Ting Richard, Thomas Jason M, Lermer Leonard, Perrin David M
Chemistry Department, 2036 Main Mall, University of British Columbia, Vancouver, BC V6T 1Z1, Canada.
Nucleic Acids Res. 2004 Dec 29;32(22):6660-72. doi: 10.1093/nar/gkh1007. Print 2004.
This work addresses the binding, cleavage and dissociation rates for the substrate and products of a synthetic RNaseA mimic that was combinatorially selected using chemically modified nucleoside triphosphates. This trans-cleaving DNAzyme, 9(25)-11t, catalyzes sequence-specific ribophosphodiester hydrolysis in the total absence of a divalent metal cation, and in low ionic strength at pH 7.5 and in the presence of EDTA. It is the first such sequence capable of multiple turnover. 9(25)-11t consists of 31 bases, 18 of which form a catalytic domain containing 4 imidazole and 6 allylamino modified nucleotides. This sequence cleaves the 15 nt long substrate, S1, at one embedded ribocytosine at the eighth position to give a 5'-product terminating in a 2',3'-phosphodiester and a 3'-product terminating in a 5'-OH. Under single turnover conditions at 24 degrees C, 9(25)-11t displays a maximum first-order rate constant, k(cat), of 0.037 min(-1) and a catalytic efficiency, k(cat)/K(m), of 5.3 x 10(5) M(-1) min(-1). The measured value of k(cat) under catalyst excess conditions agrees with the value of k(cat) observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity of 9(25)-11t was gauged in terms of k(cat) values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease k(cat) values by a factor of 4 to 250, indicating that 9(25)-11t displays significant sequence specificity despite the lack of an apparent Watson-Crick base-pairing scheme for recognition.
这项工作研究了一种合成核糖核酸酶A模拟物的底物和产物的结合、切割和解离速率，该模拟物是使用化学修饰的核苷三磷酸通过组合筛选得到的。这种反式切割DNA酶9(25)-11t在完全不存在二价金属阳离子的情况下，在pH 7.5的低离子强度以及存在乙二胺四乙酸（EDTA）的条件下，催化序列特异性的核糖磷酸二酯水解。它是第一个能够进行多次周转的此类序列。9(25)-11t由31个碱基组成，其中18个形成一个催化结构域，包含4个咪唑和6个烯丙基氨基修饰的核苷酸。该序列在位于第八位的一个嵌入核糖胞嘧啶处切割15个核苷酸长的底物S1，产生一个以2',3'-磷酸二酯结尾的5'产物和一个以5'-OH结尾的3'产物。在24摄氏度的单周转条件下，9(25)-11t显示出最大一级速率常数k(cat)为0.037 min⁻¹，催化效率k(cat)/K(m)为5.3×10⁵ M⁻¹ min⁻¹。在催化剂过量条件下测得的k(cat)值与稳态多次周转时观察到的k(cat)值一致，这意味着就多次周转而言，缓慢的产物释放不是限速步骤。9(25)-11t的底物特异性通过底物序列变体的k(cat)值来衡量。在可切割核糖和紧接下游的两个碱基上进行碱基替换会使k(cat)值降低4至250倍，这表明尽管缺乏明显的沃森-克里克碱基配对识别方案，但9(25)-11t仍表现出显著的序列特异性。
