Chemical Cross-Linking of the Human Immunodeficiency Virus Type 1 Tat Protein to Synthetic Models of the RNA Recognition Sequence TAR Containing Site-Specific Trisubstituted Pyrophosphate Analogues
A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2‘-deoxynucleoside 5‘ to the tsp was used in the ligation reaction. A t...
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Published in | Biochemistry (Easton) Vol. 36; no. 12; pp. 3496 - 3505 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
25.03.1997
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Subjects | |
Online Access | Get full text |
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Summary: | A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2‘-deoxynucleoside 5‘ to the tsp was used in the ligation reaction. A tsp linkage was found to be reasonably stable to hydrolysis but cleaved by treatment with ethylenediamine or lysine to give phosphoamidate adducts. A model human immunodeficiency virus type 1 (HIV-1) TAR RNA duplex containing an activated tsp was able to bind to HIV-1 Tat protein with only 3-fold reduced affinity and to a Tat peptide (residues 37−72) with identical affinity compared to that of an unmodified duplex. Tsps incorporated at sites previously identified as being in close proximity to Tat protein were able to cross-link to Tat peptide (37−72) to form a covalent phosphoamidate conjugate. Endopeptidase cleavage followed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometric analysis provided strong evidence that a TAR duplex containing a tsp replacing the phosphate at 38−39 had reacted specifically with Lys51 in the basic region of Tat peptide (37−72). The new chemical cross-linking method may be generally useful for identifying lysines in close proximity to phosphates in basic RNA-binding domains of proteins. |
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Bibliography: | ark:/67375/TPS-C6MLP19T-W Abstract published in Advance ACS Abstracts, March 1, 1997. This work was supported in part by INTAS grant 94/3810 of the European Union for scientific cooperation with countries of the former Soviet Union. istex:1E01CC678A644109C42A4ACB1A46E08106409991 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi962789p |