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 ...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.展开更多
基金the National Natural Science Foundation of China(Grant Nos.30221003,30720022)the Ministry of Science and Technology 973 Project(Grant No.2006CB806503)+2 种基金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)。
文摘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.
