Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain

Yang, Jing; Usman, Nassim; Chartrand, Pascal; Cedergren, Robert

doi:10.1021/bi00136a013

Cited by 113 publications

(68 citation statements)

References 29 publications

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“…Modifications such as terminal phosphorothioate groups and 2Ј-modifications lead to high stability against digestion by nucleases but are often accompanied by a decrease in catalytic efficiency. For example, replacement of U 4 within the catalytic core by its 2Ј-O-methyl analogue leads to a significant reduction of the ribozyme catalytic efficiency (28). This is in agreement with results presented in this study, as 2Ј-Omethyl substitution causes complete catalytic inactivation of ribozyme RE917 (Table IV).…”

Section: Discussionsupporting

confidence: 92%

“…Since the 2Ј-hydroxyl group plays an important role in the degradation mechanism by nucleases, ribozymes have been protected against degradation by modification of the 2Ј-hydroxyl position (25,26). For this 2Ј-deoxyribonucleotide (27,28), 2Ј-O-methyl groups (29), 2Ј-fluoro-and/or 2Ј-amino groups (30), or 2Ј-deoxyribonucleotides together with phosphorothioate linkages (31) have been used.…”

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confidence: 99%

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Specific Hammerhead Ribozyme-mediated Cleavage of Mutant N-ras mRNA in Vitro and ex Vivo

Scherr

Grez

Ganser

et al. 1997

Journal of Biological Chemistry

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Two hammerhead ribozymes targeted to point mutations in codon 13 of the N-ras oncogene were synthesized and their catalytic activity and substrate specificity evaluated in vitro and ex vivo. In vitro studies showed that these ribozymes were specific for the oncogenic form of N-ras, since cleavage was observed only in a 849-nucleotide-long transcript containing mutant but not wild-type N-ras sequences. For the ex vivo studies, the ribozymes were 2 -modified to protect them against degradation by nucleases. 2 -Fluoro-2 -deoxyuridine/ cytidine-substituted ribozymes were nearly as active as their unmodified counterparts, but had a prolonged stability in cell culture supernatant containing fetal calf serum. The stability of the modified ribozymes increased by introduction of terminal phosphorothioates groups without significant influence in their catalytic efficiency. A sensitive assay based on the use of N-ras/ luciferase fusion genes as a reporter system was established to detect ribozyme-mediated cleavage in HeLa cells. A reduction of nearly 60% in luciferase activity was observed in cells expressing mutant but not wild-type N-ras/luciferase fusion transcripts. Moreover, cleavage of N-ras transcripts in HeLa cells was directly confirmed by a semi-quantitative RT-PCR assay.

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Section: Discussionsupporting

confidence: 92%

mentioning

confidence: 99%

Specific Hammerhead Ribozyme-mediated Cleavage of Mutant N-ras mRNA in Vitro and ex Vivo

Scherr

Grez

Ganser

et al. 1997

Journal of Biological Chemistry

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“…For example, a substrate containing all DNA residues (except for a single ribonucleotide at the cleavage site) can be cleaved by the hammerhead ribozyme, albeit with reduced efficiency (23)(24)(25). From a catalytic perspective, a predominately DNA analogue of the ribozyme domain is capable of cleaving an RNA substrate (26). Other examples of RNA catalysis with DNA substrates include Group I introns (27) and RNase P (28,29).…”

