Bourquin, Rebecca A., Sorenson, Alanna E., and Schaeffer, Patrick M. (2025) High-performance hairpin-whiptail DNA target for HIV-1 integrase assay. International Journal of Biological Macromolecules, 318. 145195.

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Abstract

Human immunodeficiency virus (HIV) integrase is an important antiretroviral drug target. However, growing resistance towards integrase strand transfer inhibitors will need new drugs with distinct mechanisms of action. Highly efficient integrase activity assays are key tools for drug screening campaigns and the development of therapies addressing these resistance challenges. The affinity of the integrase for viral long terminal repeats LTR is well-understood. As such, integrase strand transfer activity assays commonly use a short DNA species mimicking LTR sequences. In contrast, the target site preference of the HIV integrase is far less understood. Here we examined the performance of different DNA target sequences and identified a new hairpin-whiptail structure that vastly improved the performance of a qPCR-based integrase activity assay and was also compatible with an isothermal fluorescent protein stability assay. Maximal strand transfer reaction yield could be achieved within 5 min. The hairpin-whiptail target DNA yielded an excellent Z' score of 0.758 with the qPCR-based integrase activity assay making it suitable for screening compound libraries and determination of IC<inf>50</inf> as demonstrated with elvitegravir. While the hairpin-whiptail target DNA was specifically designed for the qPCR integrase activity assay, it could easily be adapted to improve the performance of other integrase assays.

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