Aptamers

William James
in Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) pp. 48484871 John Wiley & Sons Ltd, Chichester, 2000

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1 INTRODUCTION
A very neat job in a small space 1

Aptamers
William James University of Oxford, Oxford, UK

1 Introduction 2 In Vitro Evolution of Nucleic Acid Ligands 2.1 Outline of Process 2.2 History of Discovery and Development 2.3 Choice of Chemistry 2.4 Practical and Theoretical Considerations 3 Properties of Published Aptamers 3.1 Structures 3.2 Aptamer Size 3.3 Aptamer Targets 3.4 Afnity 3.5 Specicity 4 Applications and Downstream Technologies 4.1 Conjugation to Detectable Moieties 4.2 Aptamers in Biosensors and Other Detectors 4.3 Aptamer Combination 4.4 Use of Aptamers In Vivo 4.5 Studies on Nucleic Acid Structure 4.6 Isolation of New Catalysts 5 Perspectives and Future Developments

1 1 1 3 3 5 8 8 9 9 10 11 11 11 12 12 13 13 15 15 15 16 16 16

Aptamers range in size from approximately 6 to 40 kDa and sometimes have complex three-dimensional structures, produced by a combination of Watson Crick and non-canonical intramolecular interactions. They bind to their targets with KD typically in the low nanomolar range and can distinguish enantiomers of small molecules or minor sequence variants of macromolecules with frequently several orders of magnitude KD ratio. They are typically composed of RNA, single-stranded DNA or a combination of these with non-natural nucleotides. Aptamers are isolated from extremely complex libraries of nucleic acids, generated by combinatorial chemistry, by an iterative process of adsorption, recovery and reamplication. Additional sequence variation can be introduced at each cycle and the process becomes an in vitro paradigm of Darwinian evolution. After sufcient enrichment, aptamers can be cloned and studied as homogeneous sequence populations. Aptamers can be used to analyze the natural processes of nucleic acid protein recognition, to generate inhibitors of enzymes, hormones and toxins with potentially pharmacological uses, to detect the presence of target molecules in complex mixtures and to generate lead compounds for medicinal chemistry. Their advantages over alternative approaches include the relatively simple techniques and apparatus required for their isolation, the number of alternative molecules that can be screened (routinely of the order of 1015 ) and their chemical simplicity. Disadvantages of aptamers include their pleiomorphism, their high molecular mass and the restricted range of target sites that appear to be suitable.

Acknowledgments Abbreviations and Acronyms Related Articles References

2 IN VITRO EVOLUTION OF NUCLEIC ACID LIGANDS

2.1 Outline of Process Aptamers are ligands derived by a process of combinatorial chemistry, in which the desired property is identied by afnity chromatography and encoded genetically. The process is illustrated in Figure 1 and described below. 2.1.1 Library Synthesis and Complexity Combinatorial chemistry is the production of a very large number of different molecules by the repeated

Aptamers are articial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acid by an iterative process of adsorption, recovery and reamplication. They have potential applications in analytical devices, including biosensors, and as therapeutic agents.

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

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5 constant T7 pro -> 36nt random 3 constant DNA library

NUCLEIC ACIDS STRUCTURE AND MAPPING

In vitro transcription RNA library RNA folding PCR amplification

Mixing Enriched pool Target

Reverse transcription

Partition

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 many forms, outside the scope of this article, but was inspired by the sequential, solid-phase synthesis of oligopeptides and oligonucleotides (oligos: Combinatorial Chemistry Libraries, Analysis of). An oligo of dened sequence is commonly synthesized on a solid support by a cycle of deprotection of the acceptor end of the growing chain and its derivitization by using an excess of the next activated monomer in the mobile phase. By modifying this process so that the activated monomer is a mixture, rather than a single species, oligos of randomized sequence may be readily obtained. For sequences of randomized length N and y alternative monomers at each position, a library of oligos of diversity yN can, in principle, be synthesized. For example, a nucleic acid library of randomized length 40 has a maximum theoretical diversity of 440 D 1.2 1024 . The theoretical diversity, otherwise known as sequence space, of such a library will often exceed by many orders of magnitude the number of molecules that can be handled in practice. Convenience and expense generally restrict the initial library to a sample of in the region of 1013 1015 molecules taken, presumably randomly, from the larger sequence space. 2.1.2 Selection of the Desired Property The desired property is, typically, the ability to bind to a molecule of interest. Depending on the anticipated application, the desired binding properties may be a fast association rate, slow dissociation rate, high afnity, low afnity to closely related molecules, or a combination of these. This property will be a function of the threedimensional structure of the folded nucleic acid and will be a combination of its van der Waals surface contacts, hydrogen bonds, stacking interactions and other noncovalent bonds that can form between the aptamer and its target. It is a necessary assumption that, to a reasonable approximation, the three-dimensional structure of an aptamer is uniquely determined by the sequence of its bases. These assumptions will be explored below. By mixing the solution-phase library with the target molecule and subsequently retrieving the target and removing unbound or loosely bound nucleic acids, the few nucleic acids that have the desired property can be recovered from the library. This partitioning step is a form of afnity chromatography and the methodology can be varied depending on the nature of the target and the exact property desired. This will be discussed in detail below. The selection step differs from screening methods used in other branches of combinatorial chemistry because of the numbers of candidate molecules that can be reviewed at one time. Conventional libraries of potential drugs may consist of the order of 106 molecules, which can be screened for activity by robotic means in a few months. Combinatorial libraries of greater than about 105 are impractical to screen by conventional, iterative methods, as vanishingly few molecules of each species would be present in each assay. Perhaps the most sensitive methods involve afnity partitioning followed by mass spectrometry (MS). 2

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they coined the term SELEX (selective expansion of ligands by exponential enrichment), was able to identify the natural target of the enzyme as the predominant, high-afnity ligand, with one major variant emerging with similar afnity. The authors patented their process. Less than a month later, the Szostak group reported the use of in vitro selection to isolate molecules with specic ligand-binding activities. 5 More radically than the previous reports, they began with a library that was structurally unrelated to any known nucleic acid, having 100 nucleotides of randomized sequence, and chose targets that had no previously identied nucleic acid ligands. They used afnity chromatography to isolate RNAs with specic and selective binding characteristics for a number of organic dyes (chosen because of their potential as H-bond partners and their planar structures that might be expected to form stacking interactions with the nitrogenous bases of RNA). It was this group that coined the term aptamer for such nucleic acid ligands. 2.3 Choice of Chemistry Aptamers are nucleic acids that can be composed of naturally occurring monomers or chemically synthesized derivatives. For a review of this eld, see Eaton and Pieken. 6 As outlined above, the nucleotides must be compatible with RNA-dependent DNA polymerases (reverse transcriptase) and with DNA-dependent RNA polymerases (such as T7 RNA polymerase) for the enzymatic steps required in the production of RNA-based aptamers, or with thermostable DNA-dependent DNA polymerases (such as Taq polymerase) for the production of DNA-based aptamers. 7,8 This restricts the available chemistry more strictly than is the case with purely synthetic oligonucleotides. Nevertheless, researchers have adopted many modications of natural nucleotides in order to overcome two substantial problems posed by natural nucleic acids. 2.3.1 Phosphodiester Bond Hydrolysis This is a particular problem in natural RNA, as the hydroxyl at the 20 -position is reactive, particularly at higher than neutral pH, and will attack the neighboring phosphodiester bond to produce a cyclic 20 ,30 phosphate, thereby breaking the nucleic acid backbone (see Figure 2). This reaction is catalyzed by many transition metals ions, particularly lead and iron, and by a range of ribonucleases found ubiquitously in biological samples. To overcome this problem, one has turned to modications of either the 20 -position of the ribose moiety or modications of the phosphodiester backbone. By far the most common approach is the use of nucleotides substituted at the 20 -position with either an amino group or uorine atom. These modications were pioneered

