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Enzymatic Synthesis Of Oligoribonucleotides Of Defined Base Sequence, Bronwyn Geraldine Hughes

Theses and Dissertations

A possible method for the synthesis of ordered oligoribonucleotides involves primer-dependent polynucleotide phosphorylase (PNPase) synthesis using the RNase A or T1 resistant N-cyclohexyl-N'-β(4-methylmorpholinium)ethylcarbodiimide chloride (CMC-Cl) derivatives of uridine or guanosine containing primers. Thus, CMC-UpA, CMC-GpA, CMC-UpCpC, CMC-GpApC, and ApApApApU-CMC were prepared and studied as primers for PNPase in 15 min 14C-ADP polymerization reactions and also in 6-10 hr synthesis reactions. The CMC-primers were not suitable primers for PNPase catalyzed synthesis reactions for either time. The PNPases used in such studies were contaminated with nuclease that showed the following order of susceptibility: UpA > CpC, GpA > ApU, ApA. Even a highly purified …