Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatically synthesized 2',5'-phosphorothioate dimer and trimer: unequivocal structural assignment and activation of 2',5'-oligoadenylate-dependent endoribonuclease.
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 X 10(-7) M, compared to an IC50 of 5 X 10(-9) M for authentic p3A3. Further, p3A2 alpha S activates RNase L to hydrolyze poly(U)-3'-[32P]pCp (20% at 2 X 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Karikó
K
K
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Sobol
R W
RW
Suhadolnik
L
L
Li
S W
SW
Reichenbach
N L
NL
Suhadolnik
R J
RJ
Charubala
R
R
Pfleiderer
W
W
D015088:2',5'-Oligoadenylate Synthetase / Q000097:blood; D000227:Adenine Nucleotides / Q000138:chemical synthesis* / Q000494:pharmacology; D000818:Animals; D004722:Endoribonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007739:L Cells / Q000201:enzymology; D051379:Mice; D009843:Oligoribonucleotides / Q000138:chemical synthesis* / Q000494:pharmacology; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000378:metabolism; D012156:Reticulocytes / Q000201:enzymology; D013329:Structure-Activity Relationship; D013873:Thionucleotides / Q000138:chemical synthesis / Q000494:pharmacology
D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; D012335:RNA, Ribosomal; D013873:Thionucleotides; C023505:2',5'-oligoadenylate; D015088:2',5'-Oligoadenylate Synthetase; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease