Phosphorothioate analogs of 2',5'-oligoadenylate. Activation of 2',5'-oligoadenylate-dependent endoribonuclease by 2',5'-phosphorothioate cores and 5'-monophosphates

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Abstract
The preceeding paper in this issue described the synthesis and structural elucidation of the phosphorothioate analogues of 2'',5''-oligoadenylate (2-5A) dimer and trimer cores [Kariko, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., and Pfleiderer, W. (1987) Biochemistry (preceeding paper in this issue)]. In this report, the binding and activation processes of 2-5A-dependent endoribonuclease (RNase L) have been examined by using four diastereomeric 2'',5''-phosphorothioate trimer core analogues and their 5''-monophosphates. These 2'',5''-phosphorothioates have revealed a distinct separation of the structural parameters that govern binding vs activation of RNase L. Radiobinding assays have demonstrated that extensive stereochemical modification of the internucleotide linkages of 2-5A is possible without adversely affecting its ability to bind to RNase L. However, a marked difference was observed in the activation of RNase L by the stereochemically modified 2-5A molecules as determined in core-cellulose and rRNA cleavage assays. Three of the four 2'',5''-phosphorothioate trimer cores (with RpRp, SpRp, and Rpsp internucleotide linkages) are the first 2-5A core molecules able to activate RNase L. For example, the RpRp,SpRp, and RpRp, SpRp, and RpSp diastereoisomers activate RNase L to hydrolyze poly(U)-3''[32P]pCp 65%, 20%, and 15%, respectively, at 5 .times. 10-5 M. The SpSp diastereomer cannot activate RNase L. The order of RNase L activation was the same for the core analogues and their 5''-monophosphates (RpRp > SpRp > RpSp). The binding/activation ability of the 2'',5''-phosphorothioate cores could result from the combined effect of stereoconfigurational and electronic alterations brought about by the replacement of oxygen by sulfur in the internucleotide linkages. The SpSp and pSpSp 2,''5''-phosphorothioates are capable of binding to RNase L as well as the corresponding authentic A3 and pA3 but are unable to activate the enzyme at concentrations as high as 10-3 and 10-5 M, respectively. The SpSp and pSpS pS analogues inhibit the activation process as shown in competition assays. This new 2-5A analogue (pSpSp) is the most effective inhibitor of RNase L reported to date and will be useful in in vivo studies. The results reported here are consistent with the hypothesis that RNase L is a functionally stereoselective enzyme and that the binding process is independent of the activation process. RNase L cannot distinguish between Rp and Sp chirality in the internucleotide linkages of 2-5A for binding but can distinguish between different stereoconfigurations for activation. The binding and activation processes are discussed in terms of their spatial homology and their accommodation in the binding domain of RNase L.