2.1.3 Genetic Encoding In contrast to other forms of combinatorial chemistry, where a variety of methods have been devised to encode the structure of each agent, or at least its synthetic recipe, in the solid-phase support, aptamers contain within themselves the genetic code for their own amplication and synthesis. Nucleic acid polymerase enzymes are used to convert the target-bound aptamer, if it was based on RNA, into DNA, to amplify the copy number exponentially (by a factor of at least 1010 ) and, again if the aptamer is RNA, to transcribe the amplied DNA template back into RNA. 2.1.4 Enrichment and Evolution Because the afnity-based partitioning methods are imperfect, the cycle of partitioning and amplication is normally repeated 6 12 times, sometimes more, before molecules of the desired property predominate in the population of nucleic acids. The process, as described above, represents a reduction in sequence diversity from the initial sample of sequence space (e.g. 1015 out of a space of 1024 ) to a population of the order of typically 10 distinct sequences that show appreciable binding to the target. In addition, however, the error-prone nature of the enzymes used to amplify the selected sequences leads to the introduction of mutations that effectively allow the procedure to sample a greater proportion of the sequence space than was initially sampled. This leads to the appearance of clearly related but evolutionary divergent sequences within the nal aptamer population, in a process akin to Darwinian evolution by natural selection. 2.2 History of Discovery and Development While experiments involving the isolation of nucleic acids from articial libraries on the basis of their biochemical properties were being widely discussed during 1988 and 1989, three groups independently published their results in 1990. First, the Joyce group reported the use of in vitro mutation, selection and amplication to isolate RNAs that were able to cleave DNA. 3 They began with a natural RNA-cleaving ribozyme, rather than a purely random library, and were looking for a novel enzymic activity rather than a selective ligand. However, their experiment had most of the essential features described in this article, including the repeated cycles of reaction performed in a single vessel. Second, the Gold group described experiments designed to identify the sequence requirements of T4 DNA polymerase, in which the library was based on the natural target hairpin structure but with the eight loop nucleotides randomized. 4 The process of in vitro selection, for which

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OH O P O O O P O O O P O O
4 5

NUCLEIC ACIDS STRUCTURE AND MAPPING

NH2 N cytosine O
3 2

N
1

O O N NH guanosine N NH2

O OH O P O O (a) OH O P O O O P O O O P O

N O

OH OH

NH2 N

O O O O P O HO O N N N NH NH2

(b) OH O P O O O P O O O P OO O N NH2 N

OH OH

Where they have been compared, 20 -deoxy and 20 amino chemistries have been found to produce aptamers with similar afnity, although different structure (for an example, against immunoglobulin E (IgE), see Wiegand et al. 12 ). However, 20 -uoro chemistry produces aptamers with greater thermal stability and probably higher afnity than 20 -deoxy or 20 -amino chemistry. 13 Although one cannot generally change the chemistry of the nucleotides after selection without changing the structure of the aptamer and frequently abolishing its properties as a ligand, this can often be done in a more selective manner (A. Tahiri-Alaoui, unpublished work). For example, further modications at the 20 -position that give additional stability can be incorporated synthetically into aptamers with 20 -OH, -H, -NH2 or -F that have been identied through in vitro evolution. For example, 20 -O-methyl has been introduced at certain purines in 20 -F-pyrimidine aptamers against the vascular endothelial growth factor (VEGF), with benecial effects. 14 The alternative approach, to modify the phosphodiester backbone, is more challenging to the enzymology of in vitro evolution. Nevertheless, by using a-thio-substituted deoxynucleoside triphosphates (dNTPs), a phosphorothioate DNA library was successfully screened for aptamers against the transcription factor NF-IL6, 15 and some progress has been made toward the use of analogous, thio-RNA aptamers. 16,17 Another way to produce nuclease-resistant aptamers is to select an aptamer that binds the enantiomer of the eventual target, then synthesize the enantiomer of the aptamer as a nuclease-insensitive ligand of the normal target. Such spiegelmers have been made in L-DNA against the peptide hormone, vasopressin 18 and in LRNA against L-adenosine and L-arginine. 19,20 2.3.2 Absence of Hydrophobic or Basic Residues

O O N NH N NH2

O F O P O O (c)

N O

OH OH

Figure 2 Representative RNA components. (a) Diagram of

the structure of a CG dinucleotide, showing the numbering convention for carbons in the ribose moiety. (b) The 20 ,30 -cyclic phosphate that forms as the result of nucleolysis. (c) A CG dinucleotide containing 20 -uorine.

by the Eckstein group 9,10 and are compatible with T7 RNA polymerase for efcient in vitro transcription. 11

In contrast to proteins, nucleic acids are strikingly uniform in their hydrophilicity and low pI. In spite of these limitations, a surprising range of enzymatic activities are possible for nucleic acids and the hydrogen bond and stacking interactions of their component bases provide a diverse toolbox of structural motifs. 6 Nevertheless, this uniformity almost certainly limits the ease with which aptamers can be made to certain targets and a greater range of side chains, including some with basic or hydrophobic character would be welcome additions to the armamentarium. The challenge, as with substitutions giving nuclease resistance, is to discover approaches that are consistent with the enzymes used during aptamer isolation. One approach, using 5-(1-pentynyl)-20 -deoxyuridine, succeeded in producing additional aptamers to thrombin, 21 though no reports

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not be repeated here. A mathematical description of the process, under assumptions of equilibration, was produced by Irvine et al. 29 and supplemented by a model for simultaneous selection against multiple targets by Vant-Hull et al. 30 We shall conne ourselves to a discussion of some of the most relevant details. 2.4.1 Library Complexity When designing a library, the desire is to produce sequences that can be amplied and transcribed with high efciency, of as great a diversity of structures as is practicable and at reasonable cost. One is bound to use substantial xed, anking regions for primerbased amplication and transcription, so one question is how long to make the randomized region (N) and how to ensure that the library produced contains as near to the theoretical maximum of sequences possible. First, although the sequence space available to the library is 4N , the maximum number of molecules that can be manipulated in standard molecular biology laboratories (M) is of the order of 1015 , and errors of synthesis and workup have been estimated to reduce this diversity to 1013 1014 . N need only be around 22 24 to reach this practical limit. However, in order to produce a structure capable of specic interaction with a target molecule, the RNA needs to be large enough to fold into a complex tertiary structure. It appears that many classes of RNA fold need to composed of signicantly longer stretches of nucleic acid than this (see above), so most people use libraries where N 35 and sometimes much more. As N increases, one is able to sample progressively a smaller fraction of the theoretical sequence space. For example, if M D 1014 and N D 24, we could sample 1014 /2.81 1014 D 36% of the sequence space, but if N D 25, this reduces to 8.9%. However, if we consider each 25-mer to be composed of two overlapping 24-mers which differ only at their termini, then the N D 25 library effectively contains 2 1014 24-mers. This suggests that one should aim for larger rather than smaller values of N. Two considerations effectively limit the practical size of N. First, although the efciency of oligonucleotide synthesis is constantly improving, errors accumulate with each cycle and a point is reached at which incorrectly synthesized molecules begin to have a signicant impact on the quality of the library. The limit is imprecise but it seems unwise at present to make large-scale libraries using oligo lengths much greater than about 120 nt, suggesting a practical limit of around N D 70 80. Second, if RNA folds are typically between 25 and 50 nt long, libraries with N greater than approximately 50 nt may well contain sequences comprising two independent folds, complicating the process of in vitro selection. The actual diversity of a library can be compromised by variations in the efciency of coupling phosphoramidite

have emerged showing that this approach has produced aptamers against more refractory targets. It has long been speculated that the precursor to our DNA-genome, protein-catalyst world of organisms was a world in which RNA functioned for genomes, ligands and enzymes 22 and this was consolidated by the discovery of ribozymes. 23,24 (In his paper on the Evolution of the Genetic Apparatus, Orgel 22 speculated on life based on nucleic acids without a genetic code and said that it seems to me quite possible that polynucleotide chains could make primitive selection among organic molecules such as amino acids by forming stereospecic complexes stabilized by hydrogen-bonding and hydrophobic interactions. He also speculated that polynucleotides might have the ability in early stages of life to catalyze chemical reactions but that this function would subsequently have been taken over by the much more versatile polypeptides.) Indeed, it is proposed by some that the restricted range of amino acids extant in the current world of biology is dictated by those homologous with natural, prebiotic derivatives of 5-hydroxymethyluracil that had previously been a feature of the RNA world. 25 Further, one should note that many biological RNAs of the present world have additional methyl groups added post-transcriptionally to their purines, which increase the local hydrophobicity of the nucleic acid (reviewed by Levy and Miller 26 ). None of these potentially helpful modications is reproduced during enzymatic replication of nucleic acids, though some of them, rather than acting as chain terminators, are ignored. For example, 5-methylation of cytosine is a common postreplication modication of eukaryotic DNA and 5-methyldeoxycytidine is replicated routinely to dG during the S phase of the eukaryotic cell cycle. Instead of attempting to broaden the repertoire of nucleotides used during the replication and transcription phases of in vitro selection of aptamers, one might modify one or more nucleotides post-transcriptionally. It was found that derivitization of 20 -NH2 groups on aminopyrimidine-RNA with succinimide did not prevent reverse transcription. 27 Although this might usefully open up a wide range of functional adducts, the efciency was low, with only two out of 23 amino groups per molecule, on average, being successfully derivatized. In order to preserve the genetic encoding of aptamers, it is essential that such processes are either fully efcient or reproducibly inefcient (i.e. the same nucleotides being derivatized each cycle). 2.4 Practical and Theoretical Considerations A comprehensive and practical introduction to the methodology of in vitro evolution of RNA ligands was provided by Fitzwater and Polisky 28 and so will

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monomers during oligonucleotide synthesis, which in turn can depend on the particular practices of the manufacturer chosen. Although standard methods use equimolar mixtures at each N stage during synthesis, the Szostak group has reported the use of an A : C : G : T ratio of 3 : 3 : 2 : 2 and we have found it necessary to use ratios of 6 : 5 : 5 : 4 in order to achieve random incorporation. Gross bias during synthesis and enzymic manipulation can be estimated by checking the sequence of a dozen or so clones made from the library after a single round of amplication, transcription, reverse transcription and reamplication, without selective partitioning. The clones should be sequenced and the overall base composition within the random region tested for divergence from expectation. In addition, one should look for bias in the dinucleotide and trinucleotide composition and at base composition at each position. What are the chances of nding a target-binding sequence during the in vitro exploration of sequence space? The answer seems to depend on ones target and the criteria (e.g. afnity threshold) used to identify binding. For example, within the N D 100 library described by Ellington and Szostak, 5 the authors estimated that 10% of the library contained aptamers for organic dye molecules. In contrast, Burke and Gold 31 estimated that perhaps one in 1011 sequences within their library contained adenosine-binding motifs. Our own experience with libraries of N D 36 is that protein-binding aptamers sequences exist at an initial frequency of typically 1 in 1011 1012 . If these gures are representative, it shows that care needs to be taken during the early stages of library production and manipulation not to reduce the sequence complexity much below its theoretical maximum. Tuerk 32 has made calculations based on computer modeling of the SELEX process and suggested that binding reactions that yield 6% of the total RNA bound to target would be optimal. Schneider et al. 33 showed that when reactions were arranged to select 10% of the RNA population bound to r protein (low stringency) or 1% (high stringency) during SELEX, ligands of similar sequences were produced by both selection strategies, although the speed of afnity enhancement of the pool was increased somewhat for the high stringency selection. This illustrates that the ne tuning of ligand target ratios is not crucial for success in obtaining optimal binding ligands. Tuerk 32 suggests that the rst binding reaction be conducted in large volumes (50 mL) with target molecule concentrations at 0.2 times the KD of the original nucleic acid pool target interaction and all of the starting nucleic acid. This would yield about 9% of the original nucleic acid population bound to a large number of target molecules, decreasing the chance that low-abundant, unique sequences of high afnity are lost in the rst selection.

NUCLEIC ACIDS STRUCTURE AND MAPPING

2.4.2 Mutagenesis and Afnity Maturation Thus far, we have been considering merely the selection of ligands from an initially very small and approximately random sample of sequence space. However, the phrase in vitro evolution implies that we are able to explore a much greater fraction of sequence space by allowing mutation to generate additional diversity and selection to retain a small fraction with favorable properties. By repeatedly selecting for the desired property, the path of exploration should tend towards optimum ligands; those that occupy segments of sequence space that correspond to afnity maxima. However, we can imagine many ways in which this favorable result would not have been reached. First, the initial sample of the library may not have contained any members that were within striking distance of a high-afnity region of sequence space. This will depend on the complexity of the library and the nature of the target, as described above. Second, the amount of mutation at each level may have been too little to allow signicant progress towards the afnity maximum. Conversely, mutation might have been so high that the peaks would have been missed or overshot. Third, selection might not have been stringent enough, allowing the minority of high-afnity ligands to be swamped by their low-afnity brethren. Conversely, selection might have been too stringent, eliminating sequences of intermediate afnity before high-afnity sequences had been arrived at. This is a particularly acute problem during in vitro selection because one rarely has any way of knowing either the afnity topology of sequence space or the properties of early-cycle oligonucleotides. There has been surprisingly little experimental analysis of this problem. Although we know that aptamers selected against a particular target usually fall into sequence groups which suggest phylogenetic relatedness, the polyphyletic origin of aptamers means that two similar sequences may have arisen by convergence from two independent sequences sampled in the initial library as easily as by divergence from a common ancestor. Nevertheless, in one study, explicit evolution was measured from an aptamer that had been previously selected against L-citrulline to others which could bind alternative amino acids. 34 Mutation was introduced at a single step by chemical resynthesis of the original aptamer to give an average of 30% mutation at each position. Binding to citrulline was recovered after three rounds of selection and to arginine after four rounds. However, no lysineor glutamine-binding aptamers were obtained. The more usual method of introducing mutation is to encourage misincorporation of nucleotides during amplication by increasing the concentration of dNTPs and Mg2C and using enzymes lacking proofreading activity. 28

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300 250 Strataclean Activated Sepharose

2.4.3 Partitioning Methodology The separation of desired from undesired sequences is of critical importance in the selection and evolution of aptamers (see the discussion above and a mathematical analysis by Vant-Hull et al. 30 ). Ideally, one would be able to allow the nucleic acid pool and the target to interact freely, be able to monitor the progress of the interaction and retain those nucleic acids whose desired property lay above a threshold determined in part by the properties of the pool. Most methods of partitioning fall short of these ideals. First, tethering the target to a solid support facilitates the separation of bound from unbound nucleic acids and facilitates procedures such as competitive elution which can be helpful in setting afnity and specicity parameters. This is the approach used in some of the earliest experiments, and has been particularly used in the identication of aptamers to small molecules. 5,34 36 Immobilization of target proteins to Sepharose has also been used successfully to facilitate partitioning 37,38 but mixing the nucleic acid and target protein in the soluble phase has signicant advantages. First, many protein immobilization regimes involve derivitization of the e-amino groups of lysine, thereby destroying a key feature of many aptatopes. Second, immobilization reduces the mobility of the protein, impeding its mixing with nucleic acid and may produce direct steric hindrance to aptamer binding. Third, a progressive reduction in the molar ratio of target protein to nucleic acid in successive cycles, in order to increase the stringency of selection, is very hard to achieve in an immobilized system. Consequently, one of the common approaches is to use an insoluble matrix that selectively adsorbs protein following nucleic acid protein interaction in the uid phase. Nitrocellulose lters are a cheap and convenient matrix that is widely used for this purpose. 4,39,40 However, we have found that nitrocellulose preparations are not as selective for protein as would be desired. We have compared the use of nitrocellulose lters, poly(vinylidene diuoride) (PVDF) membranes, activated Sepharose, octyl Sepharose and the deproteinizing matrix Strataclean resin for their ability to select for CD4-binding aptamers via interaction with the protein (F. Kesten, personal communication). We found that nitrocellulose and PVDF trapped substantial amounts of 20 -F-RNA nonspecically and that octyl Sepharose was not an efcient protein binder under these circumstances (results not shown). However, CNBractivated Sepharose and Strataclean resin were both efcient at pulling down 20 -F aptamer RNA in a targetdependent fashion (see Figure 3). Models of the selection process also assume that equilibrium between RNA and protein has been reached before partitioning. 29 However, this is very unlikely to be the case during early rounds, when the concentration

A 260

200 150 100 50 0

HA + Control CD4 + RNA Control RNA

CD4 + CD4 Aptamer RNA

Figure 3 Selective partitioning of ligand RNA using either activated Sepharose or Strataclean resin. of ligands is extremely small or at any stage when incubation times of the order of 10 min are used routinely and the most desirable aptamers have dissociation t1/2 of the order of 1 h (J. Ibrahim and L. Frigotto, personal communication). In a recent study, we were able to show that increasing the incubation time from 30 min to 2.5 h substantially reduced the number of cycles required to isolate high-afnity aptamers. 41 Recent improvements in robotics technology have enabled the process of in vitro selection to be reduced to a matter of days. 42 2.4.4 Structural Pleiomorphism A central but unspoken assumption of the methods for in vitro selection and evolution of aptamers is that there is a direct and unique relationship between the sequence of an aptamer and its shape: the one sequence, one structure assumption. In other words, if one selects a nucleic acid sequence s in round n on the basis that it binds to protein X, then one would expect that all copies of the amplied sequence s will bind to X in round n C 1. However, if sequence s can fold into more than one conformation, only a proportion of the reamplied s will bind to X in round n C 1. Worse, if each sequence is present at low copy number, for example during the early rounds of selection, the copy of sequence s in round n may not be in the right conformation to bind X and thus not be selected at all. How pleiomorphic should we expect most RNAs to be? If we were to answer this on the basis of the known folding of functional RNAs such as the Tetrahymena intron or the hammerhead ribozyme, which fold rapidly into their native states, we might be misled. Instead, if we look at most monoclonal aptamers, it is not uncommon to discover that they comprise distinct conformers, only a minority of which are competent ligands (A. TahiriAlaoui and E. Kraus, personal communication). During selection, one would expect the selective process to favor those RNAs that are minimally pleiomorphic, so the persistence of this property in aptamers suggests that

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pleiomorphism is common in unselected oligonucleotides. RNA folding algorithms, admittedly still only able to predict approximately 75% of experimentally determined base pairings, 43,44 indicate that a given sequence is frequently capable of folding into several structures, each with similar G. If the free energy barriers between any of these folded forms are great enough, multiple conformers would be expected. Natural evolution of, for example, ribozymes, would have imposed a selective pressure for sequences whose folding pathways are more uniform or for an association with chaperone-like proteins that would favor one conformation over another. Those working with aptamers have adopted a variety of strategies to minimize this problem from snap-cooling nucleic acid pools from the denatured state to a slow refolding procedure but none is entirely successful. Although technically challenging, it is becoming possible to analyse co-existing structures within a population of RNA molecules with identical sequence. 45 Conformational exibility has been observed in protein aptamer interactions, for example, an induced t in the target polypeptide following the binding of aptamers raised against the HIV-1 regulatory protein. 46 Conversely, the conformation of the nucleic acid was seen to change upon binding of an RNA aptamer to its amino acid target. 47

NUCLEIC ACIDS STRUCTURE AND MAPPING

TT G G G G T G

TT G G G GT

Figure 4 Representation of a thrombin-binding aptamer. The phosphodiester backbone is shown with solid gray lines. The noncanonical hydrogen bonds forming the G tetrads are shown as dotted lines. between the two highly basic exosites of thrombin, making no contact with the active site at the base of the cleft between the two exosites. The X-ray and NMR models were very similar, but the polarity of the strands was different, with the two TT loops spanning the major groove in the X-ray model and the minor groove in the NMR model. These two structures are technically very hard to discriminate from the crystallographic data, and it is probable that the NMR model is, in fact, correct. 53 Another GTrich oligo, d(Gs TGGTGGGTGGGTGGGs T) (where the rst and last phosphodiester bonds are replaced with phosphorothioate), was found to be an inhibitor of the integrase enzyme of HIV-1. 54 The structure of this aptamer is again four strands, stabilized by a pair of G quartets but in which the joining loops are all TG. 55 The G octet is not the only kind of structure adopted by DNA aptamers, however. For example, an aptamer against argininamide is a hairpin loop, which undergoes a rearrangement upon binding to its target, in which the loop is reexed to trap the amino acid between the stem and the stabilized loop by a combination of H-bonds and stacking interactions. 56 3.1.2 RNA Aptamers Aptamers based on RNA, or nucleotides with predominantly RNA-like properties, are capable of adopting a seemingly greater variety of structures (reviewed by Patel et al. 57 and Ferre-DAmare and Doudna 58 ). For example, a avin mononucleotide (FMN)-binding aptamer consists of an asymmetric loop that binds the target, anked by two A-form helices. 59 The asymmetric loop itself forms a widened, colinear helix comprising an anti purine pair and a base triple at one side, two anti purine pairs at the other of the intercalated isoalloxasine moeity of FMN (see Figure 5). Essentially, the target is acting as a highly abnormal intercalated base in a modied double helix. Another nucleotide-binding aptamer, which recognizes adenosine 50 -monophosphate (AMP), 60 incorporates its target even more intimately into its structure. Tetraloops of consensus sequence GNRA (where N is any nucleotide and R is a purine) are

3 PROPERTIES OF PUBLISHED APTAMERS

3.1 Structures The structure of a large number of aptamers has been determined by enzymatic or chemical probing, nuclear magnetic resonance (NMR) and X-ray crystallography (see reviews by Feigon et al. 48 and Patel 49 ). They are relatively amenable to NMR methodology because of their small size and their rigidity when complexed with target. Although the fundamental repertoire of nucleic acid secondary structures is radically different from that of proteins, the similarities between the interaction sites between protein ligands and their receptors can be strikingly similar to those of aptamers. 50 A few of the salient features are described below. 3.1.1 DNA Aptamers One of the earliest aptamers studied structurally was the 15mer DNA aptamer against thrombin, d(GGTTGGTGTGGTTGG), isolated by Bock. 7 Using two-dimensional proton NMR, it was shown 51 that this short oligo folded tightly into a four-stranded structure, stabilized by two stacked G tetramers, with each of the two pairs of strands having a TT dinucleotide loop (see Figure 4). X-ray crystallography of the aptamer complexed with its target 52 showed that the aptamer was sandwiched

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9
A A G G U A U G C A G G A A G C G G U U C

3.2 Aptamer Size It has already been noted that the sequence space available to most libraries used in aptamer isolation exceeds the practicable limit of molecules that can be handled. Moreover, for many applications it is desirable that the aptamer should be as small as possible, on costs grounds, reasons of target accessibility and so on. Nevertheless, because functional RNA folds have a nite size, the minimum length of aptamers is often larger than the size that generates a manageable sequence space. Further, the size of aptamer that is initially recovered from a process of in vitro selection is inated by the anking, xed sequence regions, required for amplication and transcription, which may add up to 50 nt. A combination of deletion analysis, footprinting and in vitro synthesis can be used to determine the shortest stretch of nucleic acid that can bind to the target. The size range of minimal aptamers is fairly wide and the following give some indication. The minimum motif within VEGF aptamers was between 23 and 35 nt (depending on sequence family); 14 minimum xanthine- and guanine-binding aptamers were 32 nt long; 72 streptomycin-binding aptamers were 46 nt long; 73 and the minimum region of an aptamer that binds a serine protease of the blood (protein C) may be as much as 99 nt long. 74 This gives an Mr range of 7.5 32 kDa for aptamers, with 10 kDa being typical. The solvent-exposed surface area for a typical aptamer would be expected to be 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 diversity among nucleic acids be compensated by the size of the sequence space that can be sampled and the resultant structural diversity of aptamers? The evidence is that aptamers can be generated against small ions, such as Zn2C , 36 to nucleotides such as adenosine triphosphate (ATP), 60,75 oligopeptides 76 and large glycoproteins such as CD4, 38 spanning the size range 65 Da 150 kDa, with no theoretical upper limit. The chemical classes of targets are reasonably diverse, including organic dyes, 5,77 neutral disaccharides, 78 aminoglycoside antibiotics (see Figure 7), 79 dopamine, 35 a porphyrin 80 and biotin. 71 Nevertheless, there is evidence for a strong degree of bias, among protein-binding aptamers, for a limited number of sites. First, a surprisingly high proportion of proteins against which aptamers have been described are themselves ligands for polyanions such as nucleic acids or glycosaminoglycans. These include thrombin and other proteases of the clotting cascade, 7,74,81 a number of heparin-binding growth factors, 13,82 84 cellular transcription factors 85,86 and viral regulatory

5 G G C G U G U 3 C C G C A C A

Figure 5 Secondary structure representation of an FMNbinding aptamer. Noncanonical base pairs are indicated by single solid lines and the base triplet by a set of three radiating lines. The FMNs isoalloxasine moiety is indicated by a triple hexagon.

common in natural RNAs and are particularly stable. 61,62 The AMP-binding aptamer was found to recruit the free AMP and thereby form a GNRA-like loop from a region that is unpaired in the absence of a target. 63 65 The structures of protein-binding aptamers show further variations on these themes. For example, aptamers that bind the HIV-1 regulatory protein, Rev, accommodate an a-helix of their target within a modied major groove that is widened by the presence of a number of non-Watson Crick base pairs. 66 68 In contrast, a class of aptamers that bind the reverse transcriptase of HIV1 possess a compact form of pseudoknot structure 69,70 (see Figure 6) which is believed to occupy the groove within the enzyme normally occupied by its kinked template. A biotin-binding aptamer was also found to have a rigid pseudoknot structure. 71 The pseudoknot represents just one class of structural element or fold exhibited by functional RNAs. The common feature of these folds is that they allow the molecule to be more compact than the classical A-form double helix (reviewed by FerreDAmare and Doudna 58 ) and thereby take on a more globular form, which is perhaps more suited to ligand recognition.
5 G A U C C A A Loop 1 G Stem 1 G S S a N N

N Loop 2 A

N N Stem 2 X N N X X

Figure 6 A reverse transcriptase-binding pseudoknot aptamer. 69 S S0 represents a G : C or C : G base pair, X represents optional nucleotides and N N0 represents any base pair.

10

NUCLEIC ACIDS STRUCTURE AND MAPPING

(a)

aptamers against reverse transcriptase all bound the same region of the enzyme. 70 More strikingly, when ve different sequence classes of aptamer against CD4 were analysed, all bound to a single region of one of the four immunoglobulin (Ig)-like domains, 38 in contrast to monoclonal antibodies, which dene several epitopes in each domain. This focusing of aptamer reactions on a small fraction of a large macromolecular target suggests that the process of in vitro selection does not proceed entirely according to simple models of multitarget partitioning. 30 Rather, it suggests that favorable and unfavorable aptatopes are distinguished by differences of afnity of many orders of magnitude. It seems likely that a major obstacle is that the Coulombic repulsion between the phosphatecontaining backbone of nucleic acids and negatively charged amino acid and sugar residues at the surface of many proteins and glycoproteins produces extremely low association rates. This would mean that only a small proportion of the surface of the macromolecule would be visible to aptamers, probably regions of high solvent exposure in which positively charged residues provided a degree of electrostatic steerage towards the aptatope. This has been most closely studied in the case of antithrombin DNA aptamers. By mutating a critical arginine to glutamate, the binding of aptamers was abolished. 91 The mutant form of thrombin was then used to raise further aptamers and these were found still to bind regions homologous to that recognized by the rst-generation aptamers. 92 3.4 Afnity The afnity of published aptamers varies very widely. Generally, aptamers against small molecules have afnities in the micromolar range. For example, aptamers against amino acids such as citrulline and arginine range from 0.3 to 65 M, 34,93 those against ATP and xanthine were 6 and 3.3 M, respectively, 72,75 those against dopamine were 2.8 M and those against vitamin B12 were 90 nM. 94 Aptamers to nucleic acid-binding molecules typically have afnities in the nanomolar range. For example, aptamers against retroviral integrase, reverse transcriptase and nucleocapsid proteins were 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 Afnities in the nanomolar to subnanomolar range are found against heparin-binding proteins. Examples include platelet-derived growth factor (PDGF) (0.1 nM), 99 basic broblast growth factor (low nanomolar), 83 thrombin (25 nM), 7 VEGF (approximately 100 pM), 14 keratinocyte growth factor (approximately 1 pM) 13 and

(b)

Figure 7 NMR-derived structure of a tobramycin-binding

aptamer, bound to its target. (Data derived from conformer 1 of 13 published by L. Jiang and D.J. Patel, Solution Structure of the Tobramycin RNA Aptamer Complex, Nature Struct. Biol., 5(9), 769 774 (1998).) (a) The phosphodiester backbone of the aptamer is depicted as an orange cylinder and the tobramycin is shown in ball-and-stick form. (b) The aptamer is shown as a wireframe model with the base identities and positions labeled.

proteins. 39,87 89 It has even been reported that some heparin-binding proteins, such as thrombin, may have natural plasma aptamers. 90 Second, when the aptamer-binding sites (or aptatopes) on large target proteins are mapped, it is usually found that they are coincident, even if the aptamers fall into unrelated sequence families. For example, all six of the distinct classes of aptamer against VEGF were found to bind to the same region, competing with heparin and other natural ligands and being cross-linkable to the same cysteine, 14,84 and four structurally distinct

