Chemical synthesis and biological investigation of a 77-mer oligoribonucleotide with a sequence corresponding to E-coli tRNA(Asp)

A 77-mer RNA with the sequence of Escherichia coli tRNA(ASp) has been chemi cally synthesised using standard automated phosphoramidite chemistry with t he coupling reagent 4,5-dicyanoimidazole (DCI). The synthesis was carried o ut on a 1000 A CPG-column and, after deprotection and gel purification, a y ield of about 7 nmol with a purity of >95% was reproducibly obtained. By co mparing automated synthesis of the 77-mer RNA using 1H-tetrazole and DCI as activator, DCI is advantageous in producing longer RNAs. However, for shor ter 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 modifi cation at C48. The three tRNAs were tested for their aminoacylation efficie ncy and high affinity binding to E. coli RNase P RNA. The results demonstra te that chemically synthesised 77-mer oligoribonucleotides can be successfu lly used for structure-function studies. (C) 2000 Elsevier Science Ltd. All rights reserved.