We previously used in vitro selection to identify several classes of deoxyribozymes that mediate RNA ligation by attack of a hydroxyl group at a 5′-triphosphate. In these reactions, the nucleophilic hydroxyl group is located at an internal 2′-position of an RNA substrate, leading to 2′,5′-branched RNA. To obtain deoxyribozymes that instead create linear 3′-5′-linked (native) RNA, here we strategically modified the selection approach by embedding the nascent ligation junction within an RNA:DNA duplex region. This approach should favor formation of linear rather than branched RNA because the two RNA termini are spatially constrained by Watson-Crick base pairing during the ligation reaction. Furthermore, because native 3′-5′ linkages are more stable in a duplex than isomeric non-native 2′-5′ linkages, this strategy is predicted to favor the formation of 3′-5′ linkages. All of the new deoxyribozymes indeed create only linear 3′-5′ RNA, confirming the effectiveness of the rational design. The new deoxyribozymes ligate RNA with kobs values up to 0.5 h-1 at 37°C and 40 mM Mg2+, pH 9.0, with up to 41% yield at 3 h incubation. They require several specific RNA nucleotides on either side of the ligation junction, which may limit their practical generality. These RNA ligase deoxyribozymes are the first that create native 3′-5′ RNA linkages, which to date have been highly elusive via other selection approaches.
ASJC Scopus subject areas
- Colloid and Surface Chemistry