APTAMERS

11
4 APPLICATIONS AND DOWNSTREAM TECHNOLOGIES
In comparison with the comparable eld of monoclonal antibodies, aptamers suffer from a relatively modest range of downstream technology which would enable them to be used more routinely in diagnostic and analytical assays or other practical applications. Accordingly, in the following sections, some of the advances that have been made in these areas are reviewed. 4.1 Conjugation to Detectable Moieties Often, the rst step in exploiting an antibody is its conjugation to a detectable moiety, such as a uorochrome, an enzyme or a generic ligand such as biotin. Conjugation is most usually done by derivatizing e-amino groups of lysines on the antibody molecule, but care has to be taken not to block lysine residues near or within the complementarity-determining regions. Conjugation can be done through carbohydrate side chains, but this is much less usual. Even more commonly, secondary antibodies, which recognize conserved epitopes on antibodies, irrespective of their antigen, are conjugated to detectable moieties, thereby simplifying the development of assays for new antigens. The challenge of aptamers is to develop a generic labeling system that does not tend to disrupt aptamer structure or hinder interaction with target. Nucleic acids can be radioactively body labeled during transcription or replication by the use of modied nucleotides. Where these are 3 H or 32 P body labeled, this does not affect the chemistry of the aptamer and thus does not affect its properties as a ligand. This is a very common way of quantitating the binding of an aptamer or a pool of nucleic acids to the target molecule. 28 In addition to body labeling, site-specic labeling with a radionuclide can be useful, for example where the aptamer is synthesized chemically. For example, in one case, a stannyl nucleotide was incorporated at the 50 end of a DNA aptamer during chemical synthesis and tin was subsequently replaced by 123 I in an oxidation reaction. 110 The aim was to produce a ligand that can be used for in vivo imaging. Aptamers can also be labeled using uorescent or reactive groups. Here, the challenge is to introduce the detectable groups at positions that do not interfere with ligand properties. For example, a chemically synthesized aptamer against human neutrophil elastase was labeled at either the 30 or 50 end with uorescein or biotin. 111 It was found that incorporation of the label reduced the afnity of the aptamer unless a substantial spacer was used. In vitro transcribed aptamers can have biotin or uorochromes incorporates as body labels but this is generally at the expense of their properties as ligands. Labeling at the 30 end can be achieved in a number of ways:

the heparin-binding non-pancreatic, secretory phospholipase A2 (1.7 nM). 100 Aptamers against proteins that do not bind heparin or nucleic acids have typically lower afnities. For example, those against the oncoproteins K-Ras and Raf-1 were 0.14 1 M and 300 nM, respectively, 101,102 one to substance P had a KD of 190 nM 76 and one to the NS3 protein of hepatitis C virus had a KD of 650 nM. 103 Immunoglobulin class G (IgG) superfamily domain-containing proteins seem to be able to elicit aptamers in the 2 40 nM range of afnity 12,38,104 and this might relate to their ability to interact with other cell-surface glycoproteins. 3.5 Specicity When measured, it has been found that those targets that give rise to very high-afnity aptamers have measurable afnity for unselected RNA. For example, the E. coli r protein has an afnity for unselected RNA in the 1 M range and can be used to isolate specic aptamers that bind with an afnity of approximately 1 nM. 33 This observation suggests that the assertion that high afnity necessarily produces high specicity 105 is unsafe, but the evidence suggests that aptamers can show a great deal of specicity. First, different enzymes with similar activity can be distinguished. For example, a-thrombin could be differentiated from g-thrombin; 106 the reverse transcriptase of feline immunodeciency virus could be distinguished from the homologous enzyme of three other retroviruses; 107 two isozymes of protein kinase C differing in just 23 residues could be readily distinguished; 108 and the CD4 glycoproteins of rat and mouse could be distinguished (73% identity). 38 Perhaps more impressive is the ability to discriminate between enantiomers, such as the threefold discrimination between L- and Dcitrulline cit (G D 2.5 kJ mol 1 ), sixfold difference in KD between L- and D-arginine (G D 4.6 kJ mol 1 ) 34 and, subsequently, the 12 000-fold ratio in KD between L- and D-arginine reported by Geiger et al. 93 Aptamers against aminoglycoside antibiotics have been particularly specic, with anti-tobramycin aptamers having 3 4 orders of magnitude lower afnity to other aminoglycosides 98 and one against streptomycin having four orders of magnitude lower afnity to the closely related bluensomycin. 73 Strikingly, aptamers against caffeine had a 10 000-fold lower afnity for theophylline, which differs in just a methyl group. 109 However, there are limits to specicity, as one would expect. For example, aptamers against coenzyme A also recognize AMP, 90 those against xanthine recognize guanine, but not adenosine cytosine or uracil, 72 and those against the disaccharide cellobiose recognize cellulose, a polysaccharide containing the same repeating unit, but not the disaccharides lactose and maltose. 78

12
O In vitro transcription with GDP--S S

NUCLEIC ACIDS STRUCTURE AND MAPPING

O O O OH O P O O O base

P P O O O O

base

H O N H N S O N H

H N O Iodoacetyl-LC-biotin I +

O ...

H O N H N S O N H

H N O

O P S O O O O O P O

base

Biotinylated RNA

OH O P O O O O ...

base

Figure 8 50 -Labeling of in vitro RNA transcripts using GTP-b-S (see text). by templated extension using Klenow polymerase, 112 by T4 RNA ligase-mediated ligation 113 and by terminal deoxynucleotidyl transferase. 114 Labeling at the 50 end can be achieved by the supplementation of the in vitro transcription mix with an excess of GTP-b-S, the thiol of which can then be used to attach biotin (see Figure 8). 115 This approach was used successfully to multimerize an anti-CD4 aptamer via streptavidin, which in turn allowed a uorochrome to be attached to the aptamer for use in ow cyometry. 115 We have already discussed the problems associated with inefcient derivitization of internal 20 -amino groups. 27 4.2 Aptamers in Biosensors and Other Detectors As high-afnity, high-selectivity ligands, aptamers have potential in a range of detection systems, including biosensors (for reviews, see Osborne et al. 116 and Bier and Furste 117 ). We have already discussed their application in ow cytometry 115,118 and they can also be used in sandwich assays akin to enzyme-linked immunosorbent assay (ELISA). 119 Fluorescently labeled aptamers have also been used to quantitate IgE and thrombin using a rapid method based on capillary electrophoresis/laserinduced uorescence (CE/LIF), in which detection down to 50 pM was reported. 120 Unlabeled aptamers can also be used in certain analytical methods. For example, surface plasmon resonance (SPR) methods, such as those exploited by the BIAcore 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 was immobilized. However, with appropriate derivitization and immobilization, aptamers could be used to detect specic molecules in complex mixtures using SPR. Perhaps the most exciting method so far described also depends on an optical ow cell system and the evanascent wave from a total internal reection event at the optical surface. However, in the application described by Potyrailo et al., 122 the effect detected is not on plasmon resonance angle but on uorescence anisotropy. They reported that uorescent aptamers immobilized at the optical surface could be used to detect as little as 0.7 amol of thrombin in a 140-pL test volume in just a few minutes. Other methods that might be applicable to aptamerbased detection in the future include thin-layer interference techniques which have been used to measure DNA small molecule interactions in real time. 123 4.3 Aptamer Combination One approach to increasing the usefulness of aptamers is to combine them with another ligand. For example, a DNA aptamer to human neutrophil elastase was noninhibitory to the enzymes protease activity but improved the Ki of a weak peptide inhibitor by ve orders

