A common speed limit for RNA-cleaving ribozymes and deoxyribozymes

Breaker, Ronald R.; Emilsson, Gail Mitchell; Lazarev, Denis; Nakamura, Shingo; Puskarz, Izabela; Roth, Adam; Sudarsan, Narasimhan

doi:10.1261/rna.5670703

Cited by 128 publications

(137 citation statements)

References 27 publications

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“…Considering the relatively high background rate for reaction of a cyclic phosphate, these rate enhancements are considerable. In addition, k obs values of ~0.5 min −1 approach the recently postulated "speed limit" for naturally occurring nucleic acid enzymes (74,75), suggesting that Zn 2+ permits deoxyribozyme reaction rates that are fast on an absolute scale.…”

Section: Using Zn 2+ As the Cofactor Instead Of Mg 2+ : Effects On Rnmentioning

confidence: 75%

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: Using Zn 2+ As the Cofactor Instead Of Mg 2+ : Effects On Rnmentioning

confidence: 75%

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

et al. 2005

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“…The estimated rate constants (k obs ) for twister is 1,000 per minute, and the experimental k obs for hammerhead is 870 per minute (1, 8). These two ribozymes are ∼100-to 500-fold faster than other small self-cleaving ribozymes (2-10 per minute under the similar in vitro reaction conditions) (1,(8)(9)(10)). Twister constructs previously tested exhibited a maximum cleavage rate at 1 mM Mg 2+ and pH 7.4 (1).…”

mentioning

confidence: 86%

Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme

Eiler¹,

Wang²,

Steitz

2014

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

142

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Twister is a recently discovered RNA motif that is estimated to have one of the fastest known catalytic rates of any naturally occurring small self-cleaving ribozyme. We determined the 4.1-Å resolution crystal structure of a twister sequence from an organism that has not been cultured in isolation, and it shows an ordered scissile phosphate and nucleotide 5′ to the cleavage site. A second crystal structure of twister from Orzyza sativa determined at 3.1-Å resolution exhibits a disordered scissile phosphate and nucleotide 5′ to the cleavage site. The core of twister is stabilized by base pairing, a large network of stacking interactions, and two pseudoknots. We observe three nucleotides that appear to mediate catalysis: a guanosine that we propose deprotonates the 2′-hydroxyl of the nucleotide 5′ to the cleavage site and a conserved adenosine. We suggest the adenosine neutralizes the negative charge on a nonbridging phosphate oxygen atom at the cleavage site. The active site also positions the labile linkage for in-line nucleophilic attack, and thus twister appears to simultaneously use three strategies proposed for small self-cleaving ribozymes. The twister crystal structures (i) show its global structure, (ii) demonstrate the significance of the double pseudoknot fold, (iii) provide a possible hypothesis for enhanced catalysis, and (iv) illuminate the roles of all 10 highly conserved nucleotides of twister that participate in the formation of its small and stable catalytic pocket.X-ray crystallography | RNA structure | weak derivative phasing | samarium derivatives | cesium derivatives T he twister RNA motif was identified by bioinformatic searches and then validated biochemically to be a small self-cleaving ribozyme (1). This recently discovered class of ribozymes is called twister because its conserved secondary structure resembles the ancient Egyptian hieroglyph "twisted flax." Representatives of the twister ribozyme class are found in all domains of life, but its biological role has yet to be determined. In addition to twister, the small self-cleaving ribozyme family includes the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), and glmS ribozymes (the glmS ribozyme is upsteam of the the glmS gene that codes for the enzyme that catalyzes glucosamine-6-phosphate production) (1-6).The small self-cleaving ribozyme family can be split into two groups based on whether their active site is formed by an irregular helix (hammerhead, hairpin, and VS) or a double pseudoknot (PK) structure (HDV and glmS) (7). The structures of HDV and glmS are known, whereas twister was predicted from representative sequences to use two PKs to form its active site. It was expected that twister would be smaller in size than either HDV or glmS and more comparable in size and complexity to hammerhead (1).The self-cleavage rate constant of twister is estimated to be as rapid as or slightly more rapid than the hammerhead ribozyme. The estimated rate constants (k obs ) for twister is 1,000 per minute, and the experime...

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“…RNAs with phosphodiester linkages positioned to some extent in an in-line orientation have been found to cleave with a rate enhancement of more than 10-fold above unstructured linkages (Usher and McHale 1976;Soukup and Breaker 1999). Furthermore, in-line geometry is unlikely to contribute much more than 100-fold to the rate enhancement of a catalyst (Soukup and Breaker 1999;Breaker et al 2003;Min et al 2007). Therefore, the rate enhancement that can be derived by a catalyst that fully employs α catalysis by perfectly pre-organizing the RNA linkage for in-line attack is very modest.…”

Section: The Catalytic Strategies Used By Hatchet Ribozymesmentioning

confidence: 99%

Biochemical analysis of hatchet self-cleaving ribozymes

Lünse

Harris

et al. 2015

RNA

Self Cite

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Hatchet RNAs are members of a novel self-cleaving ribozyme class that was recently discovered by using a bioinformatics search strategy. The consensus sequence and secondary structure of this class includes 13 highly conserved and numerous other modestly conserved nucleotides interspersed among bulges linking four base-paired substructures. A representative hatchet ribozyme from a metagenomic source requires divalent ions such as Mg 2+ to promote RNA strand scission with a maximum rate constant of ∼4 min −1 . As with all other small self-cleaving ribozymes discovered to date, hatchet ribozymes employ a general mechanism for catalysis involving the nucleophilic attack of a ribose 2 ′ -oxygen atom on an adjacent phosphorus center. Kinetic characteristics of the reaction demonstrate that members of this ribozyme class have an essential requirement for divalent metal ions and that they might have a complex active site that employs multiple catalytic strategies to accelerate RNA cleavage by internal phosphoester transfer.

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A common speed limit for RNA-cleaving ribozymes and deoxyribozymes

Cited by 128 publications

References 27 publications

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

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

Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme

Biochemical analysis of hatchet self-cleaving ribozymes

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A common speed limit for RNA-cleaving ribozymes and deoxyribozymes

Cited by 128 publications

References 27 publications

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

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

Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme

Biochemical analysis of hatchet self-cleaving ribozymes

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

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