The regioisomeric integrity of the internucleotide phosphate linkage in synthetic RNA using 2′-tertbutyldimethylsilyl protection was examined using enzymatic and NMR techniques. Two sets of DNA-RNA hybrid nonamers, T3XT5 and T5XT3 (where X = rA, rC, rG and U) and the tetramer AGCU were analyzed. Enzyme catalyzed hydrolysis of the nonamers with ribonuclease T2 showed that the linkage at the ribonucleotide was the desired 3′-5′. A control nonamer with a 2′-5′ linkage was subjected to the enzyme, and showed no cleavage. High-resolution proton NMR of the tetramer also gave a favorable comparison with the same molecule obtained by non-chemical means.
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