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 Eschlerichia coli tRNA(Asp) 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 A CPG-column and. after deprotection and gel purification, a yield of about 7 mmol 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 tRNA(Asp) 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.
Persson
T
T
Max-Planck Institut für Experimentelle Medizin, Göttingen, Germany. persson@biochem.mu-luebeck.de
Kutzke
U
U
Busch
S
S
Held
R
R
Hartmann
R K
RK
D004926:Escherichia coli / Q000235:genetics*; D007093:Imidazoles; D007700:Kinetics; D009154:Mutation; D009843:Oligoribonucleotides / Q000138:chemical synthesis* / Q000235:genetics; D012343:RNA, Transfer / Q000138:chemical synthesis*; D012349:RNA, Transfer, Asp / Q000737:chemistry* / Q000235:genetics
C110830:4,5-dicyanoimidazole; D007093:Imidazoles; D009843:Oligoribonucleotides; D012349:RNA, Transfer, Asp; D012343:RNA, Transfer