Zn²⁺-dependent deoxyribozymes that form natural and unnatural RNA linkages

We report Zn²⁺-dependent deoxyribozymes that ligate RNA. The DNA enzymes were identified by in vitro selection and ligate RNA with k _obs up to 0.5 min^-1 at 1 mM Zn²⁺ and 23 °C, pH 7.9, which is substantially faster than our previously reported Mg ²⁺-dependent deoxyribozymes. Each new Zn²⁺-dependent deoxyribozyme mediates the reaction of a specific nucleophile on one RNA substrate with a 2′,3′-cyclic phosphate on a second RNA substrate. Some of the Zn²⁺-dependent deoxyribozymes create native 3′-5′ RNA linkages (with k_obs up to 0.02 min ^-1), whereas all of our previous Mg²⁺-dependent deoxyribozymes that use a 2′,3′-cyclic phosphate create non-native 2′-5′ RNA linkages. On this basis, Zn²⁺-dependent deoxyribozymes have promise for synthesis of native 3′-5′-linked RNA using 2′,3′-cyclic phosphate RNA substrates, although these particular Zn²⁺-dependent deoxyribozymes are likely not useful for this practical application. Some of the new Zn²⁺-dependent deoxyribozymes instead create non-native 2′-5′ linkages, just like their Mg²⁺ counterparts. Unexpectedly, other Zn²⁺- dependent deoxyribozymes synthesize one of three unnatural linkages that are formed upon the reaction of an RNA nucleophile other than a 5′-hydroxyl group. Two of these unnatural linkages are the 3′-2′ and 2′-2′ linear junctions created when the 2′-hydroxyl of the 5′-terminal guanosine of one RNA substrate attacks the 2′,3′-cyclic phosphate of the second RNA substrate. The third unnatural linkage is a branched RNA that results from attack of a specific internal 2′-hydroxyl of one RNA substrate at the 2′,3′-cyclic phosphate. When compared with the consistent creation of 2′-5′ linkages by Mg²⁺-dependent ligation, formation of this variety of RNA ligation products by Zn²⁺-dependent deoxyribozymes highlights the versatility of transition metals such as Zn²⁺ for mediating nucleic acid catalysis.