Santoro S W, Joyce G F
Department of Chemistry, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4262-6. doi: 10.1073/pnas.94.9.4262.
An in 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 (kcat/Km) of approximately 10(9) 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-1 gag/pol, env, vpr, tat, and nef mRNAs.
一种体外筛选程序被用于开发一种DNA酶，该酶在模拟生理条件下能够切割几乎任何靶向RNA底物。该酶由一个15个脱氧核苷酸的催化结构域组成，两侧各有一个七到八个脱氧核苷酸的底物识别结构域。RNA底物通过沃森-克里克碱基配对结合，并在一个未配对嘌呤和一个配对嘧啶残基之间的特定磷酸二酯键处被切割。尽管其尺寸小，但该DNA酶在多次周转条件下的催化效率（kcat/Km）约为10(9) M-1.min-1，超过任何其他已知核酸酶。其活性依赖于Mg2+离子的存在。通过改变底物识别结构域的序列，该DNA酶可被设计用于靶向不同的RNA底物。例如，在本研究中，它被定向切割与HIV-1 gag/pol、env、vpr、tat和nef mRNA起始密码子区域相对应的合成RNA。
