Photochemical crosslinking in oligonucleotide-protein complexes between a bromine-substituted 2-5A analog and 2-5A-dependent RNase by ultraviolet lamp or laser.
2',5'-oligoadenylates known as 2-5A [px(A2'p)nA; chi = 2 or 3, n greater than or equal to 2] are produced in interferon-treated cells in response to double-stranded RNA. 2-5A binds with high affinity to a 2-5A-dependent RNase resulting in the cleavage of single-stranded RNA. An efficient, rapid, and extremely sensitive photoaffinity labeling method was developed to facilitate detection of 2-5A-dependent RNase. A bromine-substituted and radioactive derivative of 2-5A, the 5'-monophosphate, p(A2'p)2(br8A2'p)2A3'-[32P]Cp, was synthesized as probe for 2-5A-dependent RNase. Even though this bromine-substituted analog of 2-5A bore no 5'-terminal triphosphate or diphosphate, it bound to 2-5A-dependent RNase with the same high affinity as did 2-5A per se but it was a less effective activator of the RNase under the present assay conditions. The presence of bromine atoms in the 2-5A analog enhanced by more than 200-fold crosslinking to 2-5A-dependent RNase under a uv lamp; many additional polypeptides were also labeled but at much lower levels. Furthermore, using high-intensity uv laser irradiation (308 nm) covalent attachment of the bromine-substituted 2-5A analog to 2-5A-dependent RNase was readily achieved within 10(-6) s.
Nolan-Sorden
N L
NL
Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Lesiak
K
K
Bayard
B
B
Torrence
P F
PF
Silverman
R H
RH
D000227:Adenine Nucleotides / Q000378:metabolism / Q000528:radiation effects*; D000241:Adenosine / Q000031:analogs & derivatives; D000345:Affinity Labels; D004722:Endoribonucleases / Q000378:metabolism / Q000528:radiation effects*; D004789:Enzyme Activation; D007834:Lasers; D009843:Oligoribonucleotides / Q000378:metabolism / Q000528:radiation effects*; D010777:Photochemistry; D011485:Protein Binding; D014466:Ultraviolet Rays
D000227:Adenine Nucleotides; D000345:Affinity Labels; D009843:Oligoribonucleotides; C036038:8-bromoadenosine; C023505:2',5'-oligoadenylate; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease; D000241:Adenosine