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.