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Proceedings Paper

Quenched near-infrared fluorescent peptide substrate for HIV-1 protease assay
Author(s): Xinzhan Peng; Daniel R. Draney; William M. Volcheck
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Paper Abstract

The HIV-1 protease enzyme is an excellent target for drug therapy of HIV infection/AIDS. To measure the protease activity and screen for potent protease inhibitors, homogeneous protease assays based on quenched fluorescent peptide substrates have been widely used as a high-throughput screening methods. The major problem in these assays is the compound interference or assay artifacts from colored or insoluble materials in the assay, e.g. assay components, screening library compounds, etc. We report in this paper a near-infrared fluorescence resonance energy transfer (NIRFRET) based HIV-1 protease assay that can dramatically reduce or completely eliminate these assay artifacts by using a novel near-IR donor-quencher pair and long wavelength excitation (780 nm) and detection (820±10 nm). In this assay, a HIV-1 protease peptide substrate is conjugated with a near-IR fluorescent donor (IRDyeTM 800CW), and a novel near-IR non-fluorescent quencher (QC1) on opposite sides of the proteolytic cleavage site. The quencher, QC1, has extremely good spectral overlap of its absorption spectrum with the donor emission spectrum to ensure the efficient quenching of the donor's fluorescence. In the HIV-1 protease assay, this NIR-FRET system shows a large dynamic range, high signal to noise ratio, excellent Z'-factors, a wide range of DMSO tolerance, and no compound interference. This system provides a sensitive, robust assay for high-throughput screening (HTS) and can be readily adapted to other therapeutically significant protease targets.

Paper Details

Date Published: 14 February 2006
PDF: 12 pages
Proc. SPIE 6097, Optical Molecular Probes for Biomedical Applications, 60970F (14 February 2006); doi: 10.1117/12.669174
Show Author Affiliations
Xinzhan Peng, LI-COR Biosciences (United States)
Daniel R. Draney, LI-COR Biosciences (United States)
William M. Volcheck, LI-COR Biosciences (United States)

Published in SPIE Proceedings Vol. 6097:
Optical Molecular Probes for Biomedical Applications
Samuel I. Achilefu; Darryl J. Bornhop; Ramesh Raghavachari, Editor(s)

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