’. He also speculated thatpolynucleotides might have the ability in early stagesof life to catalyze chemical reactions but that this‘function would subsequently have been taken over bythe much more versatile polypeptides’.) Indeed, it isproposed by some that the restricted range of aminoacids extant in the current world of biology is dictatedby those homologous with natural, prebiotic derivativesof 5-hydroxymethyluracil that had previously been afeature of the ‘RNA world’..25/ Further, one should notethat many biological RNAs of the present world haveadditional methyl groups added post-transcriptionally totheir purines, which increase the local hydrophobicity ofthe nucleic acid (reviewed by Levy and Miller.26/). Noneof these potentially helpful modifications is reproducedduring enzymatic replication of nucleic acids, thoughsome of them, rather than acting as chain terminators,are ignored. For example, 5-methylation of cytosine is acommon postreplication modification of eukaryotic DNAand 5-methyldeoxycytidine is replicated routinely to dGduring the S phase of the eukaryotic cell cycle.

Instead of attempting to broaden the repertoire ofnucleotides used during the replication and transcriptionphases of in vitro selection of aptamers, one mightmodify one or more nucleotides post-transcriptionally.It was found that derivitization of 20-NH2 groups onaminopyrimidine-RNA with succinimide did not preventreverse transcription..27/ Although this might usefullyopen up a wide range of functional adducts, the efficiencywas low, with only two out of 23 amino groups permolecule, on average, being successfully derivatized. Inorder to preserve the genetic encoding of aptamers, it isessential that such processes are either fully efficient orreproducibly inefficient (i.e. the same nucleotides beingderivatized each cycle).

2.4 Practical and Theoretical Considerations

A comprehensive and practical introduction to themethodology of in vitro evolution of RNA ligandswas provided by Fitzwater and Polisky.28/ and so will

not be repeated here. A mathematical description ofthe process, under assumptions of equilibration, wasproduced by Irvine et al..29/ and supplemented by amodel for simultaneous selection against multiple targetsby Vant-Hull et al..30/ We shall confine ourselves to adiscussion of some of the most relevant details.

2.4.1 Library Complexity

When designing a library, the desire is to producesequences that can be amplified and transcribed withhigh efficiency, of as great a diversity of structures asis practicable and at reasonable cost. One is boundto use substantial fixed, flanking regions for primer-based amplification and transcription, so one questionis how long to make the randomized region (N) andhow to ensure that the library produced contains asnear to the theoretical maximum of sequences possible.First, although the sequence space available to the libraryis 4N , the maximum number of molecules that can bemanipulated in standard molecular biology laboratories(M) is of the order of 1015, and errors of synthesis andworkup have been estimated to reduce this diversity to1013 –1014. N need only be around 22–24 to reach thispractical limit. However, in order to produce a structurecapable of specific interaction with a target molecule, theRNA needs to be large enough to fold into a complextertiary structure. It appears that many classes of RNAfold need to composed of significantly longer stretchesof nucleic acid than this (see above), so most peopleuse libraries where N ½ 35 and sometimes much more.As N increases, one is able to sample progressively asmaller fraction of the theoretical sequence space. Forexample, if M D 1014 and N D 24, we could sample.1014/2.81/ð 1014 D 36% of the sequence space, but ifN D 25, this reduces to 8.9%. However, if we considereach 25-mer to be composed of two overlapping 24-merswhich differ only at their termini, then the N D 25 libraryeffectively contains 2ð 1014 24-mers. This suggests thatone should aim for larger rather than smaller valuesof N. Two considerations effectively limit the practicalsize of N. First, although the efficiency of oligonucleotidesynthesis is constantly improving, errors accumulate witheach cycle and a point is reached at which incorrectlysynthesized molecules begin to have a significant impacton the quality of the library. The limit is imprecisebut it seems unwise at present to make large-scalelibraries using oligo lengths much greater than about120 nt, suggesting a practical limit of around N D 70–80.Second, if RNA folds are typically between 25 and 50 ntlong, libraries with N greater than approximately 50 ntmay well contain sequences comprising two independentfolds, complicating the process of in vitro selection.

The actual diversity of a library can be compromised byvariations in the efficiency of coupling phosphoramidite

6 NUCLEIC ACIDS STRUCTURE AND MAPPING

monomers during oligonucleotide synthesis, which inturn can depend on the particular practices of themanufacturer chosen. Although standard methods useequimolar mixtures at each N stage during synthesis, theSzostak group has reported the use of an A : C : G : T ratioof 3 : 3 : 2 : 2 and we have found it necessary to use ratiosof 6 : 5 : 5 : 4 in order to achieve random incorporation.Gross bias during synthesis and enzymic manipulationcan be estimated by checking the sequence of a dozenor so clones made from the library after a single roundof amplification, transcription, reverse transcription andreamplification, without selective partitioning. The clonesshould be sequenced and the overall base compositionwithin the random region tested for divergence fromexpectation. In addition, one should look for bias in thedinucleotide and trinucleotide composition and at basecomposition at each position.

What are the chances of finding a target-bindingsequence during the in vitro exploration of sequencespace? The answer seems to depend on one’s target andthe criteria (e.g. affinity threshold) used to identify bind-ing. For example, within the N D 100 library describedby Ellington and Szostak,.5/ the authors estimated that10% of the library contained aptamers for organic dyemolecules. In contrast, Burke and Gold.31/ estimated thatperhaps one in 1011 sequences within their library con-tained adenosine-binding motifs. Our own experiencewith libraries of N D 36 is that protein-binding aptamerssequences exist at an initial frequency of typically 1 in1011 –1012. If these figures are representative, it shows thatcare needs to be taken during the early stages of libraryproduction and manipulation not to reduce the sequencecomplexity much below its theoretical maximum.

