Improving the Stability of Aptamers by Chemical Modification

Abstract

Ever since the invention of SELEX (systematic evolution of ligands by exponential enrichment), there has been rapid development for aptamers over the last two decades, making them a promising approach in therapeutic applications as either drug candidates or diagnostic tools. For therapeutic purposes, a durable performance of aptamers in biofluids is required, which is, however, hampered by the lack of stability of most aptamers. Not only are the nucleic acid aptamers susceptible to nucleases, the peptide aptamers are also subjective to degradation by proteases. With the advancement of chemical biology, numerous attempts have been made to overcome this obstacle, many resulting in significant improvements in stability. In this review, chemical modifications to increase the stability of three main types of aptamers, DNA, RNA and peptide are comprehensively summarized. For nucleic acid aptamers, development of modified SELEX coupled with mutated polymerase is discussed, which is adaptive to a number of modifications in aptamers and in a large extent facilitates the research of aptamer-modifications. For peptide aptamers, approaches in molecular biology with introduction of stabilizing protein as well as the switch of scaffold protein are included, which may represent a future direction of chemical conjugations to aptamers.

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165toward pharmaceutical applications

“…Although several post-SELEX modification methods to stabilize aptamers have been reported (12–17), there are fewer opportunities for modifying DNA aptamers to confer increased resistance against nucleases without a loss of target affinity and an increase in cost. The most established method is the modification of the 2′-position of the ribose moieties in aptamers with fluoro and methoxy groups (18–20).…”

Mapping the affinity landscape of Thrombin-binding aptamers on 2′F-ANA/DNA chimeric G-Quadruplex microarrays

“…Considering the essential role of chemically-modified nucleosides in modern aptamer discovery (10,64), the prospect of extending the chemical repertoire of nucleic acids microarrays is highly appealing. We present here the fabrication of the first 2΄F-ANA microarrays and their use in exploring the binding landscape of TBA1 to its target thrombin and in identifying novel 2΄F-ANA aptamers.…”

“…TBA2, a 29mer showing structural features from both duplex and quadruplex conformations, binds to thrombin with even higher affinity ( K d = 0.5 nM (8)) and, relevantly, to a different epitope than TBA1 (9). As sequences whose structural, biophysical and binding properties are well characterized, TBA1 and TBA2 have become model aptamers and, as such, have been widely investigated and subjected to a plethora of chemical modifications (10,11), for the most part in TBA1, however, with little success in improving the thrombin-binding properties of the original oligomer.…”

Aptamers: A New Technological Platform in Cancer Immunotherapy

“…Deep sequencing, together with new IT packages, allows for the identification of high-affinity aptamers in early rounds of selection, thus reducing the time and cost of selection, but, most importantly, minimizing the introduction of undesirable bias accumulated along the selection process in the amplification step, which is carried out by Polymerase Chain Reaction (PCR) [11,12,13,14]. The nature of the aptamer-oligonucleotide backbone can be made of DNA or RNA, and there are several modifications available that can be used to increase the stability of the aptamer in serum, by substitution of ribonucleotides with 2′-amino, 2′-fluoro, or 2′- O -alkyl nucleotides, among others [15]. Some research groups are using nucleotide analogs in order to improve the affinity and specificity of the aptamer, supposedly increasing the number of different types of ribonucleotides, which could increase the aptamer structural complexity [16,17].…”