Total Synthesis of the Structural Gene for the Precursor of a Tyrosine Suppressor Transfer RNA from Escherichia coli 8. ENZYMATIC JOINING OF THE CHEMICALLY SYNTHESIZED SEGMENTS TO FORM DNA DUPLEXES

Polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxyribopolynucleotide segments (Fig. 1) comprising the nucleotide sequence 23-66 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been systematically investigated. Joining was studied using all possible combinations of 3, 4, and 5 and larger numbers of segments at a time. The extent of joining varied widely (0 to about 90%) in three component systems. The “self-structure” of some of the components evidently inhibited the joining. Addition of a fourth segment in general enhanced the extent of joining and optimal yields were obtained in systems containing six or more segments. A comparison of the T,induced ligase and the E. coli polynucleotide ligase for joining of the chemically synthesized segments showed the E. coli enzyme to be inferior to the T,-induced ligase. Satisfactory syntheses of the duplexes [IIa] and [IIb] comprising, respectively, eight and seven segments were achieved in single steps. Of the two terminal segments carrying 5’.OH groups in the duplexes, only one (segment 7) was used in the prephosphorylated form. The duplexes were isolated pure and characterized by enzymatic degradations and by electrophoresis.