Tuerk.32/ has made calculations based on computermodeling of the SELEX process and suggested thatbinding reactions that yield 6% of the total RNA boundto target would be optimal. Schneider et al..33/ showedthat when reactions were arranged to select 10% of theRNA population bound to r protein (low stringency) or1% (high stringency) during SELEX, ligands of similarsequences were produced by both selection strategies,although the speed of affinity enhancement of the poolwas increased somewhat for the high stringency selection.This illustrates that the fine tuning of ligand–targetratios is not crucial for success in obtaining optimalbinding ligands. Tuerk.32/ suggests that the first bindingreaction be conducted in large volumes (50 mL) withtarget molecule concentrations at 0.2 times the KD ofthe original nucleic acid pool–target interaction and allof the starting nucleic acid. This would yield about 9% ofthe original nucleic acid population bound to a largenumber of target molecules, decreasing the chance thatlow-abundant, unique sequences of high affinity are lostin the first selection.

2.4.2 Mutagenesis and Affinity Maturation

Thus far, we have been considering merely the selectionof ligands from an initially very small and approximatelyrandom sample of sequence space. However, the phrase‘in vitro evolution’ implies that we are able to explorea much greater fraction of sequence space by allowingmutation to generate additional diversity and selectionto retain a small fraction with favorable properties. Byrepeatedly selecting for the desired property, the path ofexploration should tend towards optimum ligands; thosethat occupy segments of sequence space that correspondto affinity maxima. However, we can imagine manyways in which this favorable result would not have beenreached. First, the initial sample of the library may nothave contained any members that were within strikingdistance of a high-affinity region of sequence space.This will depend on the complexity of the library andthe nature of the target, as described above. Second, theamount of mutation at each level may have been toolittle to allow significant progress towards the affinitymaximum. Conversely, mutation might have been so highthat the peaks would have been missed or overshot. Third,selection might not have been stringent enough, allowingthe minority of high-affinity ligands to be swampedby their low-affinity brethren. Conversely, selectionmight have been too stringent, eliminating sequencesof intermediate affinity before high-affinity sequenceshad been arrived at. This is a particularly acute problemduring in vitro selection because one rarely has any wayof knowing either the affinity topology of sequence spaceor the properties of early-cycle oligonucleotides.

There has been surprisingly little experimental analy-sis of this problem. Although we know that aptamersselected against a particular target usually fall intosequence groups which suggest phylogenetic relatedness,the polyphyletic origin of aptamers means that two simi-lar sequences may have arisen by convergence from twoindependent sequences sampled in the initial library aseasily as by divergence from a common ancestor. Nev-ertheless, in one study, explicit evolution was measuredfrom an aptamer that had been previously selected againstL-citrulline to others which could bind alternative aminoacids..34/ Mutation was introduced at a single step bychemical resynthesis of the original aptamer to give anaverage of 30% mutation at each position. Binding tocitrulline was recovered after three rounds of selectionand to arginine after four rounds. However, no lysine-or glutamine-binding aptamers were obtained. The moreusual method of introducing mutation is to encouragemisincorporation of nucleotides during amplification byincreasing the concentration of dNTPs and Mg2C andusing enzymes lacking proofreading activity..28/

APTAMERS 7

2.4.3 Partitioning Methodology

The separation of desired from undesired sequences isof critical importance in the selection and evolution ofaptamers (see the discussion above and a mathematicalanalysis by Vant-Hull et al..30/). Ideally, one would beable to allow the nucleic acid pool and the targetto interact freely, be able to monitor the progress ofthe interaction and retain those nucleic acids whosedesired property lay above a threshold determined inpart by the properties of the pool. Most methods ofpartitioning fall short of these ideals. First, tetheringthe target to a solid support facilitates the separationof bound from unbound nucleic acids and facilitatesprocedures such as competitive elution which can behelpful in setting affinity and specificity parameters. Thisis the approach used in some of the earliest experiments,and has been particularly used in the identificationof aptamers to small molecules..5,34 – 36/ Immobilizationof target proteins to Sepharose has also been usedsuccessfully to facilitate partitioning.37,38/ but mixing thenucleic acid and target protein in the soluble phase hassignificant advantages. First, many protein immobilizationregimes involve derivitization of the e-amino groupsof lysine, thereby destroying a key feature of manyaptatopes. Second, immobilization reduces the mobilityof the protein, impeding its mixing with nucleic acidand may produce direct steric hindrance to aptamerbinding. Third, a progressive reduction in the molar ratioof target protein to nucleic acid in successive cycles,in order to increase the stringency of selection, is veryhard to achieve in an immobilized system. Consequently,one of the common approaches is to use an insolublematrix that selectively adsorbs protein following nucleicacid–protein interaction in the fluid phase. Nitrocellulosefilters are a cheap and convenient matrix that is widelyused for this purpose..4,39,40/ However, we have foundthat nitrocellulose preparations are not as selective forprotein as would be desired. We have compared the use ofnitrocellulose filters, poly(vinylidene difluoride) (PVDF)membranes, activated Sepharose, octyl Sepharose and thedeproteinizing matrix Strataclean resin for their abilityto select for CD4-binding aptamers via interaction withthe protein (F. Kesten, personal communication). Wefound that nitrocellulose and PVDF trapped substantialamounts of 20-F-RNA nonspecifically and that octylSepharose was not an efficient protein binder underthese circumstances (results not shown). However, CNBr-activated Sepharose and Strataclean resin were bothefficient at pulling down 20-F aptamer RNA in a target-dependent fashion (see Figure 3).

Models of the selection process also assume thatequilibrium between RNA and protein has been reachedbefore partitioning..29/ However, this is very unlikely tobe the case during early rounds, when the concentration

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Figure 3 Selective partitioning of ligand RNA using eitheractivated Sepharose or Strataclean resin.

of ligands is extremely small or at any stage whenincubation times of the order of 10 min are used routinelyand the most desirable aptamers have dissociation t1/2of the order of 1 h (J. Ibrahim and L. Frigotto, personalcommunication). In a recent study, we were able to showthat increasing the incubation time from 30 min to 2.5 hsubstantially reduced the number of cycles required toisolate high-affinity aptamers..41/ Recent improvementsin robotics technology have enabled the process of invitro selection to be reduced to a matter of days..42/

2.4.4 Structural Pleiomorphism

A central but unspoken assumption of the methods forin vitro selection and evolution of aptamers is that thereis a direct and unique relationship between the sequenceof an aptamer and its shape: the ‘one sequence, onestructure’ assumption. In other words, if one selects anucleic acid sequence s in round n on the basis that itbinds to protein X, then one would expect that all copiesof the amplified sequence s will bind to X in round nC 1.However, if sequence s can fold into more than oneconformation, only a proportion of the reamplified s willbind to X in round nC 1. Worse, if each sequence ispresent at low copy number, for example during theearly rounds of selection, the copy of sequence s inround n may not be in the right conformation to bindX and thus not be selected at all. How pleiomorphicshould we expect most RNAs to be? If we were toanswer this on the basis of the known folding offunctional RNAs such as the Tetrahymena intron orthe hammerhead ribozyme, which fold rapidly into theirnative states, we might be misled. Instead, if we lookat most monoclonal aptamers, it is not uncommon todiscover that they comprise distinct conformers, onlya minority of which are competent ligands (A. Tahiri-Alaoui and E. Kraus, personal communication). Duringselection, one would expect the selective process to favorthose RNAs that are minimally pleiomorphic, so thepersistence of this property in aptamers suggests that

8 NUCLEIC ACIDS STRUCTURE AND MAPPING

pleiomorphism is common in unselected oligonucleotides.RNA folding algorithms, admittedly still only able topredict approximately 75% of experimentally determinedbase pairings,.43,44/ indicate that a given sequence isfrequently capable of folding into several structures, eachwith similar G. If the free energy barriers betweenany of these folded forms are great enough, multipleconformers would be expected. Natural evolution of,for example, ribozymes, would have imposed a selectivepressure for sequences whose folding pathways aremore uniform or for an association with chaperone-likeproteins that would favor one conformation over another.Those working with aptamers have adopted a variety ofstrategies to minimize this problem – from snap-coolingnucleic acid pools from the denatured state to a slowrefolding procedure – but none is entirely successful.Although technically challenging, it is becoming possibleto analyse co-existing structures within a population ofRNA molecules with identical sequence..45/

Conformational flexibility has been observed in pro-tein–aptamer interactions, for example, an induced fit inthe target polypeptide following the binding of aptamersraised against the HIV-1 regulatory protein..46/ Con-versely, the conformation of the nucleic acid was seento change upon binding of an RNA aptamer to its aminoacid target..47/

3 PROPERTIES OF PUBLISHED APTAMERS

3.1 Structures

The structure of a large number of aptamers has beendetermined by enzymatic or chemical probing, nuclearmagnetic resonance (NMR) and X-ray crystallography(see reviews by Feigon et al..48/ and Patel.49/). They arerelatively amenable to NMR methodology because oftheir small size and their rigidity when complexed withtarget. Although the fundamental repertoire of nucleicacid secondary structures is radically different from thatof proteins, the similarities between the interaction sitesbetween protein ligands and their receptors can bestrikingly similar to those of aptamers..50/ A few of thesalient features are described below.

3.1.1 DNA Aptamers

One of the earliest aptamers studied structurally was the15mer DNA aptamer against thrombin, d(GGTTGGTG-TGGTTGG), isolated by Bock..7/ Using two-dimensionalproton NMR, it was shown.51/ that this short oligofolded tightly into a four-stranded structure, stabilizedby two stacked G tetramers, with each of the two pairsof strands having a TT dinucleotide loop (see Figure 4).X-ray crystallography of the aptamer complexed withits target.52/ showed that the aptamer was sandwiched

G

GG

G

G

G

G

G

T T T T

T GT

Figure 4 Representation of a thrombin-binding aptamer. Thephosphodiester backbone is shown with solid gray lines. Thenoncanonical hydrogen bonds forming the G tetrads are shownas dotted lines.

between the two highly basic exosites of thrombin,making no contact with the active site at the baseof the cleft between the two exosites. The X-rayand NMR models were very similar, but the polarityof the strands was different, with the two TT loopsspanning the major groove in the X-ray model andthe minor groove in the NMR model. These twostructures are technically very hard to discriminatefrom the crystallographic data, and it is probable thatthe NMR model is, in fact, correct..53/ Another GT-rich oligo, d(GsTGGTGGGTGGGTGGGsT) (where thefirst and last phosphodiester bonds are replaced withphosphorothioate), was found to be an inhibitor of theintegrase enzyme of HIV-1..54/ The structure of thisaptamer is again four strands, stabilized by a pair ofG quartets but in which the joining loops are all TG..55/

The G octet is not the only kind of structure adoptedby DNA aptamers, however. For example, an aptameragainst argininamide is a hairpin loop, which undergoesa rearrangement upon binding to its target, in which theloop is reflexed to trap the amino acid between the stemand the stabilized loop by a combination of H-bonds andstacking interactions..56/

3.1.2 RNA Aptamers

Aptamers based on RNA, or nucleotides with pre-dominantly RNA-like properties, are capable of adoptinga seemingly greater variety of structures (reviewed byPatel et al..57/ and Ferre-D’Amare and Doudna.58/).For example, a flavin mononucleotide (FMN)-bindingaptamer consists of an asymmetric loop that binds thetarget, flanked by two A-form helices..59/ The asymmetricloop itself forms a widened, colinear helix comprisingan anti purine pair and a base triple at one side,two anti purine pairs at the other of the intercalatedisoalloxasine moeity of FMN (see Figure 5). Essentially,the target is acting as a highly abnormal intercalated basein a modified double helix. Another nucleotide-bindingaptamer, which recognizes adenosine 50-monophosphate(AMP),.60/ incorporates its target even more intimatelyinto its structure. Tetraloops of consensus sequenceGNRA (where N is any nucleotide and R is a purine) are