APTAMERS

13
them. The possibility that aptamers might be candidate therapeutic agents has naturally received much attention (see review by Osborne et al. 116 ). The main challenge to all these applications is the pharmacokinetics of oligonucleotides in vivo: the rate at which they are degraded by plasma nucleases; the rate at which they are sequestered by reticulo-endothelial cells and plasma proteins; the rate with which they are excreted by the kidney; and the rate at which they redistribute from the blood to the tissue or body uid of interest. Natural, 20 -OH-RNA degrades very rapidly in human serum in vitro but this process can be slowed at least 1000-fold by 20 -amino modication. 135 In vivo studies on the t1/2 of single-stranded DNA aptamers give values in the range from <2 min to approximately 8 min. 136 138 Conjugation of the aptamer to either lipids or polyethylene glycol has been reported to improve the stability and distribution kinetics of DNA aptamers sufciently to produce therapeutic effects. 139,140 A wide range of potentially therapeutic targets for aptamers are summarized in Table 1, with a note of therapeutic effect or clinical benet, if any. More radically, it is conceivable that one could use aptamers in a form of gene therapy, to inhibit the function of intracellular proteins either host-derived or viral in vivo. This approach is fraught by all the problems that have dogged the gene therapy eld for many years: inefcient and intrusive delivery systems, inefcient or transient expression, safety concerns, poor selectivity, and so on (for a review, see Palu et al. 141 ). Worse, the rather low concentration of free divalent cations and the presence of high concentrations of RNA-binding proteins in the cell means that aptamers selected under the conventional in vitro conditions might well be inappropriately folded in the cell. Nevertheless, some positive reports have appeared. For example, in a study of the inducible expression of aptamers against RNA polymerase II in yeast, a signicant effect on transcription was seen as a result of aptamer expression, but only in yeast strains with abnormally low levels of polymerase II expression. 142 More promisingly, U6 snRNA and tRNAmet cassettes were used to express aptamers that bound the HIV-1 regulatory protein, Rev, in cells 143 and it was found that these, like Rev decoys derived from the natural Rev RNA ligand, were able to inhibit the expression of the virus. However, these experiments were done in vitro in a convenient but unphysiological cotransfection system, so one must be careful not to overextrapolate. 4.5 Studies on Nucleic Acid Structure One of the rst studies describing in vitro selection of nucleic acid ligands was designed to analyze the sequence requirements of a nucleic acid-binding

of magnitude when the two were conjugated through an N-methoxysuccinyl link. 124 Alternatively, one can link aptamers together to form a dimeric or multimeric ligand. This can take the form of joining identical aptamers, perhaps to increase afnity by reducing the dissociation rate of the complex from a multimeric target, or of joining aptamers against different aptatopes, in order to change specicity. An example of the former approach was the homodimerization of aptamers against L-selectin, which resulted in ligands with KD in the range of good monoclonal antibodies. 118 The procedure is not straightforward, however, as linking two aptamers in a single oligonucleotide can result in loss of function for one or both ligand moieties, either by steric hindrance or by disruption of the folding of each module. This problem was found with combinations of aptamers against coenzyme A, chloramphenicol and adenosine, but was overcome by the use of further rounds of in vitro selection on chimeric RNAs in which the junctions between aptamers were diversied. 125 A practical use for heterodimerization of aptamers has been the linking of aptamers that recognize thermostable polymerases but with different specicity. 126 The resultant chimeras had a broad specicity to enzymes from a range of thermophilic species and were potentially useful for improving the efciency of PCR from low-copy-number templates. A further potential application of heterodimerization is to link an aptamer that recognizes a target molecule of analytical interest to a second aptamer that recognizes a detectable molecule, such as a uorochrome. 127 Aptamer modules have also been linked to ribozymes (catalytic RNAs) to provide a means for controlling enzyme activity through allosteric interactions. For example, the activity of a hammerhead autocatalytic ribozyme fused to an ATP-binding aptamer was shown to be regulated >100-fold by the presence of ATP. 128 130 This effect depends on the conformational change in the aptamer following ligand binding and steric hindrance between the folded aptamer and the ribozyme. These and other authors have extended this work to construct RNAcleaving ribozymes whose activity is regulated by other small molecules, such as theophylleine and FMN, 129,131 and RNA-ligating ribozymes whose activity is regulated by ATP. 132 The general principle could form the basis of a new generation of in vitro assays for analytes. 4.4 Use of Aptamers In Vivo Since the lead given to the eld at the turn of the century by Paul Ehrlich (see, for example, Ehrlich 133 ) (translation by Brock 134 ), there has been a continuing desire to develop new classes of specic ligands, preferably by rational means, that could act as magic bullets, seeking out their molecular targets in vivo and destroying

14
Table 1 Potentially therapeutic aptamers
Target Toxins Ricin, pepocin, gypsphilin Enzymes Thrombin References 144 7 138, 145 146 81 74 69, 96 107, 147 95, 148 108 124, 149, 150 103, 151, 152 153 100 121 154 155 156 38, 115 157 83 14, 84, 140 82 99, 139 13 158, 159 76 18 86 101 102 160 161 67, 88, 143, 162 164 39 104 12 165

NUCLEIC ACIDS STRUCTURE AND MAPPING

Comments Have different sequence from natural rRNA targets First identication Use in vivo Better than heparin against clot-bound thrombin An RNA aptamer against thrombin

Activated plasma protein C HIV-1 reverse transcriptase Other retroviral reverse transcriptases HIV-1 integrase Protein kinase C Human neutrophil elastase Hepatitis C virus NS3 protease/helicase Yersinia protein tyrosine phosphatase Phospholipase A2 20 ,50 -Oligoadenylate synthetase Angiogenin (ribonuclease activity) Adhesion and recognition molecules L-selectin P-selectin CD4 Rhinovirus capsid protein Growth factors and hormones Basic broblast growth factor VEGF NGFa PDGF Keratinocyte growth factor g-Interferon Substance P Vasopressin Regulatory proteins and oncogenes E2F transcription factor K-ras Raf-1 P210bcr-abl MDM2 oncoprotein HIV-1 Rev HTLV-I Tax Miscellaneous IgG against human insulin receptor IgE Hamster PrPa
a

Identication of inhibitory aptamer when conjugated to weak peptide inhibitor of enzyme. Effective in an animal model of lung injury Inhibited contractions of guinea pig pleural strip in vitro DNA aptamer prevents angiogenesis and cell proliferation DNA aptamers inhibit cellular adhesion and rolling in vitro and trafcking in vivo Afnity (20 pM) is 106 -fold better than the natural ligand sialyl Lewis X. Unlike the latter, discriminate against Eand L-selectins by 104 105 Disrupts immune recognition in vitro Intended to block infectivity of virus Inhibit induction of permeability in vivo. 20 -F modication and lipid derivatization Improve stability and pharmacological properties Effective in vivo as a PEG conjugate

Prevents binding to DNA and entry into S phase Raised against farnesylated peptide DNA aptamer introduced into chronic myelogenous leukemia cells by electroporation reduced cell proliferation These are Rev-responsive, even though unrelated to natural Rev-response element. Are inhibitory to HIV-1 growth in cell cultures Possible approach to blocking a common form of antibody-mediated, autoimmune diabetes Bind Fc portion of antibody, preventing interaction with cell surface receptor. Possible role in allergy therapy

NGF, nerve growth factor; PrP, prion protein.

