title: A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening.
abstract: 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 coupled-enzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate (PP(i)), 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 high-throughput 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 PP(i)-generated and ATP-free enzyme reactions.
authors.affiliation: Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Tsinghua University, Beijing 100084, China.
authors.forename: Chang
authors.initials: C
authors.lastname: Zhang
authors.forename: Yang
authors.initials: Y
authors.lastname: Wu
authors.forename: Yuna
authors.initials: Y
authors.lastname: Sun
authors.forename: Chuan
authors.initials: C
authors.lastname: Hong
authors.forename: Kehui
authors.initials: K
authors.lastname: Xiang
authors.forename: Yu
authors.initials: Y
authors.lastname: Guo
authors.forename: Mark
authors.initials: M
authors.lastname: Bartlam
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Lou
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology; D003124:Colorimetry; D011756:Diphosphates / Q000032:analysis / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D006678:HIV / Q000187:drug effects / Q000201:enzymology*; D054303:HIV Reverse Transcriptase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D019829:Nevirapine / Q000494:pharmacology; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: D019380:Anti-HIV Agents; D011756:Diphosphates; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D019829:Nevirapine; D054303:HIV Reverse Transcriptase; C024157:thymidine 5'-triphosphate