Oligonucleotide structural parameters that influence binding of 5'-O-triphosphoadenylyl-(2'----5')-adenylyl-(2'----5')-adenosine to the 5'-O-triphosphoadenylyl-(2'----5')-adenylyl-(2'----5')-adenosine dependent endoribonuclease: chain length, phosphorylation state, and heterocyclic base.
A number of 2',5'-linked oligoadenylates and their analogues were prepared and evaluated for their ability to interact with the 5'-O- triphosphoadenylyl -(2'----5')-adenylyl-(2'----5')-adenosine (2-5A) dependent endoribonuclease of mouse L cells. The oligonucleotides were assayed for their ability to antagonize the action of 2-5A, to displace a radiolabeled probe from the 2-5A-dependent nuclease, or to inhibit translation in a cell-free system. These experiments demonstrated the following: (1) Three AMP residues in a 5'-phosphorylated oligonucleotide were needed for maximum interaction with the endonuclease, and higher oligomers (greater than or equal to 4 AMP residues) did not show significantly higher binding. (2) The third (2'-terminal) adenosine residue was required for optimal binding activity. (3) 5'-Phosphorylation of the oligonucleotide was necessary for maximum binding to the endonuclease, but the first (from the 5' terminus) internucleotide phosphate of higher unphosphorylated or core oligomers, such as A2'p5'A2'p5'A2'p5'A, may partly replace the requirement for a 5'-monophosphate moiety; in agreement with this, the 5'-methyl ester of 5'pA2'p5'A2'p5'A, i.e., Me-p5'A2'p5'A2'p5'A, was bound to the endonuclease as well as or better than the higher core oligomers but approximately 100 times more effectively than the trimer core, A2'p5'A2'p5'A. (4) Base-modified analogues, such as p5'C2'p5'C2'p5'C, p5'U2'p5'U2'p5'U, or p5'I2'p5'I2'p5'I, were at least 2000 times less effectively bound to the endonuclease than p5'A2'p5'A2'p5'A. (5) The triphosphate ppp5 'I2'p5'I2'p5'I was 10 000 times less active than 2-5A as an inhibitor of translation. These latter two points implied the critical role of the adenine N1-nitrogen and/or exocyclic amino group in the binding of 2-5A to the endonuclease.
D000227:Adenine Nucleotides / Q000378:metabolism*; D000818:Animals; D004722:Endoribonucleases / Q000378:metabolism*; D051379:Mice; D009841:Oligonucleotides / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism*; D010766:Phosphorylation; D013329:Structure-Activity Relationship
D000227:Adenine Nucleotides; D009841:Oligonucleotides; D009843:Oligoribonucleotides; C023505:2',5'-oligoadenylate; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease