Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation

McGregor, Alistair; Rao, M. V. R.; Duckworth, G.; Stockley, Peter G.; Connolly, Bernard A.

doi:10.1093/nar/24.16.3173

Cited by 42 publications

(25 citation statements)

References 41 publications

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“…Alternatively, RNA containing a reactive chemical group at the 5Ј or 3Ј terminus is generated by in vitro transcription, followed by the coupling of photochemical probe to RNA via the reactive moiety (Burgin and Pace 1990;Fidanza et al 1994). Additionally, a limited number of photoactivatable nucleotide analogs can also be incorporated into RNA by chemical synthesis (Shah et al 1994;McGregor et al 1996).…”

Section: Introductionmentioning

confidence: 99%

Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase

Gopalakrishna¹,

Gusti²,

Nair³

et al. 2004

RNA

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UV-induced photochemical crosslinking is a powerful approach that can be used for the identification of specific interactions involving nucleic acid-protein and nucleic acid-nucleic acid complexes. 8-AzidoATP (8-N 3 ATP) is a photoaffinity-labeling agent which has been widely used to elucidate the ATP binding site of a variety of proteins. However, its true potential as a photoactivatable nucleotide analog could not be exploited due to the lack of 8-azidoadenosine phosphoramidite, a monomer used in the synthesis of RNA, and the inability of 8-N 3 ATP to serve as an efficient substrate for bacteriophage RNA polymerase. In this study, we explored the ability of SP6, T3, and T7 RNA polymerases and metal ion cofactors to catalyze the incorporation of 8-N 3 AMP into RNA. Whereas transcription buffer containing 2.0-2.5 mM Mn 2+ supports T7 RNA polymerase-mediated insertion of 8-N 3 AMP into RNA, a mixture of 2.5 mM Mn 2+ and 2.5 mM Mg 2+ further improves the yield of 8-N 3 AMP-containing transcript. In addition, both RNA transcription and reverse transcription proceed with high fidelity for the incorporation of 8-N 3 AMP and complementary residue, respectively. Finally, we show that a high-affinity MS2 coat protein binding sequence, in which adenosine residues were replaced by 8-azidoadenosine, crosslinks to the coat protein of the Escherichia coli phage MS2.

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Section: Introductionmentioning

confidence: 99%

Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase

Gopalakrishna¹,

Gusti²,

Nair³

et al. 2004

RNA

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“…UV Cross-linking of MetJ Repressor with RNA Containing 4-Thiouridine-Cross-linking of the MetJ repressor to RNA containing 4-thiouridine was carried out as described previously (26). Samples contain-…”

Section: Methodsmentioning

confidence: 99%

“…Photocross-linking of 4-Thiouridine-containing RNA to MetJ-It has previously been shown that both oligodeoxyribonucleotides containing 4-thiothymidine and oligoribonucleotides containing 4-thiouridine can be used to photocross-link DNA-and RNA-binding proteins (26,34). We had hoped to use this approach to map the regions of MetJ in contact with the RNA ligand.…”

Section: Analysis Of a Rna-metj Repressor Complexmentioning

confidence: 99%

Secondary Structure Mapping of an RNA Ligand That Has High Affinity for the MetJ Repressor Protein and Interference Modification Analysis of the Protein-RNA Complex

McGregor

Murray

Adams

et al. 1999

Journal of Biological Chemistry

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The secondary structure of an RNA aptamer, which has a high affinity for the Escherichia coli MetJ repressor protein, has been mapped using ribonucleases and with diethyl pyrocarbonate. The RNA ligand is composed of a stem-loop with a highly structured internal loop. Interference modification showed that the bases within the internal loop, and those directly adjacent to it, are important in the binding of the RNA ligand to MetJ. Most of the terminal stem-loop could be removed with little effect on the binding. Ethylation interference suggests that none of the phosphate groups are absolutely essential for tight binding. The data suggest that the MetJ binding site on the aptamer is distinct from that of the natural DNA target, the 8-base pair Met box.Several genes involved in the biosynthesis of methionine in Escherichia coli are transcriptionally regulated by the methionine repressor, MetJ (1). The basic interaction occurs between a homodimer of the 12-kDa MetJ repressor subunits and an 8-base pair sequence that constitutes a Met box (2). The Met box is a tandem repeat that occurs between two and five times in natural operators and to which additional repressor dimers bind in a cooperative manner (3). The protein has a ␤␣␣ topology where the ␤-strands from each subunit intertwine to form an anti-parallel ␤-ribbon, which lies in the major groove of the target DNA. Binding specificity is largely determined by hydrogen bonds between the side chains of lysine 23 and threonine 25 of the ␤-strands and 4 purine bases in each Met box. Many hydrogen bonds are also made between the protein and the DNA phosphate backbone, and there is evidence that the conformation of the backbone is also important for binding (4). Binding of the MetJ repressor to DNA is modulated by Sadenosylmethionine (AdoMet), 1 which markedly increases the affinity of MetJ for its target sequence. AdoMet and the DNA are bound on opposite faces of the protein, and binding of AdoMet does not significantly perturb the protein structure. The increased affinity of the holorepressor appears to be due to a long range electrostatic effect caused by the positively charged tertiary sulfur atom of AdoMet (5, 6).Over the last decade in vitro selection and amplification techniques have been developed to isolate tight binding nucleic acid ligands for a wide range of target molecules (7,8). RNA molecules in particular have been isolated for a diverse set of targets, including proteins (9), amino acids (10), and nucleotides (11). These RNA aptamers potentially have very widespread applications as lead compounds in therapeutic situations, e.g. RNA-based inhibitors of the type I human immunodeficiency virus (HIV-I) reverse transcriptase (12, 13) or as ligands in diagnostic kits and biosensors. Despite their importance we have relatively little structural information on the ways in which aptamers interact with protein targets. Recently, one of our laboratories reported the first x-ray crystal structure for an aptamer bound to the RNA bacteriophage MS2 coat protein (14). T...

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“…The chemical synthesis of oligodeoxy-and oligoribonucleotides containing 4-thiopyrimidine and 6-thiodeoxyguanosine requires special protection of the sulfhydryl group (267,270). The EcoRV restriction endonuclease and modification methylase have been investigated with oligonucleotides containing these analogs (267).…”

Section: Photoaffinity Cross-linkingmentioning

confidence: 99%

MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users

Verma

Eckstein²

1998

Annu. Rev. Biochem.

299

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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Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation

Cited by 42 publications

References 41 publications

Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase

Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase

Secondary Structure Mapping of an RNA Ligand That Has High Affinity for the MetJ Repressor Protein and Interference Modification Analysis of the Protein-RNA Complex

MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users

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Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation

Cited by 42 publications

References 41 publications

Template-dependent incorporation of 8-N3AMP into RNA with bacteriophage T7 RNA polymerase

Template-dependent incorporation of 8-N3AMP into RNA with bacteriophage T7 RNA polymerase

Secondary Structure Mapping of an RNA Ligand That Has High Affinity for the MetJ Repressor Protein and Interference Modification Analysis of the Protein-RNA Complex

MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users

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Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase

Template-dependent incorporation of 8-N₃AMP into RNA with bacteriophage T7 RNA polymerase