Section: Resultsmentioning

confidence: 99%

The Escherichia coli tRNA-Guanine Transglycosylase Can Recognize and Modify DNA

Nonekowski¹,

Kung²,

Garcia

2002

Journal of Biological Chemistry

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tRNAs contain a large number of modified nucleosides (1). One of the more elaborately modified nucleosides is queuine (7-(4,5-cis-dihydroxy-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanine). tRNA-guanine transglycosylase (TGT) 1 catalyzes the exchange of queuine (or a precursor) for guanine 34 in the anticodon of certain tRNAs. The minimal RNA recognition motif for TGT has been found to involve a UGU sequence in the anticodon loop of the queuine-cognate tRNAs (tyrosine, aspartate, asparagine, and histidine) (2, 3). The UGU sequence is undeniably the major determinant for tRNA recognition by TGT. However, provided that TGT can position the UGU sequence in the active site in the proper orientation, the UGU sequence need not reside in the anticodon loop to be recognized. Recent studies have shown that TGT can recognize the UGU sequence in at least 2 additional minihelical contexts, at the base of the T⌿C stem in yeast tRNA Phe and in the anticodon position in the absence of U33 (4, 5). Thus, tRNA recognition by TGT is more flexible than previously believed. This observation prompted an examination of the ability of TGT to recognize RNAs containing modifications of the UGU sequence. The initial studies that identified the UGU sequence as the major identity element utilized only canonical base (C, G, A, U) replacements (2, 3). Previous experiments have demonstrated that a DNA analogue of an RNA minihelix corresponding to the anticodon arm of Escherichia coli tRNA Tyr , ECYMH (dEC-YMH) was inactive.2 However, there are two fundamental differences between RNA and DNA, the lack of the 2Ј-hydroxyls and the presence of thymidine rather than uracil in DNA. Therefore, dECYMH has a TGT sequence rather than a UGU sequence.Given the importance of the UGU sequence in TGT recognition, it is possible that the inactivity of dECYMH was due to the presence of thymidine rather than the loss of the 2Ј-hydroxyls. To investigate the role of the 2Ј-hydroxyl in TGT recognition and catalysis, we have studied a deoxyguanosine 34 analogue of ECYMH (Fig. 1, dG 34 ECYMH). This analogue is a substrate for TGT with less than a 10-fold reduction in activity. To further probe the ability of TGT to recognize RNA analogues lacking the 2Ј-hydroxyl, modified DNA analogues of the previously described minihelix ECYMH (2, 6) ( Fig. 1, dUdECYMH) and the alternative TGT minihelical substrates UGU ϩ1 (dUdUGU ϩ1 ) (4) and SCFMH(T⌿C) (dUdSCFMH(T⌿C)) (5) were synthesized and characterized. These analogues (containing deoxyuracil (dU) bases rather than thymidine bases) all serve as substrates for TGT, indicating that the tRNA-guanine transglycosylase from E. coli can recognize and modify DNA.

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“…The 2 -hydroxyl modifications introduced to probe hammerhead ribozyme catalysis include deoxy (197,198); fluoro, amino (199,200); methoxy, allyloxy (201); and 2 -C-allyl substituents (202). 2 -Deoxy-or 2 -O-methyl sugar modifications have been introduced to identify the positions of 2 -hydroxyl groups necessary for the hairpin ribozyme activity (203).…”

Section: Sugar Modificationmentioning

confidence: 99%

MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users

Verma

Eckstein²

1998

Annu. Rev. Biochem.

303

190

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Synthetic oligonucleotide analogs have greatly aided our understanding of several biochemical processes. Efficient solid-phase and enzyme-assisted synthetic methods and the availability of modified base analogs have added to the utility of such oligonucleotides. In this review, we discuss the applications of synthetic oligonucleotides that contain backbone, base, and sugar modifications to investigate the mechanism and stereochemical aspects of biochemical reactions. We also discuss interference mapping of nucleic acid-protein interactions; spectroscopic analysis of biochemical reactions and nucleic acid structures; and nucleic acid cross-linking studies.The automation of oligonucleotide synthesis, the development of versatile phosphoramidite reagents, and efficient scale-up have expanded the application of modified oligonucleotides to diverse areas of fundamental and applied biological research. Numerous reports have covered oligonucleotides for which modifications have been made of the phosphodiester backbone, of the purine and pyrimidine heterocyclic bases, and of the sugar moiety; these modifications serve as structural and mechanistic probes. In this chapter, we review the range, scope, and practical utility of such chemically modified oligonucleotides. Because of space limitations, we discuss only those oligonucleotides that contain phosphate and phosphate analogs as internucleotidic linkages. CONTENTS

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Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain

Cited by 113 publications

References 29 publications

Specific Hammerhead Ribozyme-mediated Cleavage of Mutant N-ras mRNA in Vitro and ex Vivo

Specific Hammerhead Ribozyme-mediated Cleavage of Mutant N-ras mRNA in Vitro and ex Vivo

The Escherichia coli tRNA-Guanine Transglycosylase Can Recognize and Modify DNA

MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users

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