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 trypsin treated PNPase had nuclease. A method for the removal of nuclease was not found. CMC-Cl and CMC-UDP exhibited a mixed type of inhibition for PNPase unless low inhibitor or high substrate concentrations were plotted exclusively, and then uncompetitive inhibition resulted. CMC-UDP did not form either homopolymer or copolymer with unmodified UDP.
College and Department
Chemistry and Biochemistry
BYU ScholarsArchive Citation
Hughes, Bronwyn Geraldine, "Enzymatic synthesis of oligoribonucleotides of defined base sequence" (1972). Theses and Dissertations. 8235.