Complementary addressed alkylation of the Escherichia coli 16S rRNA with 2',3'-O-[4-N-methyl-N-(2-chloroethyl)amino]benzylidene derivatives of oligodeoxyribonucleotides. IV. Identification of 16S rRNA binding sites with benzylidene derivative of d(pACCTTGTT)rA, d(pTTACGACT)rU, d(pTTTGCTCCCC)rA
M. A. Zenkova, G. G. Karpova, A. S. Levina, S. V. Mamaev, V. V. Soloviev
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Academy of Sciences of the USSR: * Institute of Cytology and Genetics, Siberian Division, Academy of Sciences of the USSR, Novosibirsk
Abstract: By site-directed alkylation of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA (II), d(pTTACGACT)rU (III), d(PTTTGCTCCCC)rA (IV) (reagents (II) —(TV)) followed by the RNase H treatment a number of IBS rRNA fragments have been obtained. Hybridisation of these fragments with restriction fragments of plasmid pKK 3535, containing operon rrnB of E. coli rRNAs, led to the identification of all reagents' binding sites in 16S rRNA. Good correlation is found between estimated stability of non-perfect 16S rRNA-oligodcoxyribonucleotide duplexes and the level of modification of this site with alkylating derivative of the same oligodeoxyribonucleotide. With high concentration of the reagents (II) – (IV) ((2–5)-10-5 M) the site-directed alkylation proceeds not only at the desired site but also at other sites corresponding to non-perfect duplexes between 16S rRNA and the reagents. It should be noted that the modification mainly occurs in the non-perfect duplexes, carrying mismatched bases at the termini. Influence of the secondary structure of 16S rRNA on the site-directed modification is discussed.
Russian Journal of Bioorganic Chemistry 1990, 16 (6):788-800