In Vitro Evolution of an RNA-Cleaving DNA Enzyme into an RNA Ligase Switches the Selectivity from 3‘−5‘ to 2‘−5‘

Flynn-Charlebois, Amber; Prior, Tracey K.; Hoadley, Kelly A.; Silverman, Scott

doi:10.1021/ja0340331

Cited by 49 publications

(59 citation statements)

References 28 publications

(53 reference statements)

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“…In round 6, the ligation activity of the pool as a whole was ~21%, which is comparable to the activity of the pools used to clone our previous Mg 2+ -dependent RNA ligase deoxyribozymes (46,47). For the BB selection, twelve rounds were performed, each with a 3-h incubation during the key step; the ligation activity was ~35% at round 12.…”

Section: In Vitro Selection Of Zn 2+ -Dependent Deoxyribozymes That Lmentioning

confidence: 64%

“…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: In Vitro Selection Of Zn 2+ -Dependent Deoxyribozymes That Lmentioning

confidence: 64%

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

confidence: 99%

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

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“…It was shown that divalent metal ions accelerate the template-directed nonenzymatic ligation of two oligonucleotides possessing the terminal 5 -hydroxyl and 2 ,3 -cyclic phosphate (Flynn-Charlebois et al, 2003;Lutay et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

The nonenzymatic template-directed ligation of oligonucleotides

et al. 2006

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Abstract. The nonenzymatic template-directed ligation of oligonucleotides containing 2 ,3 -cyclic phosphate was investigated in the presence of divalent cations. Ligation of the oligonucleotides readily occurred in the presence of Mg 2+ , Mn 2+ , Co 2+ , Zn 2+ , Pb 2+ . Efficacy of the metal ion catalysts inversely correlated with pKa values of the metal-bound water molecules. The intermolecular transesterification reaction yielded at least 95% of 2 ,5 -phosphodiester bonds independently on the nature of the metal ion. Relatively high reaction yields (up to 15%) suggest, that RNA fragmentation to oligonucleotides with 2 ,3 -cyclic phosphates, followed by reactions of those oligonucleotides could provide a source of new RNA molecules under prebiotic conditions.

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“…The 2′-5′-linked nucleic acids can form duplex structures by themselves or by pairing with natural nucleic acids (10)(11)(12)(13)(14)(15). The 2′-5′ linkage substitutions in some functional RNAs retain molecular recognition and catalytic properties (16)(17)(18)(19). These discoveries suggested that the 2′-5′ linkage in nucleic acids could be an important alternative linkage during early evolution.…”

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Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

Zhang

Chong

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Nonenzymatic RNA polymerization in early life is likely to introduce backbone heterogeneity with a mixture of 2′-5′ and 3′-5′ linkages. On the other hand, modern nucleic acids are dominantly composed of 3′-5′ linkages. RNA polymerase II (pol II) is a key modern enzyme responsible for synthesizing 3′-5′-linked RNA with high fidelity. It is not clear how modern enzymes, such as pol II, selectively recognize 3′-5′ linkages over 2′-5′ linkages of nucleic acids. In this work, we systematically investigated how phosphodiester linkages of nucleic acids govern pol II transcriptional efficiency and fidelity. Through dissecting the impacts of 2′-5′ linkage mutants in the pol II catalytic site, we revealed that the presence of 2′-5′ linkage in RNA primer only modestly reduces pol II transcriptional efficiency without affecting pol II transcriptional fidelity. In sharp contrast, the presence of 2′-5′ linkage in DNA template leads to dramatic decreases in both transcriptional efficiency and fidelity. These distinct effects reveal that pol II has an asymmetric (strand-specific) recognition of phosphodiester linkage. Our results provided important insights into pol II transcriptional fidelity, suggesting essential contributions of phosphodiester linkage to pol II transcription. Finally, our results also provided important understanding on the molecular basis of nucleic acid recognition and genetic information transfer during molecular evolution. We suggest that the asymmetric recognition of phosphodiester linkage by modern nucleic acid enzymes likely stems from the distinct evolutionary pressures of template and primer strand in genetic information transfer during molecular evolution.2′-5′ phosphodiester linkage | trigger loop | synthetic nucleic acid analogues T he remarkable capacity of RNA as the functional catalyst as well as the genetic information carrier provides a strong support for the RNA world hypothesis, in which RNA is proposed to play a key role in the early evolution of primitive life before DNA and protein (enzyme) evolved (1, 2). One critical issue for RNA replication in early life is backbone heterogeneity because RNA contains both 2′-OH and 3′-OH, in which both can form phosphodiester bond during RNA polymerization. Indeed, previous studies revealed that nonenzymatic RNA replication would lead to a mixture of 2′-5′ and 3′-5′ linkages (Fig. 1A) (3-9). The 2′-5′-linked nucleic acids can form duplex structures by themselves or by pairing with natural nucleic acids (10-15). The 2′-5′ linkage substitutions in some functional RNAs retain molecular recognition and catalytic properties (16)(17)(18)(19). These discoveries suggested that the 2′-5′ linkage in nucleic acids could be an important alternative linkage during early evolution.This backbone heterogeneity problem is largely eliminated during evolution. Modern nucleic acids are dominated by 3′-5′ linkages. The 2′-5′ RNA linkages are only present in a few specific processes. One example is the formation of lariat RNA intron during splicing, where the 2′-OH of an...

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In Vitro Evolution of an RNA-Cleaving DNA Enzyme into an RNA Ligase Switches the Selectivity from 3‘−5‘ to 2‘−5‘

Cited by 49 publications

References 28 publications

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

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

The nonenzymatic template-directed ligation of oligonucleotides

Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

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In Vitro Evolution of an RNA-Cleaving DNA Enzyme into an RNA Ligase Switches the Selectivity from 3‘−5‘ to 2‘−5‘

Cited by 49 publications

References 28 publications

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

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

The nonenzymatic template-directed ligation of oligonucleotides

Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

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Product

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

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