INCORPORATION OF 2'-MODIFIED NUCLEOTIDES INTO TRANSFER-RNA TO TEST IMPORTANCE FOR CHARGING

Incorporation of 2'-deoxy nucleotides into tRNA is described using two complementary strategies: (a) in vitro transcription with a mutant T7 RNA polymerase and (b) enzymatic Ligation of chemically synthesised t RNA fragments. The former permits incorporation at a low to a moderate substitution level for all nucleotides while the latter allows a comp lete exchange of ribonucleotides to deoxynucleotides as well as incorp oration of point mutations. Both methods can be used for studying the influence of 2'-hydroxy groups at specific positions important for tRN A aminoacyl synthetase recognition.