Sensitive detection of endonuclease activity and inhibition using gold nanorods.

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 1.0×10(-3) to 1.0×10(-1) U μL(-1), with a limit of detection of 6.5×10(-4) U μL(-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.

[1]  Kemin Wang,et al.  Real-time monitoring of restriction endonuclease activity using molecular beacon. , 2007, Analytical biochemistry.

[2]  N. Grindley,et al.  Mechanisms of site-specific recombination. , 2003, Annual review of biochemistry.

[3]  S. Sligar,et al.  Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Yingfu Li,et al.  Enzymatic cleavage of nucleic acids on gold nanoparticles: a generic platform for facile colorimetric biosensors. , 2008, Small.

[5]  L. Marnett,et al.  Differential DNA recognition and cleavage by EcoRI dependent on the dynamic equilibrium between the two forms of the malondialdehyde-deoxyguanosine adduct. , 2005, Biochemistry.

[6]  Enzymatic DNA processing on gold nanoparticles , 2004 .

[7]  D. Low,et al.  An essential role for DNA adenine methylation in bacterial virulence. , 1999, Science.

[8]  R. Geiger,et al.  Changing the hydrogen-bonding potential in the DNA binding site of EcoRI by site-directed mutagenesis drastically reduces the enzymatic activity, not, however, the preference of this restriction endonuclease for cleavage within the site-GAATTC-. , 1989, Biochemistry.

[9]  Cheulhee Jung,et al.  A gold nanorod-based optical DNA biosensor for the diagnosis of pathogens. , 2010, Biosensors & bioelectronics.

[10]  B. Liu,et al.  Peptide-mediated energy transfer between an anionic water-soluble conjugated polymer and Texas red labeled DNA for protease and nuclease activity study. , 2009, Analytical chemistry.

[11]  F. Eckstein,et al.  Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides. , 1988, Nucleic acids research.

[12]  X. Qu,et al.  A simple, universal colorimetric assay for endonuclease/methyltransferase activity and inhibition based on an enzyme-responsive nanoparticle system. , 2009, ACS nano.

[13]  A. Jeltsch,et al.  Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[14]  S. Wada,et al.  FRET-based kinetic analysis of highly reactive heterochiral DNA toward EcoRI endonuclease. , 2009, Biochemical and biophysical research communications.

[15]  Zhenfeng Chen,et al.  Ultrasensitive endonuclease activity and inhibition detection using gold nanoparticle-enhanced fluorescence polarization. , 2011, Chemical communications.

[16]  Xiaohua Huang,et al.  Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods. , 2006, Journal of the American Chemical Society.

[17]  C. Mirkin,et al.  A gold-nanoparticle-based real-time colorimetric screening method for endonuclease activity and inhibition. , 2007, Angewandte Chemie.

[18]  Jian Wang,et al.  End-to-end assembly of gold nanorods by means of oligonucleotide-mercury(II) molecular recognition. , 2010, Chemical communications.

[19]  Catherine J Murphy,et al.  Seeded high yield synthesis of short Au nanorods in aqueous solution. , 2004, Langmuir : the ACS journal of surfaces and colloids.

[20]  Xuchuan Jiang,et al.  Gold nanorods : Limitations on their synthesis and optical properties , 2006 .

[21]  Matthew M. Rex,et al.  Pushing the limits of mercury sensors with gold nanorods. , 2006, Analytical chemistry.

[22]  A. Jeltsch,et al.  A fast and accurate enzyme-linked immunosorbent assay for the determination of the DNA cleavage activity of restriction endonucleases. , 1993, Analytical biochemistry.

[23]  W. Tan,et al.  Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA. , 2000, Nucleic acids research.

[24]  Ronghua Yang,et al.  Gold nanoparticle-based colorimetric and "turn-on" fluorescent probe for mercury(II) ions in aqueous solution. , 2008, Analytical chemistry.

[25]  J. Holland,et al.  Correction of right-angle fluorescence measurements for the absorption of excitation radiation , 1977 .

[26]  C. Stein,et al.  Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences , 2000, Nature Biotechnology.

[27]  Joseph Irudayaraj,et al.  Multiplex biosensor using gold nanorods. , 2007, Analytical chemistry.

[28]  P. Modrich,et al.  The Kinetic Mechanism of EcoRI Endonuclease* , 1999, The Journal of Biological Chemistry.

[29]  C. Murphy,et al.  Quantitation of metal content in the silver-assisted growth of gold nanorods. , 2006, The journal of physical chemistry. B.

[30]  Wang Li,et al.  Label-free colorimetric assay for methyltransferase activity based on a novel methylation-responsive DNAzyme strategy. , 2010, Analytical chemistry.

[31]  T. Bergström,et al.  Genotyping of Clinical Herpes Simplex Virus Type 1 Isolates by Use of Restriction Enzymes , 2006, Journal of Clinical Microbiology.

[32]  L. McLaughlin,et al.  Effects of functional group changes in the EcoRI recognition site on the cleavage reaction catalyzed by the endonuclease. , 1987, Biochemistry.

[33]  J. Thorson,et al.  A continuous assay for DNA cleavage: the application of "break lights" to enediynes, iron-dependent agents, and nucleases. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Zhenxin Wang,et al.  Enzymatic disassembly of DNA-gold nanostructures. , 2007, Small.

[35]  A. Wolffe,et al.  DNA methylation in health and disease , 2000, Nature Reviews Genetics.

[36]  A. Jeltsch,et al.  On the divalent metal ion dependence of DNA cleavage by restriction endonucleases of the EcoRI family. , 2009, Journal of molecular biology.

[37]  Jian Wang,et al.  One-step label-free optical genosensing system for sequence-specific DNA related to the human immunodeficiency virus based on the measurements of light scattering signals of gold nanorods. , 2008, Analytical chemistry.

[38]  Hong Liang,et al.  Multiplex detection of endonucleases by using a multicolor gold nanobeacon. , 2011, Chemistry.

[39]  A. Jeltsch,et al.  Structure and function of type II restriction endonucleases. , 2001, Nucleic acids research.

[40]  H. Goodman,et al.  Specificity of substrate recognition by the EcoRI restriction endonuclease. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[41]  K. Torimitsu,et al.  Anisotropic assembly of gold nanorods assisted by selective ion recognition of surface-anchored crown ether derivatives. , 2007, Chemical communications.

[42]  O. Fleck,et al.  DNA repair nucleases , 2004, Cellular and Molecular Life Sciences CMLS.

[43]  K. G. Thomas,et al.  Selective detection of cysteine and glutathione using gold nanorods. , 2005, Journal of the American Chemical Society.

[44]  J. Ma.  Genes, greens, and vaccines , 2000, Nature Biotechnology.