Enhancement of Probe Signal for Screening of HIV-1 Protease Inhibitors in Living Cells.docVIP

Sensors 2012, 12, 16759-16770; doi:10.3390/s121216759 OPEN ACCESS sensors ISSN 1424-8220 /journal/sensors Article Enhancement of Probe Signal for Screening of HIV-1 Protease Inhibitors in Living Cells Huantong Yao and Sha Jin * Department of Biomedical Engineering, College of Engineering, 4188 Bell Engineering, University of Arkansas, Fayetteville, AR 72701, USA; E-Mail: hxy009@ * Author to whom correspondence should be addressed; E-Mail: sjin@; Tel.: +1-479-575-5298; Fax: +1-479-575-7696. Received: 9 October 2012; in revised form: 18 November 2012 / Accepted: 30 November 2012 / Published: 6 December 2012 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 F?rster 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 devel