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SYBR Green II Dye-Based Real-Time Assay for Measuring Inhibitor Activity Against HIV-1 Reverse Transcriptase

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Abstract

There are arrays of in vitro assays to quantify the activity of HIV-1 reverse transcriptase (HIV-1 RT). These assays utilize either chemically customized/labelled nucleotides, or TaqMan probes, or radiolabeled nucleotides/primers. Although several real-time PCR assays exist commercially for measuring the RT activity, which are usually used for quantifying the viral titres, these assays are not optimized for measuring the inhibitory concentrations (IC50) of HIV-1 RT inhibitors. Moreover, a recently established inorganic pyrophosphate-coupled enzyme assay cannot be employed for studying nonphosphorylated nucleoside reverse transcriptase inhibitors (NRTIs). In the present study, we have developed a novel one-step assay with native nucleotide substrates and SYBR Green II dye to determine IC50 values of triphosphorylated NRTIs against HIV-1 RT. Using exact batches of wild-type and mutant RT, and triphosphorylated NRTIs, we showed that our method gave IC50 values for inhibitors similar to that of an earlier published colorimetric assay with BrdUTP substrate (CABS). Our assay should be suitable for high-throughput screening of antiretroviral drugs and could also be suitable for studying drug resistance profiles. Additionally, we also used our assay to study inhibition by AZT in its nonphosphorylated form by supplementing the reaction mixture with necessary kinases and ATP.

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Acknowledgments

We thank Sarah Lam for her grammatical assistance. This work was supported in part by the Swedish Physicians against AIDS Research Fund.

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Author notes

  1. Chakradhar Kokkula

    Present address: Bharat Biotech International Limited, Genome Valley Shameerpet, Hyderabad, Telangana, 500078, India

  2. Navaneethan Palanisamy

    Present address: Synthetic Biology Group, HBIGS, University of Heidelberg, BioQuant, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany

Authors and Affiliations

  1. Section of Virology, Department of Medical Sciences, Uppsala University, Dag Hammarskjölds väg 17, 75185, Uppsala, Sweden

    Chakradhar Kokkula, Navaneethan Palanisamy, Malin Ericstam & Johan Lennerstrand

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  1. Chakradhar Kokkula
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  2. Navaneethan Palanisamy
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  4. Johan Lennerstrand
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Correspondence to Johan Lennerstrand.

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Conflict of Interest

J. L. acts as a consultant to Cavidi AB. C. K. is currently an employee at Bharat Biotech International Limited. But, these companies are not involved in this project in any ways.

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Chakradhar Kokkula and Navaneethan Palanisamy have Contributed equally to the work.

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Kokkula, C., Palanisamy, N., Ericstam, M. et al. SYBR Green II Dye-Based Real-Time Assay for Measuring Inhibitor Activity Against HIV-1 Reverse Transcriptase. Mol Biotechnol 58, 619–625 (2016). https://doi.org/10.1007/s12033-016-9961-y

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  • DOI: https://doi.org/10.1007/s12033-016-9961-y

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