Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates.
Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.
Vorobjev
Pavel E
PE
Laboratory of Nucleic Acid Chemistry, Institute of Bioorganic Chemistry, Russian Academy of Sciences, Novosibirsk 630090, Russia.
Smith
Janet B
JB
Pyshnaya
Inna A
IA
Levina
Asya S
AS
Zarytova
Valentina F
VF
Wickstrom
Eric
E
D001665:Binding Sites; D001761:Bleomycin / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D018076:DNA, Complementary; D006801:Humans; D015394:Molecular Structure; D012313:RNA / Q000378:metabolism*; D013379:Substrate Specificity
D018076:DNA, Complementary; D001761:Bleomycin; D012313:RNA; D004247:DNA