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.

Important note: Information in this article was accurate in 1997. The state of the art may have changed since the publication date.

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.

Biochemistry. 1997 Mar 25;36(12):3496-505. Unique Identifier : AIDSLINE MED/97238549
Naryshkin NA; Farrow MA; Ivanovskaya MG; Oretskaya TS; Shabarova ZA; Gait MJ; Laboratory of Molecular Biology, Medical Research Council, Cambridge,; U.K.

Abstract: 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.

Keywords: *Cross-Linking Reagents/PHARMACOLOGY *Diphosphates/CHEMISTRY *Gene Products, tat/METABOLISM *HIV Long Terminal Repeat *HIV-1 *Models, Chemical *RNA, Viral/METABOLISM
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M9772153

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