Phosphorothioate analogs of 2',5'-oligoadenylate. Enzymatically synthesized 2',5'-phosphorothioate dimer and trimer: unequivocal structural assignment and activation of 2',5'-oligoadenylate-dependent endoribonuclease
- 1 November 1987
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 26 (22) , 7127-7135
- https://doi.org/10.1021/bi00396a039
Abstract
In continued studies to elucidate the requirements for binding to and activation of the 2'',5''-oligoadenylate-dependent endoribonuclease (RNase L), chirality has been introduced into the 2'',5''-oligoadenylate (2-5A, p3An) molecule to give the RP configuration in the 2'',5''-internucleotide backbone and the SP configuration in the .alpha.-phosphorus of the pyrophosphoryl moiety of the 5''-terminus. This was accomplished by the enzymatic conversion of (SP)-ATP.alpha.S to the 2'',5''-phosphorothioate dimer and trimer by the 2-5A synthetase from lysed rabbit reticulocytes. The most striking finding reported here is the ability of the 2'',5''-phosphorothioate dimer 5''-triphosphate (i.e., p3A2.alpha.S) to bind to and activate RNase L. p3A2.alpha.S displaces the p3A4[32P]pCp probe from RNase L with an IC50 of 5 .times. 10-7 M, compared to an IC50 of 5 .times. 10-9 M for authentic p3A3. Further, p3A2.alpha.S activates RNase L to hydrolyze poly(U)-3''-[32P]pCp (20% at 2 .times. 10-7 M), whereas authentic p3A2 is unable to activate the enzyme. Similarly, the enzymatically synthesized p3A2.alpha.S at 10-6 M activated RNase L to degrade 18S and 28S rRNA, whereas authentic p3A2 was devoid of activity. p3A3.alpha.S was as active as authentic p3A3 in the core-cellulose and rRNA cleavage assays. The absolute structural and configurational assignment of the enzymatically synthesized p3A2.alpha.S and p3A3.alpha.S was accomplished by high-performance liquid chromatography, charge separation, enzymatic hydrolyses, and comparison to fully characterized chemically synthesized (RP)- and (SP)-2'',5''-phosphorothioate dimer and trimer cores. The absolute structural assignment for p3A2.alpha.S is 5''-O-[(SP)-1-P-thiotriphosphoryl]-(RP)-P-thioadenylyl(2''-5'')adenosine, and for p3A3.alpha.S it is 5''-O-[(SP)-1-P-thiotriphosphoryl]-(RP)-P-thioadenylyl(2''-5'')-(RP)-P-thioadenylyl(2''-5'')adenosine. These assignments confirm the previous suggestion of an RP configuration at the 2''-5''-internucleotide linkages of enzymatically synthesized p3A3.alpha.S [Lee, C., and Suhadolnik, R. J. (1985) Biochemistry 24, 551-555].This publication has 36 references indexed in Scilit:
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