Selection of DNA aptamers with two modified bases

Significance Aptamers are now used ubiquitously as binding agents for a broad range of applications. Natural (unmodified) DNA and RNA aptamers have considerably less chemical diversity than protein-based ligands such as antibodies, limiting their utility. Aptamers possessing a single chemical modification have helped bridge this diversity gap. We report the selection and identification of aptamers with two diversity-enhancing chemical modifications that bind and inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9), a representative human therapeutic protein target. The addition of a second modification, especially in certain pairwise combinations, resulted in significant improvements in affinity, ligand efficiency, epitope coverage, metabolic stability, and inhibitory activity. Extensively chemically functionalized aptamers have the potential to become the next generation of nucleic-acid–based ligands. The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid–like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications.

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