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Sequence-specific alkylation of 16S rRNA Escherichia coli with 2',3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]-benzylidene derivative of oligodeoxyribonucleotides. V. Investigation of the factors affecting the selectivityof modification
M. A. Zenkova, G. G. Karpova, A. S. Levina, S. V. Mamaev, Yu. N. Nazarova, O. S. Fedorova
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Academy of Sciences of the USSR
Abstract: We studied the effect of different factors (reagent concentration, temperature, presence of oligonucleotide-effector (3',5'-diphenazinium derivative of oligodeoxyribomicleotide) stabilizing duplex RNA-reagent) on the selectivity of the site-directed modification of 16S rRNA with 2,3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]-benzylidene derivative of oligonucleotide p(dTTTGCTCCCC)rA (reagent I) under conditions of secondary structure stability. The constant of cooperative binding of the reagent and oligonucleotide-effector with 16S rRNA was determined. The temperature rise from 20 to 40°C brought about a 1.5-fold increase in the relative extent of modification at the target site 771–781. In the presence of oligonucleotide-effector, which is a full complementary copy of the 782–789 fragment of 16S rRNA (reagent concentration is 1 10-6M), the selectivity of the RNA modification at the target site is doubled and a high level of the modification is retained. When the reagent concentration in the reaction mixture was decreased down to 1·10-7M, the same level of selectivity was achieved without the oligonucleotide-effector. Under these conditions, however, a drastic (20-fold), drop of the level of the 16S rRNA alkylation was observed.
Russian Journal of Bioorganic Chemistry 1991, 17 (4):470-481