Optimization and Generality of a Small Deoxyribozyme that Ligates RNA

Ricca, Benjamin L; Wolf, Amanda C.; Silverman, Scott

doi:10.1016/s0022-2836(03)00654-5

Cited by 19 publications

(22 citation statements)

References 37 publications

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“…The use of Zn 2+ was key to this outcome, because all previous Mg 2+ -dependent deoxyribozymes that ligate RNA by reaction at a cyclic phosphate form 2′-5′ linkages (46)(47)(48)(49)(50). Others have described a Zn 2+ -dependent deoxyribozyme that cleaves 3′-5′ linkages (with formation of a cyclic phosphate), but no ligation activity was detected (17).…”

Section: Synthesis Of Native 3′-5′ Rna Linkages By Zn 2+ -Dependent Dmentioning

confidence: 99%

“…Of course, the attacking nucleophile is not necessarily a 5′-hydroxyl, but this functional group is typically the most nucleophilic of those present in RNA. Indeed, in our previous studies, all of the Mg 2+ -dependent deoxyribozymes that use a 2′,3′-cyclic phosphate RNA substrate were found to create only non-native 2′-5′ linkages (path ii in Figure 1), although the selection strategy itself did not compel this high selectivity against 3′-5′ linkages (46)(47)(48)(49)(50). Therefore, understanding the distribution of ligation products upon DNAcatalyzed reaction of a 2′,3′-cyclic phosphate RNA substrate is of particular interest.…”

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confidence: 94%

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Zn²⁺-Dependent Deoxyribozymes That Form Natural and Unnatural RNA Linkages

et al. 2005

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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 °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 several unnatural linkages arising from 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 resulting 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 like Zn 2+ for mediating nucleic acid catalysis. † This research was supported by the Burroughs Wellcome Fund (New Investigator Award in the Basic Pharmacological Sciences to S.K.S.), the March of Dimes Birth Defects Foundation Many of the Mg 2+ -dependent deoxyribozymes mediate RNA ligation via reaction of a 2′,3′-cyclic phosphate, which may be opened by an attacking nucleophile with cleavage of either the P-O 2′ bond or the P-O 3′ bond. If the nucleophilic group is a 5′-hydroxyl, then these two reaction pathways lead to the isomeric native 3′-5′ linkage or non-native 2′-5′ linkage, respectively ( Figure 1). Of course, the attacking nucleophile is not necessarily a 5′-hydroxyl, but this functional group is typically the most nucleophilic of those present in RNA. Indeed, in our previous studies, all of the Mg 2+ -dependent deoxyribozymes that use a 2′,3′-cyclic phosphate RNA substrate were found to create only non-native 2′-5′ linkages (path ii in Figure 1), although the selection strategy itself did not compel this high selectivity against 3′-5′ linkages (46-50). Therefore, understanding the distribution of ligation products upon DNAcatalyzed reaction of a 2′,3′-cyclic phosphate RNA substrate i...

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Section: Synthesis Of Native 3′-5′ Rna Linkages By Zn 2+ -Dependent Dmentioning

confidence: 99%

mentioning

confidence: 94%

Zn²⁺-Dependent Deoxyribozymes That Form Natural and Unnatural RNA Linkages

et al. 2005

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“…1). Because RNA-ligating deoxyribozymes that require numerous unpaired RNA substrate nucleotides typically have substantial sequence requirements for those nucleotides,3,6,9–11 we did not consider any of the 12BB deoxyribozymes that create native 3′–5′ linkages as likely candidates for high RNA substrate generality. In contrast, because a smaller number of overhanging RNA nucleotides has been more compatible with substrate sequence generality, the new 7CX and 8CW deoxyribozymes (which have only one-nucleotide overhangs) are more reasonable candidates for generality.…”

Section: Resultsmentioning

confidence: 99%

“…The reduced overhang length should help to suppress nucleophilic reactivity by the internal 2′-OH of R, because this nucleotide is now physically constrained within a DNA:RNA Watson-Crick base pair; this change should therefore contribute to controlling the site-selectivity of ligation. Another benefit of reducing the RNA overhang length on both substrates is an anticipated increase in RNA substrate sequence generality of the resulting deoxyribozymes, because we have observed that long single-stranded regions of the RNA substrates often lead to deoxyribozymes that have undesired requirements for specific RNA nucleotides in these non-duplex regions 3,6,9–11. RNA substrate generality is an important practical feature of useful RNA-ligating deoxyribozymes.…”

Section: Introductionmentioning

confidence: 99%

Controlling the direction of site-selectivity and regioselectivity in RNA ligation by Zn2+-dependent deoxyribozymes that use 2′,3′-cyclic phosphate RNA substrates

et al. 2008

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Our previous efforts have used in vitro selection to identify numerous Zn 2+ -dependent deoxyribozymes that ligate two RNA substrates with reaction at a 2′,3′-cyclic phosphate. Each deoxyribozyme creates one of several different RNA linkages, including native 3′-5′ and non-native 2′-5′ phosphodiester bonds as well as many unnatural linkages. In this report, we describe experiments to reveal design aspects of the selection strategy that favor site-selective and regioselective synthesis of native 3′-5′ RNA linkages. The results also reveal that an explicit selection pressure for RNA substrate sequence generality must be developed if the deoxyribozymes are to have practical generality.

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“…Since the 1994 report on DNAzymes that catalyze the cleavage of RNA phosphodiester bonds, 1 many more DNAzymes have been reported 2,3 to catalyze more than a dozen different types of chemical transformations such as porphyrin metallation, 4 DNA phosphorylation, 5 RNA ligation, 6 thyminethymine dimer repair, 7 carbon-carbon bond formation, 8 and, more recently, hydrolytic cleavage of DNA. 9 However, RNA-cleaving DNAzymes remain the best characterized and likely the most significant class of catalytic DNA molecules currently available, given their potential as therapeutic agents and diverse molecular tools.…”

Section: Introductionmentioning

confidence: 99%