Abstract
The global human immunodeficiency virus infection/acquired immuno-deficiency syndrome (HIV/AIDS) epidemic is one of the biggest threats to human life. Mutation of the virus and toxicity of the existing drugs necessitate the development of new drugs for effective AIDS treatment. Previously, we developed a molecular probe that utilizes the Förster resonance energy transfer (FRET) principle to visualize HIV-1 protease inhibition within living cells for drug screening. We explored using AcGFP1 (a fluorescent mutant of the wild-type green fluorescent protein) as a donor and mCherry (a mutant of red fluorescent protein) as an acceptor for FRET microscopy imaging measurement of HIV-1 protease activity within living cells and demonstrated that the molecular probe is suitable for the High-Content Screening (HCS) of anti-HIV drugs through an automated FRET microscopy imaging measurement. In this study, we genetically engineered a probe with a tandem acceptor protein structure to enhance the probe’s signal. Both in vitro and in vivo studies revealed that the novel structure of the molecular probe exhibits a significant enhancement of FRET signals, reaching a probe FRET efficiency of 34%, as measured by fluorescence lifetime imaging microscopy (FLIM) measurement. The probe developed herein would enable high-content screening of new anti-HIV agents.
Highlights
Human immunodeficiency virus (HIV) was discovered in the early 1980s and has been responsible for an estimated 25 million deaths
We anticipated a shortened distance between the donor and acceptors of the Förster resonance energy transfer (FRET) pair, and the two acceptors would lead to enhanced energy transfer efficiency, which will subsequently lead to an amplified FRET signal for screening anti-HIV protease agents
The effect of HIV-1 protease concentration on normalized FRET signal was examined by adjusting protease concentrations up to 45 ng per mL in the reaction buffer, while the concentration of the probe protein GcCC was held constant at 50 μg per mL
Summary
Human immunodeficiency virus (HIV) was discovered in the early 1980s and has been responsible for an estimated 25 million deaths. (a fluorescent mutant of the wild-type green fluorescent protein) served as a donor and mCherry acted as an acceptor for FRET microscopy imaging measurement of HIV-1 protease activity inhibitors within living cells [17]. We demonstrated that AcGFP1 is more tolerant of photobleaching, which makes quantitative analysis using AcGFP1 more reliable, and the molecular probe designated as GcC is suitable for high-content screening (HCS) of anti-HIV drugs through an automated FRET microscopy imaging measurement [17]. We genetically engineered a probe with a tandem acceptor protein structure and designated it as GcCC Both in vitro and in vivo studies demonstrated that the novel structure of the molecular probe exhibits a significant enhancement in FRET signals. The FRET efficiency of the probe developed increased greatly, as measured by fluorescence lifetime imaging microscopy measurement (FLIM)
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