An aptamer (from the Latin aptus, to fit, and the Greek meros, meaning part or region) is a single-stranded oligonucleotide that folds into a unique three-dimensional structure and, as a result of this spatial structure, is able to bind with high affinity and specificity to a target molecule such as a protein. Aptamers are larger than small molecule drugs but smaller than antibodies. They are obtained via in vitro selection from combinatorial oligonucleotide libraries, a process that allows for the selection of aptamers with specificity for almost any protein target. Since 1990, aptamers have been generated against hundreds of molecular targets, from small molecules and peptides to many proteins of therapeutic interest and even to cells and tissues (1,2).

Aptamers are typically 15 to 40 nucleotides in length and can be made of DNA, RNA, or nucleotides with a chemically modified sugar backbone. The secondary structure of aptamers is defined by complementary base pairing, which generates stable tertiary structure. The large amount of possible tertiary structures enables aptamers to bind with high affinity via van der Waals, hydrogen bonding, and electrostatic interactions to most small-molecule, peptide, or protein targets (3).

Aptamers have the potential for superior binding capabilities compared to naturally occurring RNA or DNA molecules because they are selected solely on the basis of their binding affinity. Furthermore, high-resolution,
three-dimensional structural analyses have shown that adaptive recognition (involving conformational alteration of either the protein or the aptamer) is possible (4,5). This adaptive recognition produces an even tighter fit between the aptamer and protein, further increasing binding affinity. Aptamers display dissociation constants in the picomolar to low nanomolar range (6,9).

In addition to high binding affinity, aptamers are able to recognize their targets with great specificity. They are able to distinguish between protein isoforms as well as between different functional or conformational forms of the same protein (7). Many aptamers that are selected to bind to a specific protein also inhibit the protein’s function. Most, but not all, therapeutically useful aptamers are inhibitors, binding and inhibiting enzyme activities or protein-protein interactions, and thereby function as antagonists (8,13).

Aptamers are randomly synthesized by an iterative process known as SELEX (systematic evolution of ligands by exponential enrichment), which was concurrently developed by the Gold and Szostak laboratories in 1990 (2).

The SELEX method begins with a population of randomly synthesized RNA or DNA oligonucleotides, usually 20 to 40 nucleotides in length. In vitro selection allows the specificity and affinity of the aptamer to be tightly controlled. The randomized oligomer region is flanked by specific sequences needed for enzymatic manipulation (8). These regions contain binding sequences for reverse transcriptase (RT) and polymerase chain reaction (PCR) primers, a promoter sequence for T7 RNA polymerase, and possibly restriction endonuclease sites for cloning (9).

In practice, a standard randomized 40-nucleotide library generates as many as 10¹⁵ distinct aptamers (9). During the selection process, the library is enriched in sequences possessing increased affinity to the target. The sequences in the library that do not bind to the target ligand are removed, typically by affinity chromatography (10). Aptamers that bind to a target are selected, amplified by RT-PCR and then reselected. This process can be repeated if necessary to achieve the desired affinity and specificity. Depending on the dissociation constant values of the aptamer-target complex, five to fifteen rounds of selection (in which the stringency of the elution conditions is increased to identify the tightest-binding sequences) are typically performed to obtain aptamers (11). After cloning and sequencing, selected aptamer sequences can be synthesized chemically using solid-phase phosphoramidite chemistry (9).

Most of the targets for therapeutic aptamers are in solution in the blood plasma or on the surface of cells that are accessible from the blood plasma. These aptamers are subject to nuclease degradation, renal filtration, and uptake by the liver, as well as other tissues such as the spleen (8,12). As aptamers are chemically synthesized and lack many of the functional groups commonly present in proteins, a single functional group can be site-specifically introduced and used as a unique site for conjugation of other molecules to the aptamer without disrupting structure or function. Thus, aptamers can be modified in ways that enhance pharmacokinetic properties, avoiding the losses of activity that are often seen for stochastically modified therapeutic protein conjugates.

Unmodified RNA and DNA molecules are too susceptible to nuclease-mediated degradation to be useful for most therapeutic applications and therefore require some manner of chemical modification, before, after, or during selection. For example, the phosphodiester backbone of RNA-based aptamers is vulnerable to serum ribonucleases at pyrimidine residues, and the 5′ and 3′-termini are vulnerable to exonucleases. To avoid degradation, chemically synthesized aptamers can be capped with modified or inverted nucleotides (13). Modified nucleotides can be introduced into libraries before initiation of SELEX by polymerases that can accept modified nucleotide triphosphates. By incorporating these modified nucleotides into aptamers, they become more resistant to nucleases, thereby improving their biological efficacy. To date, the highest affinity aptamers that have been reported contain modified nucleotides that bind in the single-digit picomolar range (8).

