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

Chang Zhang1,Chuan Hong1,Kehui Xiang1,Zhiyong Lou1,Yang Wu2,Yuna Sun2,Yu Guo3,Mark Bartlam3,

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Protein Cell ›› 2010, Vol. 1 ›› Issue (3) : 284-290. DOI: 10.1007/s13238-010-0031-0
Research articles

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

  • Chang Zhang1,Chuan Hong1,Kehui Xiang1,Zhiyong Lou1,Yang Wu2,Yuna Sun2,Yu Guo3,Mark Bartlam3,
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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 (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 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 PPi-generated and ATP-free enzyme reactions.

Keywords

HIV-RT (RdDp) / assay / non-radioactive / high throughput drug screening

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Chang Zhang, Chuan Hong, Kehui Xiang, Zhiyong Lou, Yang Wu, Yuna Sun, Yu Guo, Mark Bartlam,. A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening. Protein Cell, 2010, 1(3): 284‒290 https://doi.org/10.1007/s13238-010-0031-0
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