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Sensors 2013, 13(12), 16330-16346; doi:10.3390/s131216330
Article

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

1,2
,
1,2
,
1,2
 and
1,2,*
1 Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia 2 Center of Excellence EN-FIST, Ljubljana 1000, Slovenia
* Author to whom correspondence should be addressed.
Received: 18 October 2013 / Revised: 11 November 2013 / Accepted: 13 November 2013 / Published: 28 November 2013
(This article belongs to the Special Issue Fluorescent Biosensors)
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Abstract

To effectively fight against the human immunodeficiency virus infection/ acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity.
Keywords: mCerulean-mCitrine FRET-HIV protease sensor; ratiometric flow cytometry; sensitize emission FRET mCerulean-mCitrine FRET-HIV protease sensor; ratiometric flow cytometry; sensitize emission FRET
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Gaber, R.; Majerle, A.; Jerala, R.; Benčina, M. Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry. Sensors 2013, 13, 16330-16346.

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