Effect of Chemical Modifications on Aptamer Stability in Serum

Abstract

There is increasing interest in the use of aptamers for the development of therapeutics. However, as oligonucleotides, aptamers are susceptible to nuclease degradation; poor serum stability is likely to negatively affect in vivo function. Modified nucleotides have been used to thwart nuclease degradation. However, few studies report the serum stability of selected aptamers. In this study, we examined the effect of various chemical modifications (2′-deoxy, 2′-hydroxyl, 2′-fluoro, and 2′-O-methyl) on the stability of a control oligonucleotide sequence following incubation in frozen human, fresh mouse, and fresh human serum. We also assessed the effect of the 3′ inverted dT cap on stability. Surprisingly, we found that fYrR (2′-fluoro RNA) is only roughly as stable as DNA (2′-deoxy). Interestingly, the inclusion of a 3′ inverted dT cap had only a modest effect on serum stability, if any. In one instance, the addition of a 3′ inverted dT cap rendered a molecule composed of DNA more stable than its fYrR counterpart. By far, fully modified oligonucleotides (100% 2-O-Methyl or 2′-O-methyl A, C, and U in combination with 2′-fluoro G, termed fGmH) had the longest half-lives. These compositions demonstrated little degradation in human serum even after prolonged incubation. Together these results support the need for using fully modified aptamers for in vivo applications and should encourage those in the field to exploit newer polymerase variants capable of directly generating such polymers.