Li Y, Breaker R R
Department of Molecular, Cellular, and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520-8103, USA.
Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2746-51. doi: 10.1073/pnas.96.6.2746.
Nearly 50 individual DNAs with polynucleotide kinase-like activity were isolated from a random-sequence pool by using in vitro selection. Each self-phosphorylating deoxyribozyme makes use of one or more of the eight standard NTPs or dNTPs as a source of activated phosphate. Although most prototypic deoxyribozymes poorly differentiate between the ribose and deoxyribose moieties, further optimization by in vitro selection produced variants that display up to 100-fold discrimination between related NTP and dNTP substrates. An optimized ATP-dependent deoxyribozyme uses ATP >40,000-fold more efficiently than CTP, GTP, or UTP. This enzyme operates with a rate enhancement of nearly one billion-fold over the uncatalyzed rate of ATP hydrolysis. A bimolecular version of the ATP-dependent deoxyribozyme was further engineered to phosphorylate specific target DNAs with multiple turnover. The substrate-recognition patterns and rate enhancements intrinsic to these DNAs are characteristic of naturally occurring RNA and protein enzymes, supporting the hypothesis that DNA has sufficient catalytic potential to function as an enzyme in biological systems.
通过体外筛选，从一个随机序列库中分离出了近50种具有多核苷酸激酶样活性的单个DNA。每个自磷酸化脱氧核酶利用八种标准NTP或dNTP中的一种或多种作为活化磷酸盐的来源。尽管大多数原型脱氧核酶对核糖和脱氧核糖部分的区分能力较差，但通过体外筛选进行进一步优化后，产生的变体对相关NTP和dNTP底物的区分能力提高了100倍。一种优化的依赖ATP的脱氧核酶利用ATP的效率比CTP、GTP或UTP高出40000倍以上。该酶的催化速率比ATP水解的非催化速率提高了近十亿倍。依赖ATP的脱氧核酶的双分子形式经过进一步改造，可对特定靶DNA进行多次周转磷酸化。这些DNA固有的底物识别模式和速率增强是天然存在的RNA和蛋白质酶的特征，支持了DNA具有足够的催化潜力在生物系统中作为酶发挥作用的假说。
