Abstract

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.

Degree

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

http://lib.byu.edu/about/copyright/