APTAMERS

15
This RNA catalyst is therefore the rst nucleic acid with carbon carbon bond-forming activity.

protein. 4 Aptamers have continued to be a powerful method for analyzing nucleic acid sequence binding requirement of a range of viral proteins. For example, aptamers have been used to dissect the nucleic acid binding sites of the coat proteins of bacteriophages R17, 166 MS2 167 and f29, 168 the RNA-dependent RNA polymerase of bacteriophage Qb, 169 the Epstein Barr virus EBER1 RNA, 170 the regulatory, Rev-response element of HIV-1 136,171 and the HIV-1 nucleocapsid protein. 97 The approach has been used productively to examine the interaction between components of the translational machinery and natural RNAs, e.g. the elongation factors EF-Tu, 172 elF-4B 173 and SelB, 174 the S8 175 and S1 176 ribosomal proteins from E. coli, the L32 ribosomal protein of yeast 177 and the decoding end of 16S rRNA. 178 Finally, transcription and post-transcriptional processing have been amenable to the aptamer approach, e.g. the requirements for the E. coli transcriptional terminator r, 33 the eukaryotic splicing factors SF2 and SC35 179 and GU-rich sequences for efcient polyadenylation. 180 It has been proposed that these methods could be used to identify all the protein RNA interactions encoded in the genome, 181 although this is not yet an established approach. 4.6 Isolation of New Catalysts The very rst paper describing directed in vitro evolution of nucleic acids described the isolation of a totally new enzyme, a nuclease composed of DNA. 3,182 The possibility was thereby opened up that one could develop any form of useful new catalyst that could be conceived and for which a selection procedure could be devised. To an extent, this has been realized, with the isolation of enzymes that can ligate DNA in a Zn/Cu-dependent fashion 183 or ligate RNA with a similar mechanism to that of protein enzymes. 184 187 These new enzymes are not restricted to phosphodiester bond formation and breakage. For example, in vitro-selected nucleic acid enzymes have been described that cleave amide bonds 188 and alkylate halogenated peptides. 189 The combination of a uorochrome aptamer and its target was found to have low-level oxidative activity, when coupled to its target, 77 and a hemin aptamer complex was found to have levels of peroxidase activity comparable with protein peroxidases, and much higher than those of catalytic antibodies. 190 More signicantly, RNA enzymes have been selected with amide bond-forming activity 191 in which a uridine was replaced by a 50 -imidazole derivative of uridine, capturing the chemical characteristics of histidine in nucleic acid. Also, a ribozyme was isolated that catalyzed a Diels Alder cycloaddition reaction in which uridine is replaced by a 5-pyridyl derivative of uridine. 192

5 PERSPECTIVES AND FUTURE DEVELOPMENTS

Clearly, the rst decade of research using aptamers has opened up some very exciting avenues in basic and applied research. The power of the technique has surprised many and, even though the chemistry of nucleic acids is much less diverse than that of polypeptides and there are substantial restrictions to the technology as it stands, it is already a useful tool and promises to become more so. There are substantial advances in methods which use the principle of in vitro selection but apply it to the evolution of peptide ligands. For example, adaptations of the twohybrid display methodology using randomized peptides displayed on thioredoxin in E. coli has been used to generate peptide aptamers against CDK-2 with nanomolar afnity 193 which blocks G1-S transition during the cell cycle of eukaryotic cells engineered to express it. 194,195 Similar approaches display peptides within the green uorescent protein 196 or at the Cterminus of the lac repressor protein. 197 One problem with these methods, and the widely used phage display technology [see reviews by Burton 198 and Winter et al., 199 ] is that the critical step of passing through a living organism during the generation of the library reduces the number of different molecules that can be screened by many orders of magnitude. To overcome this, methods have been developed to link a nucleic acid based library with in vitro translation, thereby obviating the need for a living cell. The challenge is to preserve the genetic encoding by maintaining the association between nucleic acid and the encoded polypeptide. One approach is to use polysomes, collections of ribosomes simultaneously translating a messenger RNA (mRNA). 200 More conveniently, a method in which translation is simultaneously arrested and the RNA linked covalently to the nascent polypeptide chain has been developed, 201 in which a puromycin group is incorporated at the 30 end of the library RNA and is linked to the C-terminus of the polypeptide by the action of the ribosomes peptidyl transferase.

ACKNOWLEDGMENTS
I am grateful for stimulating discussions with Elmar Kraus, Jamal Ibrahim, Laura Frigotto and Abdessamad Tahiri-Alaoui and the opportunity to present their as yet

16
unpublished results. I thank Abdessamad for his critical reading of the manuscript.

NUCLEIC ACIDS STRUCTURE AND MAPPING

Pharmaceuticals and Drugs (Volume 8) Combinatorial Chemistry Libraries, Analysis of

ABBREVIATIONS AND ACRONYMS

AMP ATP CE/LIF dNTP ELISA FMN Ig IgE IgG mRNA MS NGF NMR PDGF PrP PVDF rRNA SELEX SPR VEGF Adenosine 50 -Monophosphate Adenosine Triphosphate Capillary Electrophoresis/Laser-induced Fluorescence Deoxynucleoside Triphosphate Enzyme-linked Immunosorbent Assay Flavin Mononucleotide Immunoglobulin Immunoglobulin E Immunoglobulin class G Messenger RNA Mass Spectrometry Nerve Growth Factor Nuclear Magnetic Resonance Platelet-derived Growth Factor Prion Protein Poly(vinylidene diuoride) Ribosomal RNA Selective Expansion of Ligands by Exponential Enrichment Surface Plasmon Resonance Vascular Endothelial Growth Factor

REFERENCES
1. 2. F.H. Crick, The Origin of the Genetic Code, J. Mol. Biol., 38(3), 367 379 (1968). S. Kaur, L. McGuire, D. Tang, G. Dollinger, V. Huebner, Afnity Selection and Mass Spectrometry-based Strategies to Identify Lead Compounds in Combinatorial Libraries, J. Protein Chem., 16(5), 505 511 (1997). D.L. Robertson, G.F. Joyce, Selection in Vitro of an RNA Enzyme that Specically Cleaves Single-stranded DNA, Nature (London), 344(6265), 467 468 (1990). C. Tuerk, L. Gold, Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase, Science, 249(4968), 505 510 (1990). A.D. Ellington, J.W. Szostak, In Vitro Selection of RNA Molecules that Bind Specic Ligands, Nature (London), 346(6287), 818 822 (1990). B.E. Eaton, W.A. Pieken, Ribonucleosides and RNA, Annu. Rev. Biochem., 64, 837 863 (1995). L.C. Bock, L.C. Grifn, J.A. Latham, E.H. Vermaas, J.J. Toole, Selection of Single-stranded DNA Molecules that Bind and Inhibit Human Thrombin, Nature (London), 355(6360), 564 566 (1992). A.D. Ellington, J.W. Szostak, Selection in Vitro of Single-stranded DNA Molecules that Fold into Specic Ligand-binding Structures, Nature (London), 355(6363), 850 852 (1992). W.A. Pieken, D.B. Olsen, F. Benseler, H. Aurup, F. Eckstein, Kinetic Characterization of Ribonucleaseresistant 20 -Modied Hammerhead Ribozymes, Science, 253(5017), 314 317 (1991). D.M. Williams, F. Benseler, F. Eckstein, Properties of 20 -Fluorothymidine-containing Oligonucleotides: Interaction with Restriction Endonuclease EcoRV, Biochemistry, 30(16), 4001 4009 (1991). R. Padilla, R. Sousa, Efcient Synthesis of Nucleic Acids Heavily Modied with Non-canonical Ribose 20 Groups Using a Mutant T7 RNA Polymerase (RNAP), Nucleic Acids Res., 27(6), 1561 1563 (1999). T.W. Wiegand, P.B. Williams, S.C. Dreskin, M.H. Jouvin, J.P. Kinet, D. Tasset, High-afnity Oligonucleotide Ligands to Human IgE Inhibit Binding to Fc Epsilon Receptor I. J. Immunol., 157(1), 221 230 (1996). N.C. Pagratis, T. Fitzwater, D. Jellinek, C. Dang, Potent 20 -Amino- and 20 -Fluoro-20 -deoxyribonucleotide RNA Inhibitors of Keratinocyte Growth Factor, Nature Biotechnol., 15(1), 68 73 (1997). J. Ruckman, L.S. Green, S. Waugh, W.L. Gillette, D.D. Henninger, L. Claesson-Welsh, N. Tanjic, 20 -Fluoropyrimidine RNA-based Aptamers to the 165-Amino Acid Form of Vascular Endothelial Growth Factor

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