Enzymatic synthesis of oligoribonucleotides of defined base sequence

• Main Author: Rowe, Mark J.
• Format: Others
• Published: BYU ScholarsArchive 1972
• Subjects: Nucleotides; Chemistry
• Online Access: https://scholarsarchive.byu.edu/etd/8344; https://scholarsarchive.byu.edu/cgi/viewcontent.cgi?article=9344&context=etd

The synthetic reaction of ribonuclease T_1 has been studied as to its potential for the synthesis of oligoribonucleotides of defined base sequence. Conditions have been determined for maximum synthesis with respect to percentage incorporation of both guanosine 2',3'-cyclic phosphate (donor), and cytidine (acceptor). With regard to maximum acceptor incorporation, a donor blocked at the 5'-hydroxyl has been used to drive the reaction. It has also been shown that acceptor incorporation decreases as the chain-length increases up to the hexanucleotide. Synthesis of a 3'-blocked substrate for polynucleotide phosphorylase for use in chain termination studies was attempted using adenylate kinase. In this regard, adenosine 2'(3'),5'-diphosphate was shown to be a noncompetitive inhibitor with respect to AMP, and a competitive inhibitor with respect to ATP, of adenylate kinase. Polynucleotide phosphorylase was incubated with a protein kinase and a protein phosphatase in an attempt to exploit a postulated phosphorylation- dephosphorylation control mechanism for the production of a primer dependent preparation. No reaction was observed.