Synthesis and characterization of composite nucleic acids containing 2', 5'-oligoriboadenylate linked to antisense DNA.
Composite nucleic acids, known as 2-5A antisense chimeras, cause the 2-5A-dependent ribonuclease (RNase L) to catalyze the specific cleavage of RNA in cell free systems and in intact cells. Such 2-5A antisense chimeras are 5'-monophosphorylated, 2,'5'-linked oligoadenylates covalently attached to antisense 3',5'-oligodeoxyribonucleotides by means of a linker containing two residues of 1,4-butanediol phosphate. Here we report a fully automated synthesis of 2-5A antisense chimeras on a solid support using phosphoramidite methodology with specific coupling time modifications and their subsequent purification by reverse-phase ion-pair and anion exchange HPLC. Purified 2-5A antisense chimeras were characterized by [1H]NMR and [31P]NMR, MALDIMS, and capillary gel electrophoresis. The synthetic 2',5'-linked oligoadenylate showed no phosphodiester isomerization to 3',5' during or after synthesis. In addition, we have developed facile methodologies to characterize the chimeras using digestion with various hydrolytic enzymes including snake venom phosphodiesterase I and nuclease P1. Finally, Maxam-Gilbert chemical sequencing protocols have been developed to confirm the entire sequence of these chimeric oligonucleotides.
Xiao
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Section on Biomedical Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0805, USA.
Player
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Li
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Zhang
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Lesiak
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Torrence
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PF
D015088:2',5'-Oligoadenylate Synthetase; D000227:Adenine Nucleotides / Q000138:chemical synthesis* / Q000191:economics / Q000302:isolation & purification; D002072:Butylene Glycols / Q000737:chemistry; D003365:Costs and Cost Analysis; D016373:DNA, Antisense / Q000737:chemistry* / Q000191:economics / Q000302:isolation & purification; D004722:Endoribonucleases; D015394:Molecular Structure; D009696:Nucleic Acids / Q000138:chemical synthesis* / Q000191:economics / Q000302:isolation & purification; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis* / Q000191:economics / Q000302:isolation & purification; D009843:Oligoribonucleotides / Q000138:chemical synthesis* / Q000191:economics / Q000302:isolation & purification; D010766:Phosphorylation; D017422:Sequence Analysis, DNA
D000227:Adenine Nucleotides; D002072:Butylene Glycols; D016373:DNA, Antisense; D009696:Nucleic Acids; D016376:Oligonucleotides, Antisense; D009843:Oligoribonucleotides; C039681:1,4-butanediol; D015088:2',5'-Oligoadenylate Synthetase; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease