Chemical synthesis and biological investigation of a 77-mer oligoribonucleotide with a sequence corresponding to E. coli tRNAAsp Original Research Article

A 77-mer RNA with the sequence of Escherichia coli tRNAAsp has been chemically synthesised using standard automated phosphoramidite chemistry with the coupling reagent 4,5-dicyanoimidazole (DCI). The synthesis was carried out on a 1000 Å CPG-column and, after deprotection and gel purification, a yield of about 7 nmol with a purity of >95% was reproducibly obtained. By comparing automated synthesis of the 77-mer RNA using 1H-tetrazole and DCI as activator, DCI is advantageous in producing longer RNAs. However, for shorter RNAs (<40 mer) no difference could be observed. In addition to the all-ribo tRNAAsp carrying the wild-type sequence, two variants were synthesised, one with a single C to G48 mutation and the second with a 2′-deoxy modification at C48. The three tRNAs were tested for their aminoacylation efficiency and high affinity binding to E. coli RNase P RNA. The results demonstrate that chemically synthesised 77-mer oligoribonucleotides can be successfully used for structure–function studies.