Even with extensive modification to block nuclease degradation, stabilized molecules must exhibit molecular weights of greater than 40 kDa (30-50 kDa is the molecular mass cutoff for the renal glomerulus) in order to remain in circulation for extended durations (3,8,14). This optimal molecular weight is most commonly realized by pegylation. To prolong renal clearance, high molecular weight polyethylene glycols (PEGs) can be covalently attached to aptamers, which typically have a molecular mass of 5 to 15 kDa (8), without significantly altering their ability to tightly bind to targets. Such modifications have a significant effect on the half-life of an aptamer, extending it from minutes to several hours (3).

This amenability to chemical modification is one property that distinguishes aptamers from monoclonal antibodies, which are only receptive to very limited chemical modification. For example, protein-PEG conjugation often results in a mixture of products and a loss of activity (8). Although aptamers are frequently referred to as “chemical antibodies,” and although both aptamers and antibodies are able to bind with high specificity and high affinity (K_d ≈ 10⁻¹⁰-10⁻⁷ M) (1) to target molecules, there are certain aptamer properties that make them preferable to antibodies in both diagnostic and therapeutic applications.

Aptamers have exhibited little or no toxicity or immunogenicity, even when administered in excess of therapeutic doses (15). In fact, producing antibodies to aptamers is extremely hard (most likely because aptamers cannot be presented by T cells via the major histocompatibility complex and the immune response is not normally programmed to recognize nucleic acid fragments). In contrast, the efficacy of many peptides and monoclonal antibodies can be severely limited by immune responses. In fact, even antibodies whose nonhuman component has been replaced with human sequence (i.e., “humanization”) can elicit immune responses (9).

Therapeutic antibodies are also more difficult to administer than aptamers. Because of their comparatively low solubility, comparatively large volumes are necessary when administrating most therapeutic monoclonal antibodies. Thus, most therapeutic antibodies require administration by intravenous infusion (generally over 2-4 hours). Conversely, aptamers have both good solubility and low molecular weight and therefore have the advantage of being able to be administered by either intravenous or subcutaneous injection (3).

Therapeutic aptamers are chemically robust. They are able to quickly regain activity following exposure to heat and denaturants. In addition, they can be stored in ambient temperatures as lyophilized powders for extended periods (more than 1 year) (3). Antibodies, however, are not as chemically stable. They are susceptible to irreversible denaturation and have a limited shelf life. The manufacturing of antibodies, which requires the use of cell-based expression systems, is far more complicated and less cost-effective than the commercial synthesis of aptamers (9). Furthermore, viral or bacterial contamination of the manufacturing process can affect the quality of the antibodies produced. The chemical production process of aptamers is not disposed to viral or bacterial contamination (8).

The first RNA aptamer to be successfully developed as a therapeutic agent in humans was pegaptanib sodium (Macugen), approved by the US Food and Drug Administration in December 2004 for the treatment of neovascular AMD (9). Pegaptanib, a 28-nucleotide RNA aptamer that is covalently linked to two PEG moieties, is an anti-vascular endothelial growth factor (anti-VEGF) RNA aptamer that is administered via intravitreal injection (4). It binds to all isoforms of human extracellular VEGF except for the smallest (VEGF121) (8). In particular, it binds with high affinity and selectivity to the VEGF165 isoform. VEGF is an endogenous proangiogenic protein involved in macular degeneration and in some cancers. Furthermore, VEGF has been associated with the leakage of blood vessels (3). Once pegaptanib is bound to VEGF, it inhibits the interaction of VEGF with its receptors, VEGFR1 and VEGFR2. Pegaptanib is used to improve the loss of visual acuity (VA) caused by the abnormal angiogenesis that is characteristic of AMD, the leading cause of blindness in people over 50 years of age in developed nations (4).

Fovista is a pegylated aptamer containing 32 monomeric units (32-mer) arranged as a linear sequence of three oligonucleotide segments connected by nonnucleotide hexaethylene glycol spacers. The aptamer terminates in a hexylamino linker to which two 20-kDa monomethoxy PEG units are covalently attached via the two amino groups on a lysine residue (16).