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 Escherichia coli Ado47-containing tRNAP41t species in which the modified nucleosides have been substituted by their unmodified parent nucleosides. The sequence was assembled on a solid-phase, controlled-pore glass support in a stepwise manner with an automated DNA synthesizer. The ribonucleotide building blocks used were fully protected 5'-monomethoxytrityl-2'-silyl-3'-N,N-diisopropylaminophosphoramidites. p-Nitrophenylethyl groups were used to protect the o' 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 that the 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, tRNA e't containing 7-methylguanosine at position 47.Ribonucleic acids (RNAs) play a wide range of important roles in living cells. These include the traditional roles in transcription and translation (mRNA, tRNA, and rRNA) of genetic information. Excitement in RNA chemistry has grown with the discovery of RNA sequences with catalytic activity (1), the formation of "lariats" during the processing of pre-RNA (2), and the development of recombinant RNA technology (3, 4). RNA sequences of the tRNA type are required in the first step of chlorophyll biosynthesis (5). tRNAs are particularly attractive as synthetic targets because their length, 70-85 nucleotides, is sufficient to demonstrate a successful chemical procedure. In addition, they have specific biological functions that can be demonstrated in the synthetic product. They are also interesting molecules in their own right, having been implicated in gene expression (6), in propagation of tumor viruses (7), and in cellular differentiation and development in cancer induction (8-10). The chemical synthesis of RNA molecules allows complete flexibility in the choice of sequence and the ability to introduce modifications at specific sites. Thus, the ability to chemically synthesize molecules of this size should permit a rapid increase in the rate of understanding of the full biological roles of RNA molecules paralleling that which occurred earlier when the chemical synthesis of DNA oligomers became routine.Our laboratory has had as a major objective the development of methods for the chemical synthesis ofRNA sequence (11,12). These methods have now progressed to the point where the synthesis of 70-to 80-unit molecules is a practical possibility. To demonstrate the chemical method, a sequence was chosen that was identical to that of an Escherichia coli Ado-47 tRNAP et species (i.e., containing adenosine instead of 7-methylguanosine at position 47), except th...

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

“…Although the aminoacylation ability of the synthetic RNA is of the order of 3% of the native tRNA, this value compares with the values of aminoacylation of semisynthetic tRNA (4% [20]) and the synthetic RNA which we reported previously (6 -10% [6]). The small amount of available ligRNA precluded a full kinetic study of this molecule, and a new sample of synRNA gave an activity which was higher than previously found [6].…”

“…239 pmol/AZ6,, when analyzed in either Tris or Hepes buffer, both rA77-tDNA and ligRNA were most active in Hepes buffer (13.8 and 7.1 pmol/A260 unit, respectively). The lower activity of ligRNA compared to synRNA reported here (however see [6]) could be due to chemical heterogeneity in the ligRNA which was prepared from the 43-nucleotide oligomer, the first long RNA oligomer synthesized. Although the charging levels are low (3 -6% of the authentic sample), this value was consistently found with different samples.…”

“…The chemical synthesis of the 43-nucleotide oligomer, representing the 3'-half molecule, has been published previously [9], as well as the 77-nucleotide oligomer (synRNA) having the complete sequence of tRNAfM"' [6]. The 34-nucleotide oligomer reported here was synthesized using the same stepwise solid-phase method [6, 91.…”

“…These procedures have also been extended recently to allow the chemical synthesis of long oligoribonucleotides including a molecule the length of a tRNA molecule [6]. In the present study, we describe how these techniques have allowed the preparation of two analogue sequences of Escherichia coli tRNAfMe", a 77-nucleotide-long RNA, by the ligation of two chemically synthesized half-molecules (ligated synthetic tRNA = IigRNA) and a 77-nucleotide-long DNA/ RNA hybrid of which the first 76 positions are filled with deoxyribonucleotides, every U in the RNA sequence is substituted by T, and the terminal 3' position is occupied by riboadenosine (rA77-tDNA).…”

Preliminary X-ray diffraction studies of an RNA pseudoknot that inhibits HIV-1 reverse transcriptase

“…Each preparation of PK26 began with four 1 ~mol column syntheses and used phosphoramidite chemistry (Ogilvie, Usman, Nicoghosian & Cedergren, 1988). The same procedures were followed for the synthesis and purification of © 1996 International Union of Crystallography Printed in Great Britain -all rights reserved the unmodified PK26 and for the molecules containing a 5-bromo-deoxyuridine at the first uridine (PKIdBr) or a 5-bromo-uridine nucleotide at the eighth or 21 st positions (PK8Br and PK2 I Br, respectively).…”