RNA cleavage by a DNA enzyme with extended chemical functionality.
In vitro selection techniques were applied to the development of a DNA enzyme that contains three catalytically essential imidazole groups and catalyzes the cleavage of RNA substrates. Nucleic acid libraries for selection were constructed by polymerase-catalyzed incorporation of C5-imidazole-functionalized deoxyuridine in place of thymidine. Chemical synthesis was used to define a minimized catalytic domain composed of only 12 residues. The catalytic domain forms a compact hairpin structure that displays the three imidazole-containing residues. The enzyme can be made to cleave RNAs of almost any sequence by simple alteration of the two substrate-recognition domains that surround the catalytic domain. The enzyme operates with multiple turnover in the presence of micromolar concentrations of Zn2+, exhibiting saturation kinetics and a catalytic rate of >1 min-1. The imidazole-containing DNA enzyme, one of the smallest known nucleic acid enzymes, combines the substrate-recognition properties of nucleic acid enzymes and the chemical functionality of protein enzymes in a molecule that is small, yet versatile and catalytically efficient.
Santoro
S W
SW
Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, USA.
Joyce
G F
GF
Sakthivel
K
K
Gramatikova
S
S
Barbas CF 3rd
D001482:Base Composition; D001483:Base Sequence; D002384:Catalysis; D002413:Cations, Divalent; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D003857:Deoxyuridine / Q000031:analogs & derivatives*; D004722:Endoribonucleases / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry*; D007700:Kinetics; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016337:RNA, Catalytic; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry*
D002413:Cations, Divalent; D007093:Imidazoles; D008670:Metals; D016337:RNA, Catalytic; D012313:RNA; D004247:DNA; D004722:Endoribonucleases; D015032:Zinc; D003857:Deoxyuridine