| Abstract
| - We previously used in vitro selection to identify several classes of deoxyribozymes that mediateRNA ligation by attack of a hydroxyl group at a 5‘-triphosphate. In these reactions, the nucleophilic hydroxylgroup is located at an internal 2‘-position of an RNA substrate, leading to 2‘,5‘-branched RNA. To obtaindeoxyribozymes that instead create linear 3‘-5‘-linked (native) RNA, here we strategically modified theselection approach by embedding the nascent ligation junction within an RNA:DNA duplex region. Thisapproach should favor formation of linear rather than branched RNA because the two RNA termini arespatially constrained by Watson−Crick base pairing during the ligation reaction. Furthermore, becausenative 3‘-5‘ linkages are more stable in a duplex than isomeric non-native 2‘-5‘ linkages, this strategy ispredicted to favor the formation of 3‘-5‘ linkages. All of the new deoxyribozymes indeed create only linear3‘-5‘ RNA, confirming the effectiveness of the rational design. The new deoxyribozymes ligate RNA withkobs 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. Theyrequire several specific RNA nucleotides on either side of the ligation junction, which may limit their practicalgenerality. These RNA ligase deoxyribozymes are the first that create native 3‘-5‘ RNA linkages, which todate have been highly elusive via other selection approaches.
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