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 degrees C, pH 7.9, which is substantially faster than our previously reported Mg2+-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'-Iinked 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.