STUDIES ON NUCLEIC ACIDS: I. THE USE OF T4 RNA LIGASE TO JOIN CHEMICALLY SYNTHESIZED OLIGO DEOXYRIBONUCLEOTIDES. II. CHEMICAL SYNTHESIS AND PURIFICATION OF PHASING PRIMERS

GREGORY KELLY, Purdue University

Abstract

Reaction conditions permitting the joining of chemically synthesized oligodeoxyribonucleotides using T4 RNA ligase have been developed. Reaction yields for the joining of a 3' ribonucleoside terminated oligodeoxyribonucleotide acceptor molecule to an oligodeoxyribonucleotide donor molecule were routinely 50-80% in 16 to 24 hours. Successful joining reactions were found to depend on the preparation of T4 RNA ligase used. The product yields for the enzymatic joining of a hexanucleotide acceptor to a decanucleotide donor was examined as a function of pH, substrate concentrations, T4 RNA ligase concentration and ATP concentration. A pancreatic ribonuclease digestion of the RNA ligase joining reaction product was used to confirm the presence and position of a ribonucleotide linkage. Several variations of the standard acceptor 5' terminus protecting group, 2',3'-O-methoxymethylidine uridine, were examined for their effect on the production of the RNA ligase joining reaction product. The feasibility of using T4 RNA ligase to produce single-stranded polynucleotides by linking chemically synthesized oligonucleotides is discussed in light of preliminary experiments on the purification of the reaction products. The phasing primer pT(pT)(,8)pdG was synthesized using a diester polycondensation reaction. The reaction products were treated with ethanedial, which adds a cis-hydroxyl system to guanosine. Guanosine terminated oligonucleotides were separated from the remaining portions of the reaction products with a phenyl boronate substituted polyacrylamide resin. The major product from a triester polycondensation reaction was shown to be 3',5' cyclic thymidine monophosphate.

Degree

Ph.D.

Subject Area

Biochemistry

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