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https://doi.org/10.1093/nar/gkv1532
Title: | FRET-based assay to screen inhibitors of HIV-1 reverse transcriptase and nucleocapsid protein | Authors: | Sharma, Kamal K Przybilla, Frederic Restle, Tobias Godet, Julien Mely, Yves |
Keywords: | Science & Technology Life Sciences & Biomedicine Biochemistry & Molecular Biology HUMAN-IMMUNODEFICIENCY-VIRUS DNA-POLYMERASE ASSAY IN-VITRO ANTIRETROVIRAL THERAPY ANGSTROM RESOLUTION CRYSTAL-STRUCTURE SINGLE-MOLECULE AIDS RESEARCH STRANDED-DNA TYPE-1 |
Issue Date: | 5-May-2016 | Publisher: | OXFORD UNIV PRESS | Citation: | Sharma, Kamal K, Przybilla, Frederic, Restle, Tobias, Godet, Julien, Mely, Yves (2016-05-05). FRET-based assay to screen inhibitors of HIV-1 reverse transcriptase and nucleocapsid protein. NUCLEIC ACIDS RESEARCH 44 (8). ScholarBank@NUS Repository. https://doi.org/10.1093/nar/gkv1532 | Abstract: | During HIV-1 reverse transcription, the single-stranded RNA genome is converted into proviral double stranded DNA by Reverse Transcriptase (RT) within a reverse transcription complex composed of the genomic RNA and a number of HIV-1 encoded proteins, including the nucleocapsid protein NCp7. Here, we developed a one-step and one-pot RT polymerization assay. In this in vitro assay, RT polymerization is monitored in real-time by Förster resonance energy transfer (FRET) using a commercially available doubly-labeled primer/template DNA. The assay can monitor and quantify RT polymerization activity as well as its promotion by NCp7. Z-factor values as high as 0.89 were obtained, indicating that the assay is suitable for high-throughput drug screening. Using Nevirapine and AZT as prototypical RT inhibitors, reliable IC50 values were obtained from the changes in the RT polymerization kinetics. Interestingly, the assay can also detect NCp7 inhibitors, making it suitable for high-throughput screening of drugs targeting RT, NCp7 or simultaneously, both proteins. | Source Title: | NUCLEIC ACIDS RESEARCH | URI: | https://scholarbank.nus.edu.sg/handle/10635/242858 | ISSN: | 0305-1048 1362-4962 |
DOI: | 10.1093/nar/gkv1532 |
Appears in Collections: | Elements Staff Publications |
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