title: Zn2+-dependent deoxyribozymes that form natural and unnatural RNA linkages.
abstract: We report Zn(2+)-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 Zn(2+) and 23 degrees C, pH 7.9, which is substantially faster than our previously reported Mg(2+)-dependent deoxyribozymes. Each new Zn(2+)-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 Zn(2+)-dependent deoxyribozymes create native 3'-5' RNA linkages (with k(obs) up to 0.02 min(-)(1)), whereas all of our previous Mg(2+)-dependent deoxyribozymes that use a 2',3'-cyclic phosphate create non-native 2'-5' RNA linkages. On this basis, Zn(2+)-dependent deoxyribozymes have promise for synthesis of native 3'-5'-linked RNA using 2',3'-cyclic phosphate RNA substrates, although these particular Zn(2+)-dependent deoxyribozymes are likely not useful for this practical application. Some of the new Zn(2+)-dependent deoxyribozymes instead create non-native 2'-5' linkages, just like their Mg(2+) counterparts. Unexpectedly, other Zn(2+)-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 Mg(2+)-dependent ligation, formation of this variety of RNA ligation products by Zn(2+)-dependent deoxyribozymes highlights the versatility of transition metals such as Zn(2+) for mediating nucleic acid catalysis.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Hoadley
authors.forename: Whitney E
authors.initials: WE
authors.lastname: Purtha
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Wolf
authors.forename: Amber
authors.initials: A
authors.lastname: Flynn-Charlebois
authors.forename: Scott K
authors.initials: SK
authors.lastname: Silverman
mesh_terms: D001483:Base Sequence; D021881:DNA, Catalytic / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D008969:Molecular Sequence Data; D015394:Molecular Structure; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006163:Ribonuclease T1 / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D021881:DNA, Catalytic; D012313:RNA; D006163:Ribonuclease T1; D015032:Zinc