APTAMERS 9

A

GA G U A U

A

AG G G G

5′ G U C UGG C G U U G3′ C A G CCC G C A A C

Figure 5 Secondary structure representation of an FMN-binding aptamer. Noncanonical base pairs are indicated bysingle solid lines and the base triplet by a set of three radiatinglines. The FMN’s isoalloxasine moiety is indicated by a triplehexagon.

common in natural RNAs and are particularly stable..61,62/

The AMP-binding aptamer was found to recruit the freeAMP and thereby form a GNRA-like loop from a regionthat is unpaired in the absence of a target..63 – 65/

The structures of protein-binding aptamers show fur-ther variations on these themes. For example, aptamersthat bind the HIV-1 regulatory protein, Rev, accommo-date an a-helix of their target within a modified majorgroove that is widened by the presence of a number ofnon-Watson–Crick base pairs..66 – 68/ In contrast, a classof aptamers that bind the reverse transcriptase of HIV-1 possess a compact form of pseudoknot structure.69,70/

(see Figure 6) which is believed to occupy the groovewithin the enzyme normally occupied by its kinked tem-plate. A biotin-binding aptamer was also found to havea rigid pseudoknot structure..71/ The pseudoknot repre-sents just one class of structural element or fold exhibitedby functional RNAs. The common feature of these foldsis that they allow the molecule to be more compact thanthe classical A-form double helix (reviewed by Ferre-D’Amare and Doudna.58/) and thereby take on a moreglobular form, which is perhaps more suited to ligandrecognition.

GG

G

N

N

NN′

N′

N′

N

A

AA

A

X

X′ X

UC

S

S′

a′

C

5′

3′

Loop 2

Loop 1

Stem 1

Stem 2

Figure 6 A reverse transcriptase-binding pseudoknot apt-amer..69/ S–S0 represents a G : C or C : G base pair, X representsoptional nucleotides and N–N0 represents any base pair.

3.2 Aptamer Size

It has already been noted that the sequence space avail-able to most libraries used in aptamer isolation exceedsthe practicable limit of molecules that can be handled.Moreover, for many applications it is desirable that theaptamer should be as small as possible, on costs grounds,reasons of target accessibility and so on. Nevertheless,because functional RNA folds have a finite size, the min-imum length of aptamers is often larger than the sizethat generates a manageable sequence space. Further,the size of aptamer that is initially recovered from aprocess of in vitro selection is inflated by the flanking,fixed sequence regions, required for amplification andtranscription, which may add up to 50 nt. A combinationof deletion analysis, footprinting and in vitro synthesiscan be used to determine the shortest stretch of nucleicacid that can bind to the target. The size range of minimalaptamers is fairly wide and the following give some indi-cation. The minimum motif within VEGF aptamers wasbetween 23 and 35 nt (depending on sequence family);.14/

minimum xanthine- and guanine-binding aptamers were32 nt long;.72/ streptomycin-binding aptamers were 46 ntlong;.73/ and the minimum region of an aptamer that bindsa serine protease of the blood (protein C) may be as muchas 99 nt long..74/ This gives an Mr range of 7.5–32 kDa foraptamers, with 10 kDa being typical. The solvent-exposedsurface area for a typical aptamer would be expected tobe in the range 50–60 nm2.

3.3 Aptamer Targets

Can aptamers be raised against any target molecule?Put another way, can the lack of chemical diver-sity among nucleic acids be compensated by the sizeof the sequence space that can be sampled and theresultant structural diversity of aptamers? The evi-dence is that aptamers can be generated against smallions, such as Zn2C,.36/ to nucleotides such as adeno-sine triphosphate (ATP),.60,75/ oligopeptides.76/ and largeglycoproteins such as CD4,.38/ spanning the size range65 Da–150 kDa, with no theoretical upper limit. Thechemical classes of targets are reasonably diverse, includ-ing organic dyes,.5,77/ neutral disaccharides,.78/ amino-glycoside antibiotics (see Figure 7),.79/ dopamine,.35/ aporphyrin.80/ and biotin..71/

Nevertheless, there is evidence for a strong degreeof bias, among protein-binding aptamers, for a limitednumber of sites. First, a surprisingly high proportion ofproteins against which aptamers have been describedare themselves ligands for polyanions such as nucleicacids or glycosaminoglycans. These include thrombinand other proteases of the clotting cascade,.7,74,81/ anumber of heparin-binding growth factors,.13,82 – 84/ cel-lular transcription factors.85,86/ and viral regulatory

10 NUCLEIC ACIDS STRUCTURE AND MAPPING

(a)

(b)

Figure 7 NMR-derived structure of a tobramycin-bindingaptamer, bound to its target. (Data derived from conformer 1of 13 published by L. Jiang and D.J. Patel, ‘Solution Structureof the Tobramycin – RNA Aptamer Complex’, Nature Struct.Biol., 5(9), 769–774 (1998).) (a) The phosphodiester backboneof the aptamer is depicted as an orange cylinder and thetobramycin is shown in ball-and-stick form. (b) The aptameris shown as a wireframe model with the base identities andpositions labeled.

proteins..39,87 – 89/ It has even been reported that someheparin-binding proteins, such as thrombin, may havenatural plasma aptamers..90/

Second, when the aptamer-binding sites (or aptatopes)on large target proteins are mapped, it is usually foundthat they are coincident, even if the aptamers fall intounrelated sequence families. For example, all six of thedistinct classes of aptamer against VEGF were foundto bind to the same region, competing with heparinand other natural ligands and being cross-linkable tothe same cysteine,.14,84/ and four structurally distinct

aptamers against reverse transcriptase all bound thesame region of the enzyme..70/ More strikingly, whenfive different sequence classes of aptamer against CD4were analysed, all bound to a single region of one of thefour immunoglobulin (Ig)-like domains,.38/ in contrast tomonoclonal antibodies, which define several epitopes ineach domain.

This focusing of aptamer reactions on a small fractionof a large macromolecular target suggests that the processof in vitro selection does not proceed entirely accordingto simple models of multitarget partitioning..30/ Rather,it suggests that favorable and unfavorable aptatopes aredistinguished by differences of affinity of many ordersof magnitude. It seems likely that a major obstacle isthat the Coulombic repulsion between the phosphate-containing backbone of nucleic acids and negativelycharged amino acid and sugar residues at the surfaceof many proteins and glycoproteins produces extremelylow association rates. This would mean that only a smallproportion of the surface of the macromolecule wouldbe ‘visible’ to aptamers, probably regions of high solventexposure in which positively charged residues provideda degree of electrostatic steerage towards the aptatope.This has been most closely studied in the case of anti-thrombin DNA aptamers. By mutating a critical arginineto glutamate, the binding of aptamers was abolished..91/

The mutant form of thrombin was then used to raisefurther aptamers and these were found still to bind regionshomologous to that recognized by the first-generationaptamers..92/

3.4 Affinity

The affinity of published aptamers varies very widely.Generally, aptamers against small molecules have affini-ties in the micromolar range. For example, aptamersagainst amino acids such as citrulline and argininerange from 0.3 to 65 µM,.34,93/ those against ATP andxanthine were 6 and 3.3 µM, respectively,.72,75/ thoseagainst dopamine were 2.8 µM and those against vita-min B12 were 90 nM..94/ Aptamers to nucleic acid-bindingmolecules typically have affinities in the nanomolarrange. For example, aptamers against retroviral inte-grase, reverse transcriptase and nucleocapsid proteinswere 10–800, 0.3–20 and 2 nM, respectively,.70,95 – 97/

and approximately 0.8 nM against the ribosomal RNA(rRNA)-binding aminoglycoside antibiotics..98/ Affini-ties in the nanomolar to subnanomolar range arefound against heparin-binding proteins. Examples includeplatelet-derived growth factor (PDGF) (0.1 nM),.99/ basicfibroblast growth factor (low nanomolar),.83/ throm-bin (25 nM),.7/ VEGF (approximately 100 pM),.14/ ker-atinocyte growth factor (approximately 1 pM).13/ and

APTAMERS 11

the heparin-binding non-pancreatic, secretory phospho-lipase A2 (1.7 nM)..100/ Aptamers against proteins thatdo not bind heparin or nucleic acids have typicallylower affinities. For example, those against the onco-proteins K-Ras and Raf-1 were 0.14–1 µM and 300 nM,respectively,.101,102/ one to substance P had a KD of190 nM.76/ and one to the NS3 protein of hepatitis C virushad a KD of 650 nM..103/ Immunoglobulin class G (IgG)superfamily domain-containing proteins seem to be ableto elicit aptamers in the 2–40 nM range of affinity.12,38,104/

and this might relate to their ability to interact with othercell-surface glycoproteins.

3.5 Specificity

When measured, it has been found that those targetsthat give rise to very high-affinity aptamers have mea-surable affinity for unselected RNA. For example, the E.coli r protein has an affinity for unselected RNA in the1 µM range and can be used to isolate specific aptamersthat bind with an affinity of approximately 1 nM..33/ Thisobservation suggests that the assertion that high affin-ity necessarily produces high specificity.105/ is unsafe, butthe evidence suggests that aptamers can show a greatdeal of specificity. First, different enzymes with similaractivity can be distinguished. For example, a-thrombincould be differentiated from g-thrombin;.106/ the reversetranscriptase of feline immunodeficiency virus could bedistinguished from the homologous enzyme of three otherretroviruses;.107/ two isozymes of protein kinase C differ-ing in just 23 residues could be readily distinguished;.108/

and the CD4 glycoproteins of rat and mouse could bedistinguished (73% identity)..38/ Perhaps more impres-sive is the ability to discriminate between enantiomers,such as the threefold discrimination between L- and D-citrulline cit (G D 2.5 kJ mol�1), sixfold difference inKD between L- and D-arginine (G D 4.6 kJ mol�1).34/

and, subsequently, the 12 000-fold ratio in KD betweenL- and D-arginine reported by Geiger et al..93/ Aptamersagainst aminoglycoside antibiotics have been particularlyspecific, with anti-tobramycin aptamers having 3–4 ordersof magnitude lower affinity to other aminoglycosides.98/

and one against streptomycin having four orders of magni-tude lower affinity to the closely related bluensomycin..73/

Strikingly, aptamers against caffeine had a 10 000-foldlower affinity for theophylline, which differs in just amethyl group..109/ However, there are limits to specificity,as one would expect. For example, aptamers againstcoenzyme A also recognize AMP,.90/ those against xan-thine recognize guanine, but not adenosine cytosine oruracil,.72/ and those against the disaccharide cellobioserecognize cellulose, a polysaccharide containing the samerepeating unit, but not the disaccharides lactose andmaltose..78/

4 APPLICATIONS AND DOWNSTREAMTECHNOLOGIES

In comparison with the comparable field of monoclonalantibodies, aptamers suffer from a relatively modest rangeof downstream technology which would enable them to beused more routinely in diagnostic and analytical assays orother practical applications. Accordingly, in the followingsections, some of the advances that have been made inthese areas are reviewed.

4.1 Conjugation to Detectable Moieties

Often, the first step in exploiting an antibody is its con-jugation to a detectable moiety, such as a fluorochrome,an enzyme or a generic ligand such as biotin. Conjuga-tion is most usually done by derivatizing e-amino groupsof lysines on the antibody molecule, but care has to betaken not to block lysine residues near or within thecomplementarity-determining regions. Conjugation canbe done through carbohydrate side chains, but this is muchless usual. Even more commonly, secondary antibodies,which recognize conserved epitopes on antibodies, ir-respective of their antigen, are conjugated to detectablemoieties, thereby simplifying the development of assaysfor new antigens. The challenge of aptamers is to developa generic labeling system that does not tend to disruptaptamer structure or hinder interaction with target.

Nucleic acids can be radioactively ‘body labeled’during transcription or replication by the use of modifiednucleotides. Where these are 3H or 32P body labeled, thisdoes not affect the chemistry of the aptamer and thus doesnot affect its properties as a ligand. This is a very commonway of quantitating the binding of an aptamer or a pool ofnucleic acids to the target molecule..28/ In addition to bodylabeling, site-specific labeling with a radionuclide can beuseful, for example where the aptamer is synthesizedchemically. For example, in one case, a stannyl nucleotidewas incorporated at the 50 end of a DNA aptamer duringchemical synthesis and tin was subsequently replaced by123I in an oxidation reaction..110/ The aim was to producea ligand that can be used for in vivo imaging.

Aptamers can also be labeled using fluorescent orreactive groups. Here, the challenge is to introduce thedetectable groups at positions that do not interfere withligand properties. For example, a chemically synthesizedaptamer against human neutrophil elastase was labeledat either the 30 or 50 end with fluorescein or biotin..111/

It was found that incorporation of the label reducedthe affinity of the aptamer unless a substantial spacerwas used. In vitro transcribed aptamers can have biotinor fluorochromes incorporates as body labels but thisis generally at the expense of their properties as ligands.Labeling at the 30 end can be achieved in a number of ways:

12 NUCLEIC ACIDS STRUCTURE AND MAPPING

O OP O

base

OHO

PO

O−O

O base

O

O

O−

O−

O

NN

IS

N

N

O

H

O

O

NN

S

N

N

O

H

O

OS O

PO

P Obase

OHO

PO

O−O

O base

O

O−

In vitro transcription with GDP-β-S

Iodoacetyl-LC-biotin

Biotinylated RNA

+

P

HH

HH

H

HO− O

...

...

S−

O

Figure 8 50-Labeling of in vitro RNA transcripts using GTP-b-S (see text).

by templated extension using Klenow polymerase,.112/ byT4 RNA ligase-mediated ligation.113/ and by terminaldeoxynucleotidyl transferase..114/ Labeling at the 50 endcan be achieved by the supplementation of the in vitrotranscription mix with an excess of GTP-b-S, the thiol ofwhich can then be used to attach biotin (see Figure 8)..115/

This approach was used successfully to multimerize ananti-CD4 aptamer via streptavidin, which in turn alloweda fluorochrome to be attached to the aptamer for usein flow cyometry..115/ We have already discussed theproblems associated with inefficient derivitization ofinternal 20-amino groups..27/

4.2 Aptamers in Biosensors and Other Detectors

As high-affinity, high-selectivity ligands, aptamers havepotential in a range of detection systems, including biosen-sors (for reviews, see Osborne et al..116/ and Bier andFurste.117/). We have already discussed their applicationin flow cytometry.115,118/ and they can also be used insandwich assays akin to enzyme-linked immunosorbentassay (ELISA)..119/ Fluorescently labeled aptamers havealso been used to quantitate IgE and thrombin using arapid method based on capillary electrophoresis/laser-induced fluorescence (CE/LIF), in which detection downto 50 pM was reported..120/

Unlabeled aptamers can also be used in certain ana-lytical methods. For example, surface plasmon resonance

(SPR) methods, such as those exploited by the BIA-core system, have been used to detect activated 20,50-oligoadenylate synthetase and CD4..38,121/ In these cases,the aptamer was in the mobile phase and the target wasimmobilized. However, with appropriate derivitizationand immobilization, aptamers could be used to detectspecific molecules in complex mixtures using SPR.

Perhaps the most exciting method so far described alsodepends on an optical flow cell system and the evanascentwave from a total internal reflection event at theoptical surface. However, in the application described byPotyrailo et al.,.122/ the effect detected is not on plasmonresonance angle but on fluorescence anisotropy. Theyreported that fluorescent aptamers immobilized at theoptical surface could be used to detect as little as 0.7 amolof thrombin in a 140-pL test volume in just a few minutes.

Other methods that might be applicable to aptamer-based detection in the future include thin-layer inter-ference techniques which have been used to measureDNA – small molecule interactions in real time..123/

4.3 Aptamer Combination

One approach to increasing the usefulness of aptamersis to combine them with another ligand. For example,a DNA aptamer to human neutrophil elastase wasnoninhibitory to the enzyme’s protease activity butimproved the Ki of a weak peptide inhibitor by five orders

APTAMERS 13

of magnitude when the two were conjugated throughan N-methoxysuccinyl link..124/ Alternatively, one canlink aptamers together to form a dimeric or multimericligand. This can take the form of joining identicalaptamers, perhaps to increase affinity by reducing thedissociation rate of the complex from a multimeric target,or of joining aptamers against different aptatopes, inorder to change specificity. An example of the formerapproach was the homodimerization of aptamers againstL-selectin, which resulted in ligands with KD in the rangeof good monoclonal antibodies..118/ The procedure is notstraightforward, however, as linking two aptamers in asingle oligonucleotide can result in loss of function forone or both ligand moieties, either by steric hindranceor by disruption of the folding of each module. Thisproblem was found with combinations of aptamers againstcoenzyme A, chloramphenicol and adenosine, but wasovercome by the use of further rounds of in vitroselection on chimeric RNAs in which the junctionsbetween aptamers were diversified..125/ A practical usefor heterodimerization of aptamers has been the linkingof aptamers that recognize thermostable polymerases butwith different specificity..126/ The resultant chimeras had abroad specificity to enzymes from a range of thermophilicspecies and were potentially useful for improving theefficiency of PCR from low-copy-number templates. Afurther potential application of heterodimerization is tolink an aptamer that recognizes a target molecule ofanalytical interest to a second aptamer that recognizes adetectable molecule, such as a fluorochrome..127/

Aptamer modules have also been linked to ribozymes(catalytic RNAs) to provide a means for controllingenzyme activity through allosteric interactions. Forexample, the activity of a hammerhead autocatalyticribozyme fused to an ATP-binding aptamer was shown tobe regulated >100-fold by the presence of ATP..128 – 130/

This effect depends on the conformational change in theaptamer following ligand binding and steric hindrancebetween the folded aptamer and the ribozyme. These andother authors have extended this work to construct RNA-cleaving ribozymes whose activity is regulated by othersmall molecules, such as theophylleine and FMN,.129,131/

and RNA-ligating ribozymes whose activity is regulatedby ATP..132/ The general principle could form the basisof a new generation of in vitro assays for analytes.

4.4 Use of Aptamers In Vivo

Since the lead given to the field at the turn of the centuryby Paul Ehrlich (see, for example, Ehrlich.133/) (transla-tion by Brock.134/), there has been a continuing desireto develop new classes of specific ligands, preferably byrational means, that could act as ‘magic bullets’, seek-ing out their molecular targets in vivo and destroying

them. The possibility that aptamers might be candidatetherapeutic agents has naturally received much attention(see review by Osborne et al..116/). The main challengeto all these applications is the pharmacokinetics of oligo-nucleotides in vivo: the rate at which they are degraded byplasma nucleases; the rate at which they are sequesteredby reticulo-endothelial cells and plasma proteins; the ratewith which they are excreted by the kidney; and the rateat which they redistribute from the blood to the tissue orbody fluid of interest. Natural, 20-OH-RNA degrades veryrapidly in human serum in vitro but this process can beslowed at least 1000-fold by 20-amino modification..135/ Invivo studies on the t1/2 of single-stranded DNA aptamersgive values in the range from <2 min to approximately8 min..136 – 138/ Conjugation of the aptamer to either lipidsor polyethylene glycol has been reported to improve thestability and distribution kinetics of DNA aptamers suf-ficiently to produce therapeutic effects..139,140/ A widerange of potentially therapeutic targets for aptamers aresummarized in Table 1, with a note of therapeutic effector clinical benefit, if any.

More radically, it is conceivable that one could useaptamers in a form of gene therapy, to inhibit thefunction of intracellular proteins – either host-derivedor viral – in vivo. This approach is fraught by all theproblems that have dogged the gene therapy field formany years: inefficient and intrusive delivery systems,inefficient or transient expression, safety concerns, poorselectivity, and so on (for a review, see Palu et al..141/).Worse, the rather low concentration of free divalentcations and the presence of high concentrations ofRNA-binding proteins in the cell means that aptamersselected under the conventional in vitro conditions mightwell be inappropriately folded in the cell. Nevertheless,some positive reports have appeared. For example, in astudy of the inducible expression of aptamers againstRNA polymerase II in yeast, a significant effect ontranscription was seen as a result of aptamer expression,but only in yeast strains with abnormally low levelsof polymerase II expression..142/ More promisingly, U6snRNA and tRNAmet cassettes were used to expressaptamers that bound the HIV-1 regulatory protein, Rev,in cells.143/ and it was found that these, like Rev decoysderived from the natural Rev RNA ligand, were ableto inhibit the expression of the virus. However, theseexperiments were done in vitro in a convenient butunphysiological cotransfection system, so one must becareful not to overextrapolate.

4.5 Studies on Nucleic Acid Structure

One of the first studies describing in vitro selec-tion of nucleic acid ligands was designed to analyzethe sequence requirements of a nucleic acid-binding

14 NUCLEIC ACIDS STRUCTURE AND MAPPING

Table 1 Potentially therapeutic aptamers

Target References Comments

ToxinsRicin, pepocin, gypsphilin 144 Have different sequence from natural rRNA targetsEnzymesThrombin 7 First identification

138, 145 Use in vivo146 Better than heparin against clot-bound thrombin

81 An RNA aptamer against thrombinActivated plasma protein C 74HIV-1 reverse transcriptase 69, 96Other retroviral reverse transcriptases 107, 147HIV-1 integrase 95, 148Protein kinase C 108Human neutrophil elastase 124, 149, 150 Identification of inhibitory aptamer when conjugated to

weak peptide inhibitor of enzyme. Effective in ananimal model of lung injury

Hepatitis C virus NS3 protease/helicase 103, 151, 152Yersinia protein tyrosine phosphatase 153Phospholipase A2 100 Inhibited contractions of guinea pig pleural strip in vitro20,50-Oligoadenylate synthetase 121Angiogenin (ribonuclease activity) 154 DNA aptamer prevents angiogenesis and cell proliferationAdhesion and recognition moleculesL-selectin 155 DNA aptamers inhibit cellular adhesion and rolling in

vitro and trafficking in vivoP-selectin 156 Affinity (20 pM) is 106-fold better than the natural ligand

sialyl Lewis X. Unlike the latter, discriminate against E-and L-selectins by 104 –105

CD4 38, 115 Disrupts immune recognition in vitroRhinovirus capsid protein 157 Intended to block infectivity of virusGrowth factors and hormonesBasic fibroblast growth factor 83VEGF 14, 84, 140 Inhibit induction of permeability in vivo. 20-F modification

and lipid derivatizationImprove stability and pharmacological properties

NGFa 82PDGF 99, 139 Effective in vivo as a PEG conjugateKeratinocyte growth factor 13g-Interferon 158, 159Substance P 76Vasopressin 18Regulatory proteins and oncogenesE2F transcription factor 86 Prevents binding to DNA and entry into S phaseK-ras 101 Raised against farnesylated peptideRaf-1 102P210bcr-abl 160 DNA aptamer introduced into chronic myelogenous

leukemia cells by electroporation reduced cellproliferation

MDM2 oncoprotein 161HIV-1 Rev 67, 88, 143, 162–164 These are Rev-responsive, even though unrelated to

natural Rev-response element. Are inhibitory to HIV-1growth in cell cultures

HTLV-I Tax 39MiscellaneousIgG against human insulin receptor 104 Possible approach to blocking a common form of

antibody-mediated, autoimmune diabetesIgE 12 Bind Fc portion of antibody, preventing interaction with

cell surface receptor. Possible role in allergy therapyHamster PrPa 165

a NGF, nerve growth factor; PrP, prion protein.

APTAMERS 15

protein..4/ Aptamers have continued to be a power-ful method for analyzing nucleic acid sequence bindingrequirement of a range of viral proteins. For exam-ple, aptamers have been used to dissect the nucleicacid binding sites of the coat proteins of bacteriophagesR17,.166/ MS2.167/ and f29,.168/ the RNA-dependent RNApolymerase of bacteriophage Qb,.169/ the Epstein–Barrvirus EBER1 RNA,.170/ the regulatory, Rev-responseelement of HIV-1.136,171/ and the HIV-1 nucleocapsidprotein..97/

The approach has been used productively to examinethe interaction between components of the translationalmachinery and natural RNAs, e.g. the elongation factorsEF-Tu,.172/ elF-4B.173/ and SelB,.174/ the S8.175/ and S1.176/

ribosomal proteins from E. coli, the L32 ribosomal proteinof yeast.177/ and the decoding end of 16S rRNA..178/

Finally, transcription and post-transcriptional processinghave been amenable to the aptamer approach, e.g. therequirements for the E. coli transcriptional terminatorr,.33/ the eukaryotic splicing factors SF2 and SC35.179/

and GU-rich sequences for efficient polyadenylation..180/

It has been proposed that these methods could be usedto identify all the protein–RNA interactions encoded inthe genome,.181/ although this is not yet an establishedapproach.

4.6 Isolation of New Catalysts

The very first paper describing directed in vitro evolutionof nucleic acids described the isolation of a totallynew enzyme, a nuclease composed of DNA..3,182/ Thepossibility was thereby opened up that one could developany form of useful new catalyst that could be conceivedand for which a selection procedure could be devised.To an extent, this has been realized, with the isolationof enzymes that can ligate DNA in a Zn/Cu-dependentfashion.183/ or ligate RNA with a similar mechanism tothat of protein enzymes..184 – 187/ These new enzymes arenot restricted to phosphodiester bond formation andbreakage. For example, in vitro-selected nucleic acidenzymes have been described that cleave amide bonds.188/

and alkylate halogenated peptides..189/ The combinationof a fluorochrome aptamer and its target was found tohave low-level oxidative activity, when coupled to itstarget,.77/ and a hemin–aptamer complex was found tohave levels of peroxidase activity comparable with proteinperoxidases, and much higher than those of catalyticantibodies..190/ More significantly, RNA enzymes havebeen selected with amide bond-forming activity.191/ inwhich a uridine was replaced by a 50-imidazole derivativeof uridine, capturing the chemical characteristics ofhistidine in nucleic acid. Also, a ribozyme was isolated thatcatalyzed a Diels–Alder cycloaddition reaction in whichuridine is replaced by a 5-pyridyl derivative of uridine..192/

This RNA catalyst is therefore the first nucleic acid withcarbon–carbon bond-forming activity.

5 PERSPECTIVES AND FUTUREDEVELOPMENTS

Clearly, the first decade of research using aptamershas opened up some very exciting avenues in basicand applied research. The power of the techniquehas surprised many and, even though the chemistryof nucleic acids is much less diverse than that ofpolypeptides and there are substantial restrictions tothe technology as it stands, it is already a usefultool and promises to become more so. There aresubstantial advances in methods which use the principleof in vitro selection but apply it to the evolution ofpeptide ligands. For example, adaptations of the two-hybrid display methodology using randomized peptidesdisplayed on thioredoxin in E. coli has been usedto generate peptide aptamers against CDK-2 withnanomolar affinity.193/ which blocks G1-S transitionduring the cell cycle of eukaryotic cells engineered toexpress it..194,195/ Similar approaches display peptideswithin the green fluorescent protein.196/ or at the C-terminus of the lac repressor protein..197/ One problemwith these methods, and the widely used phage displaytechnology [see reviews by Burton.198/ and Winteret al.,.199/] is that the critical step of passing througha living organism during the generation of the libraryreduces the number of different molecules that can bescreened by many orders of magnitude. To overcomethis, methods have been developed to link a nucleic acidbased library with in vitro translation, thereby obviatingthe need for a living cell. The challenge is to preservethe ‘genetic encoding’ by maintaining the associationbetween nucleic acid and the encoded polypeptide.One approach is to use polysomes, collections ofribosomes simultaneously translating a messenger RNA(mRNA)..200/ More conveniently, a method in whichtranslation is simultaneously arrested and the RNAlinked covalently to the nascent polypeptide chainhas been developed,.201/ in which a puromycin groupis incorporated at the 30 end of the library RNA and islinked to the C-terminus of the polypeptide by the actionof the ribosome’s peptidyl transferase.

ACKNOWLEDGMENTS

I am grateful for stimulating discussions with ElmarKraus, Jamal Ibrahim, Laura Frigotto and AbdessamadTahiri-Alaoui and the opportunity to present their as yet

16 NUCLEIC ACIDS STRUCTURE AND MAPPING

unpublished results. I thank Abdessamad for his criticalreading of the manuscript.

ABBREVIATIONS AND ACRONYMS

AMP Adenosine 50-MonophosphateATP Adenosine TriphosphateCE/LIF Capillary Electrophoresis/Laser-induced

FluorescencedNTP Deoxynucleoside TriphosphateELISA Enzyme-linked Immunosorbent AssayFMN Flavin MononucleotideIg ImmunoglobulinIgE Immunoglobulin EIgG Immunoglobulin class GmRNA Messenger RNAMS Mass SpectrometryNGF Nerve Growth FactorNMR Nuclear Magnetic ResonancePDGF Platelet-derived Growth FactorPrP Prion ProteinPVDF Poly(vinylidene difluoride)rRNA Ribosomal RNASELEX Selective Expansion of Ligands by

Exponential EnrichmentSPR Surface Plasmon ResonanceVEGF Vascular Endothelial Growth Factor

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