Action of metal ions directly involved in the cleavage reaction of hammerhead ribozymes

Nakamatsu, Yuka; Warashina, Masaki; Kuwabara, Tomoko; Tanaka, Yoshiyuki; Shibata, Atsushi; Yoshinari, Koichi; Taira, Kazunari

doi:10.1093/nass/42.1.283

Cited by 2 publications

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“…Reagents for synthesis were purchased from Glen Research and from Chem Genes (Waltham, MA). The oligonucleotide was puri®ed on a reversed-phase column (Wakosil-II5C18 AR Prep, Wako Pure Chemical Industries, Ltd, Osaka, Japan Katahira et al 1994;Nakamatsu et al 1999;Tanaka et al 2000), and then puri®ed by electrophoresis on a 20% denaturing polyacrylamide gel.…”

Section: Synthesis Of the Modi®ed Ribozymementioning

confidence: 99%

Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

et al. 2000

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Background: Several reports have appeared recently of experimental evidence for a double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes. In one case, hammerhead ribozyme-mediated cleavage was analysed as a function of the concentration of La 3 ions in the presence of a ®xed concentration of Mg 2 ions so that the role of metal ions that are directly involved in the cleavage reaction could be monitored. The resultant bell-shaped curve for activation of cleavage was used to support the proposed doublemetal-ion mechanism of catalysis. However, other studies have demonstrated that the binding of a metal ion (the most conserved P9 metal ion) to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A 9 and to the N7 of nucleotide G 10.1 is critical for ef®cient catalysis, despite the large distance (<20 A Ê ) between the P9 metal ion and the labile phosphodiester group in the ground state. In fact, it was demonstrated that an added Cd 2 ion binds ®rst to the pro-Rp phosphoryl P9 oxygen but not with the pro-Rp phosphoryl oxygen at the cleavage site.

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Section: Synthesis Of the Modi®ed Ribozymementioning

confidence: 99%

Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

et al. 2000

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“…For hammerhead ribozyme or the leadzyme a bell-shaped titration curve was observed upon titration of lanthanide in the presence of standard bivalent metal ions i.e. La 3+ titration for Mg 2+ dependent reaction of hammerhead 132 and Nd 3+ titration for Pb 2+ dependent reaction of leadzyme 133 . These observations led to the hypothesis of a two-metal ion mechanism for catalysis of RNA cleavage 134 .…”

Section: Lanthanides and Nucleic Acidsmentioning

confidence: 99%

Functional characterization and application of 2',5'- branched RNA forming deoxyribozymes using lanthanides as cofactors

Fatemeh¹

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Oral examination: October 2013.iii Affidavit I hereby declare that my doctoral thesis entitled" Functional characterization and application of 2',5'-branched RNA forming deoxyribozymes using lanthanides as cofactors" has been written independently and with no other sources and aids than quoted. I have indicated the parts which were performed by project collaborators.Fatemeh Javadi-Zarnaghi Göttingen, August 2013 IntroductionKnown natural biocatalysts are mostly proteins. However, since 1980s potential abilities of nucleic acids became a notable scientific interest. In 1981, Tomas Cech, reported conservatively on RNA-based catalysis in ciliated protozoan Tetrahymena 1 . In 1984, Sydney Altman and his coworker announced the catalytic activity of RNase P in the absence of any protein component 2 . Since then several natural ribozymes have been discovered which most of them are involved in trans-phosphorylation reactions 3 .Examples include hammerhead ribozyme 4 , hairpin ribozyme 5 and group II intron 6 . Since discovery of the ribozymes, the catalytic ability of DNA was doubted, however, in vitro selection has been used to discover artificial catalytic DNAs. The first successful discovery of deoxyribozymes was an answer to the fundamental question about the catalytic ability of the DNA in the absence of the 2'-OH functional group. In vitro selectionNaturally, DNA is responsible for genetic information storage and is double stranded. DNA lacks the properties that proteins use for catalysis such as pK a near neutral condition (as in histidines), primary alkyl amines (as in lysines), carboxyl groups (as in aspartates) and sulfhydryl groups (as in cysteines).DNA also lacks the 2'-OH group which is present in ribozymes and is anticipated to be involved in the catalytic mechanism of ribozymes 7 . Despite doubts about possibility of existence of catalytic DNAs, in vitro selections have been performed to discover catalytically active DNA sequences.In vitro selection was first used to identify artificial RNA catalysts (ribozymes). In 1990, two independent groups reported on strategies to select functional nucleic acids. Systematic evolution of ligands by exponential enrichment (SELEX) was introduced by Tuerk and Gold 8 . They report on a selection procedure to identify specific RNA ligands to bacteriophage T4 DNA polymerase. During the same year, Robertson and Joyce, had reported an in vitro selection strategy to enrich an RNA sequence with DNA cleavage activity within a randomized pool of RNA sequences 9 .In vitro selection for DNA-catalysts is performed with single stranded DNA libraries. It is anticipated that in a random library of DNA molecules there are sequences which can adapt to a three-dimensional fold that fulfills catalysis. The first in vitro selected deoxyribozyme, was a Pb 2+ dependent RNA cleaving DNA and was discovered in 1994 by Breaker and Joyce 10 . The cleavage reaction obeyed Michaelis-Menten kinetics with a multiple turnover k cat value of 1 min -1 at 23°C and pH 7.0. The deoxyribozyme discovered by B...

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Action of metal ions directly involved in the cleavage reaction of hammerhead ribozymes

Cited by 2 publications

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Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Functional characterization and application of 2',5'- branched RNA forming deoxyribozymes using lanthanides as cofactors

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Action of metal ions directly involved in the cleavage reaction of hammerhead ribozymes

Cited by 2 publications

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Significant activity of a modified ribozyme with N7‐deazaguanine at G10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Significant activity of a modified ribozyme with N7‐deazaguanine at G10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Functional characterization and application of 2',5'- branched RNA forming deoxyribozymes using lanthanides as cofactors

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Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes