4.8 Article

Sensitive detection of endonuclease activity and inhibition using gold nanorods

Journal

BIOSENSORS & BIOELECTRONICS
Volume 34, Issue 1, Pages 144-150

Publisher

ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2012.01.034

Keywords

EcoRI endonuclease; Gold nanorods; Fluorescence resonance energy transfer; Activity; Inhibition; Endonuclease inhibitor

Funding

  1. National Natural Science Foundation of China [21075079]
  2. Program for New Century Excellent Talents in University [NCET-10-0557]
  3. Fundamental Research Funds for the Central Universities [GK200902004]
  4. Program for Changjiang Scholars and Innovative Research Team in University [IRT 1070]

Ask authors/readers for more resources

It is important to develop reliable and sensitive methods for assay of nuclease activity. With this goal in mind, we report a new strategy for nuclease assay by taking advantage of efficient fluorescence resonance energy transfer (FRET) between gold nanorods (GNRs) and fluorescein-tagged single-stranded DNA (FDNA). Upon mixing with GNRs, the FRET between positively charged GNRs and negatively charged FDNA caused a decrease in fluorescence of FDNA. The formation of FDNA/cDNA duplex further improved the FRET efficiency, leading to a significant decrease in fluorescence intensity. However, fluorescence is restored when FDNA1/cDNA1 hybrid was cleaved into small fragments by EcoRI endonucleases, resulting in a decrease in FRET efficiency because of weakened electrostatic interaction between GNRs and the shortened DNA fragments. Activity of EcoRI endonuclease has been real-time studied by monitoring fluorescence change with the prolonging of interaction time. Under optimized conditions, the cleaved fraction is linear with EcoRI concentration over the range of 1.0 x 10(-3) to 1.0 x 10(-1) U mu L-1, with a limit of detection of 6.5 x 10(-4) U mu L-1 which is much better or at least comparable to previous reports. Site-specific DNA cleavage by EcoRI endonuclease has also been verified by gel electrophoresis, fluorescence anisotropy and TEM analysis, which indicated that this method is a feasible and reasonable approach to study sequence-specific protein-DNA interactions. Assay of BamHI activity demonstrated that it is a more universally applied method for studying the activity of endonuclease. Furthermore, this fluorescence assay has been also used for studying the inhibition of EcoRI endonuclease activity. Importantly, experimental results suggested that endonuclease inhibitors can be screened by monitoring the change of fluorescence change. Therefore, this FRET assay is a simple, sensitive and effective approach to study endonuclease activity and inhibition, and as such, it promises to provide a feasible method to screen nuclease inhibitors. (C) 2012 Elsevier B.V. All rights reserved.

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