High yield synthesis, purification and characterisation of the RNase L activators 5'-triphosphate 2'-5'-oligoadenylates.
Upon viral infection, double-stranded viral RNA is detected very early in the host cell by several cellular 2'-5' oligoadenylate synthetases, which synthesize 2'-5' adenylate oligonucleotides that activate the cellular RNase L, firing an early primary antiviral response through self and non-self RNA cleavage. Transfecting cells with synthetic 2'-5' adenylate oligonucleotides activate RNase L, and thus provide a useful shortcut to study the early steps of cellular and viral commitments into this pathway. Defined 2'-5' adenylate oligonucleotides can be produced in vitro, but their controlled synthesis, purification, and characterisation have not been reported in detail. Here, we report a method suitable to produce large amounts of 2-5As of defined lengths in vitro using porcine OAS1 (pOAS) and human OAS2 (hOAS). We have synthesized a broad spectrum of 2-5As at the milligram scale and report an HPLC-purification and characterisation protocol with quantified yield for 2-5A of various lengths.
Morin
B
B
Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
Rabah
N
N
Boretto-Soler
J
J
Tolou
H
H
Alvarez
K
K
Canard
B
B
D015088:2',5'-Oligoadenylate Synthetase / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004722:Endoribonucleases / Q000378:metabolism*; D020536:Enzyme Activators / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D009843:Oligoribonucleotides / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
D000227:Adenine Nucleotides; D020536:Enzyme Activators; D009843:Oligoribonucleotides; C023505:2',5'-oligoadenylate; D015088:2',5'-Oligoadenylate Synthetase; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease