Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities.
Two modified 2'-deoxynucleoside 5'-triphosphates have been used for the in vitro selection of a modified deoxyribozyme (DNAzyme) capable of the sequence-specific cleavage of a 12 nt RNA target in the absence of divalent metal ions. The modified nucleotides, a C5-imidazolyl-modified dUTP and 3-(aminopropynyl)-7-deaza-dATP were used in place of TTP and dATP during the selection and incorporate two extra protein-like functionalities, namely, imidazolyl (histidine analogue) and primary amino (lysine analogue) into the DNAzyme. The functional groups are analogous to the catalytic Lys and His residues employed during the metal-independent cleavage of RNA by the protein enzyme RNaseA. The DNAzyme requires no divalent metal ions or other cofactors for catalysis, remains active at physiological pH and ionic strength and can recognize and cleave a 12 nt RNA substrate with sequence specificity. This is the first example of a functionalized, metal-independent DNAzyme that recognizes and cleaves an all-RNA target in a sequence-specific manner. The selected DNAzyme is two orders of magnitude more efficient in its cleavage of RNA than an unmodified DNAzyme in the absence of metal ions and represents a rate enhancement of 10(5) compared with the uncatalysed hydrolysis of RNA.
Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK.
Alexander V
AV
Sidorov
Jane A
JA
Grasby
David M
DM
Williams
D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D021881:DNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000737:chemistry; D019793:Fluorescein; D005456:Fluorescent Dyes; D006639:Histidine / Q000031:analogs & derivatives*; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives*; D008670:Metals / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D021881:DNA, Catalytic; D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D008670:Metals; D006639:Histidine; D012313:RNA; D008239:Lysine; D019793:Fluorescein