Open AccessA general purpose RNA-cleaving DNA enzyme
Author(s) -
Stephen Santoro,
Gerald F. Joyce
Publication year - 1997
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.94.9.4262
Subject(s) - phosphodiester bond , rna , deoxyribozyme , cleave , dna , nucleic acid , enzyme , biochemistry , base pair , nucleotide , biology , chemistry , stereochemistry , microbiology and biotechnology , gene
Anin vitro selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson–Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (k cat /K m ) of ≈109 M−1 ⋅min−1 under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg2+ ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1gag /pol ,env ,vpr ,tat , andnef mRNAs.