Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry
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 poten...
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| Format: | Article |
| Language: | English |
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MDPI AG
2013-11-01
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| Series: | Sensors |
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| Online Access: | http://www.mdpi.com/1424-8220/13/12/16330 |
| _version_ | 1811302176764461056 |
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| author | Rok Gaber Andreja Majerle Roman Jerala Mojca Benčina |
| author_facet | Rok Gaber Andreja Majerle Roman Jerala Mojca Benčina |
| author_sort | Rok Gaber |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T07:23:20Z |
| format | Article |
| id | doaj.art-4323bef6851647b28755479129fe3123 |
| institution | Directory Open Access Journal |
| issn | 1424-8220 |
| language | English |
| last_indexed | 2024-04-13T07:23:20Z |
| publishDate | 2013-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Sensors |
| spelling | doaj.art-4323bef6851647b28755479129fe31232022-12-22T02:56:33ZengMDPI AGSensors1424-82202013-11-011312163301634610.3390/s131216330s131216330Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow CytometryRok Gaber0Andreja Majerle1Roman Jerala2Mojca Benčina3Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, SloveniaLaboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, SloveniaLaboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, SloveniaLaboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, SloveniaTo 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.http://www.mdpi.com/1424-8220/13/12/16330mCerulean-mCitrine FRET-HIV protease sensorratiometric flow cytometrysensitize emission FRET |
| spellingShingle | Rok Gaber Andreja Majerle Roman Jerala Mojca Benčina Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry Sensors mCerulean-mCitrine FRET-HIV protease sensor ratiometric flow cytometry sensitize emission FRET |
| title | Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry |
| title_full | Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry |
| title_fullStr | Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry |
| title_full_unstemmed | Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry |
| title_short | Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry |
| title_sort | noninvasive high throughput single cell analysis of hiv protease activity using ratiometric flow cytometry |
| topic | mCerulean-mCitrine FRET-HIV protease sensor ratiometric flow cytometry sensitize emission FRET |
| url | http://www.mdpi.com/1424-8220/13/12/16330 |
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