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

• Published in: Biochemistry (Easton) Vol. 36; no. 12; pp. 3496 - 3505
• Main Authors: Naryshkin, Nikolai A, Farrow, Mark A, Ivanovskaya, Marina G, Oretskaya, Tanya S, Shabarova, Zoe A, Gait, Michael J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 25.03.1997
• Subjects: AIDS/HIV; Amino Acid Sequence; Chromatography, High Pressure Liquid; Cross-Linking Reagents - pharmacology; Diphosphates - chemistry; Diphosphates - metabolism; Electrophoresis, Polyacrylamide Gel; Gene Products, tat - chemical synthesis; Gene Products, tat - metabolism; HIV Long Terminal Repeat; HIV-1; Human immunodeficiency virus 1; Models, Chemical; Molecular Sequence Data; Oligonucleotides - chemical synthesis; Oligonucleotides - chemistry; Peptide Fragments - chemistry; Peptide Fragments - metabolism; RNA, Viral - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; tat Gene Products, Human Immunodeficiency Virus; Templates, Genetic
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi962789p

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.