Total chemical synthesis of a 77-nucleotide-long RNA sequence having methionine-acceptance activity.

Abstract

Chemical synthesis is described of a 77-nucleotide-long RNA molecule that has the sequence of an Escherichi coli Ado-47-containing tRNAfMet species in which the modified nucleosides have been subsituted by their unmodified parent nucleosides. The sequence was assembled on a solid-phase, controlled-pore glass support in stepwise manner with an automated DNA synthesizer. The ribonucleotide building blocks used were fully protected 5''-monomethoxytrityl-2''-silyl-3''N,N-diisopropylaminophosphoramidites. p-Nitro-phenylethyl groups were used to protect the O6 of guanine residues. The fully deprotected tRNA analogue was characterized by polyacrylamide gel electrophoresis (sizing), terminal nucleotide analysis, sequencing, and total enzyme degradation, all of which indicated that the sequence was correct and contained only 3-5 linkages. The 77-mer was then assayed for amino acid acceptor activity by using E. coli methionyl-tRNA synthetase. The results indicated thatthe synthetic product, lacking modified bases, is a substrate for the enzyme and has an amino acid acceptance 11% of that of the major native species tRNAfMet containing 7-methylguanosine at position 47.