A novel non-radioactive assay for HIV-RT(RdDp)based on pyrosequencing for high-throughput drug screening  

作　　者：Chang Zhang Yang Wu Yuna Sun Chuan Hong Kehui Xiang Yu Guo Mark Bartlam Zhiyong Lou 

机构地区：[1]Tsinghua-Nankai-IBP Joint Research Group for Structural Biology,Tsinghua University,Beijing 100084,China [2]National Laboratory of Macromolecules,Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101,China [3]College of Life Sciences and Tianjin State Laboratory of Protein Sciences,Nankai University,Tianjin 300071,China

出　　处：《Protein & Cell》2010年第3期284-290,共7页蛋白质与细胞（英文版）

基　　金：the National Natural Science Foundation of China(Grant Nos.30221003,30720022);the Ministry of Science and Technology 973 Project(Grant No.2006CB806503);the Ministry of Science and Technology National High Technology Research and Development Program("863"Program)(Grant No.2006AA02A322);the Ministry of Science and Technology International Cooperation Project(Grant No.2006DFB32420);the Chinese Academy of Sciences Knowledge Innovation Project(Grant No.KSCX1-YW-R-05)。

摘　　要：Current in vitro assays for the activity of HIV-RT(reverse transcriptase)require radio-labeled or chemically modified nucleotides to detect reaction products.However,these assays are inherently end-point measurements and labor intensive.Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupledenzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate(PPi),which is generated during nascent strand synthesis.The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid highthroughput drug screening targeting anti-HIV therapies due to its high speed and convenience.Moreover,this assay can be used to measure primase activity in an easy and sensitive manner,which suggests that this novel approach could be wildly used to analyze the activity of PPi-generated and ATP-free enzyme reactions.

关 键 词：HIV-RT(RdDp) ASSAY non-radioactive high throughput drug screening 

分 类 号：R73[医药卫生—肿瘤]