The synthesis and functional evaluation of RNA and DNA polymers having the sequence of Escherichia coli tRNAfMet

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Abstract
Stepwise, solid-phase chemical synthesis has provided long RNA and DNA polymers related to the sequence of Escherichia coli tRNAfMet. The 34-ribonucleotide oligomer corresponding to the sequence of the 5′-half tRNA molecule has been synthesized and then characterized by gel purification, terminal nucleotide determinations and sequence analysis. This 34-nucleotide oligomer serves as an acceptor in the RNA-ligase-catalyzed reaction with a phosphorylated 43-ribonucleotide oligomer corresponding to the sequence of the 3′-half molecule of tRNAfMet. The DNA molecule having the sequence of tRNAfMet is a 76-deoxyribonucleotide oligomer with a 3′-terminal riboadenosine residue and all U residues replaced by T. These polymers have been compared with an oligodeoxyribonucleotide lacking all 2′-hydroxyl groups except for the 3′-terminal 2′-OH, an oligoribonucleotide lacking modified nucleosides and E. coli tRNAfMet. The all-RNA 77-nucleotide oligomer can be aminoacylated by E. coli methionyl-tRNA synthetase preparation from E. coli with methionine and threonylated in the A37 position using a yeast extract. In agreement with work by Khan and Roe using tDNAPhe and tDNALys, the rA77-DNAfMet can be aminoacylated, and preliminary evidence suggests that it can be threonylated to a small extent. Kinetic data support the notion that aminoacylation of tRNAfMet does not depend on the presence of 2′-hydroxyl groups with the exception of that in the 3′-terminal nucleotide.