High Yield Photocrosslinking of a 5-Iodocytidine (IC) Substituted RNA to Its Associated Protein

Meisenheimer, Kristen M.; Meisenheimer, Poncho; Willis, Michael C.; Koch, Tad H.

doi:10.1093/nar/24.5.981

Cited by 35 publications

(20 citation statements)

References 6 publications

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“…In this assay the yield of MS2 protein-RNA crosslinking is relatively low compared to the published reports where 5-bromouridine (Willis et al 1994), 5-iodouracil (Willis et al 1993), or 5-iodocytidine (Meisenheimer et al 1996) substituted RNAs were used. It is conceivable that introduction of multiple 8-N 3 AMP residues could have lowered the affinity of MS2 protein for RNA.…”

Section: Rna-protein Crosslinking With 8-azidoadenosine-containing Rnamentioning

confidence: 92%

“…However, this technique requires irradiation of the biological sample with far-UV light (254 nm), which is known to cause damage to both protein and RNA. Thus, the use of photochemical agents which are activated with near-UV light (300-360 nm) and can crosslink with efficiencies greater than standard UV-induced crosslinking has become the method of choice (Favre 1990;Willis et al 1993;Meisenheimer et al 1996;Wang and Rana 1998;Costas et al 2000). When inserted into RNA and irradiated, such agents not only identify interacting partners by the presence of site-specific crosslinking, but also provide detailed information about the molecular environment of ribonucleoprotein assemblies.…”

Section: Introductionmentioning

confidence: 99%

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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: Rna-protein Crosslinking With 8-azidoadenosine-containing Rnamentioning

confidence: 92%

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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“…Intriguingly, in our recent crosslinking studies, RRM3 was not cross-linked to 5-iodouridines incorporated at specific positions within a Py tract (Banerjee et al 2003). This could be because of limitations of the cross-linking approach such as requirements for specific amino acids in the vicinity and for proper orientation of functional groups that are involved in cross-linking (Willis et al 1993;Meisenheimer et al 2000); it is known that certain RNAs can bind with high affinity but not crosslink (Gott et al 1991;Meisenheimer et al 1996). Therefore, to determine if RRM3 has any direct role in Py-tract recognition, we tested several natural Py tracts for the binding of U2AF 65 with (RRM123) or without (RRM12) the RRM3 domain.…”

Section: Rrm3 Is Dispensable For the Binding Of U2af 65 To Several Namentioning

confidence: 96%

The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF⁶⁵) is essential in vivo but dispensable for activity in vitro

Banerjee¹,

Rahn²,

Gawande³

et al. 2004

RNA

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The general splicing factor U2AF 65 recognizes the polypyrimidine tract (Py tract) that precedes 3 splice sites and has three RNA recognition motifs (RRMs). The C-terminal RRM (RRM3), which is highly conserved, has been proposed to contribute to Py-tract binding and establish protein-protein contacts with splicing factors mBBP/SF1 and SAP155. Unexpectedly, we find that the human RRM3 domain is dispensable for U2AF 65 activity in vitro. However, it has an essential function in Schizosaccharomyces pombe distinct from binding to the Py tract or to mBBP/SF1 and SAP155. First, deletion of RRM3 from the human protein has no effect on Py-tract binding. Second, RRM123 and RRM12 select similar sequences from a random pool of RNA. Third, deletion of RRM3 has no effect on the splicing activity of U2AF 65 in vitro. However, deletion of the RRM3 domain of S. pombe U2AF 59 abolishes U2AF function in vivo. In addition, certain amino acid substitutions on the four-stranded ␤-sheet surface of RRM3 compromise U2AF function in vivo without affecting binding to mBBP/SF1 or SAP155 in vitro. We propose that RRM3 has an unrecognized function that is possibly relevant for the splicing of only a subset of cellular introns. We discuss the implications of these observations on previous models of U2AF function.

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“…The aptamers can either be selected from oligonucleotide libraries that contain photocrosslinkable pyrimidines or already selected aptamers can be modified after the SELEX process (31,34). These modified oligonucleotides coupled to a reporter molecule generate reactive groups, which when irradiated by UV light, can form covalent linkage with another molecule in close proximity (35,36). Aptamers can also be attached to radioactive or fluorescent molecules and can be used this way REVIEW ARTICLE for in vitro and in vivo imaging of target protein expression (37,38).…”

Section: Cell Selex: Discovery Of Cell Surface Biomarker Expressionmentioning

confidence: 99%

Disease‐specific biomarker discovery by aptamers

Ulrich

Wrenger

2009

Cytometry Pt A

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RNA and DNA aptamers developed by an in vitro selection process, Systematic Evolution of Ligands by EXponential enrichment (SELEX), comprise a novel class of highaffinity and specific capture agents, which can be easily modified for cytometry and in vivo applications. A novel application of this technique (Cell SELEX) explores the expression of cell surface epitopes that differ between two given cell types or between healthy and diseased cells. Using whole cells as targets, aptamer libraries can be identified that bind to biomarkers expressed by target cells and not by any other cells. Aptamers have been developed that specifically interact with cell surface epitopes of trypanosomes or distinguish between the differences in molecular signature of somatic and cancer cells. Aside from its use for target cell identification by image and flow cytometry and laser-scanning microscopy, aptamers can be used for ligand-mediated purification and identification of their binding proteins in target cell membranes. In this review, we discuss an approach for the development of aptamers targeting parasitederived surface proteins of Trypanosoma and Plasmodium. ' 2009 International Society for Advancement of CytometryKey terms aptamers; Plasmodium falciparum; deconvolution SELEX; cell and target protein identification; fluorescence-labeled aptamers for clinical and cytomics application THE SELEX TECHNOLOGYThe SELEX technique (Systematic Evolution of Ligands by EXponential enrichment) initially developed by the laboratories of Larry Gold (1) and Jack Szostak (2) uses iterative in vitro selection of combinatorial RNA or DNA pools against a target molecule for the identification of high-affinity oligonucleotide ligands, known as aptamers. This methodology has been extensively used to isolate aptamers for a wide variety of peptides and proteins of therapeutic importance, including growth and coagulation factors (3). This novel class of oligonucleotide-based ligands recognizes their targets with binding specificities and affinities comparable to those of monoclonal antibodies. Moreover, aptamers are able to distinguish between protein isoforms and different conformational forms of the same protein (4,5). In most cases, aptamer binding to its target protein inhibits its biological activity, either resulting from aptamer interference with the catalytic site of an enzyme or with sites involved in ligandreceptor recognition. However, it is also possible that aptamer-target protein binding induces allosteric effects, such as changes in conformational states resulting in loss of biological activity of the target protein.Aptamers can be identified by in vitro selection against almost any target, including toxins and antigens which do not induce immune responses in host animals for antibody production. As a result, this novel class of ligands is highly promising for the development of therapeutics and biotechnological tools. The potential utility of aptamers for in vivo applications and as therapeutic agents is considerably enhanced ...

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High Yield Photocrosslinking of a 5-Iodocytidine (IC) Substituted RNA to Its Associated Protein

Cited by 35 publications

References 6 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

The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF⁶⁵) is essential in vivo but dispensable for activity in vitro

Disease‐specific biomarker discovery by aptamers

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High Yield Photocrosslinking of a 5-Iodocytidine (IC) Substituted RNA to Its Associated Protein

Cited by 35 publications

References 6 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

The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF65) is essential in vivo but dispensable for activity in vitro

Disease‐specific biomarker discovery by aptamers

Contact Info

Product

Resources

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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

The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF⁶⁵) is essential in vivo but dispensable for activity in vitro