A Simple, Universal Colorimetric Assay for Endonuclease/Methyltransferase Activity and Inhibition Based on an Enzyme-Responsive Nanoparticle System

Abstract

An enzyme responsive nanoparticle system that uses a DNA-gold nanoparticle (AuNP) assembly as the substrate has been developed for the simple, sensitive, and universal monitoring of restriction endonucleases in real time. This new assay takes advantage of the palindromic recognition sequence of the restriction nucleases and the unique optical properties of AuNPs and is simpler than the procedure previously described by by Xu et al. (Angew. Chem. Int. Ed. Engl. 2007, 46, 3468-3470). Because it involves only one type of ssDNA modified AuNPs, this assay can be directed toward most of the endonucleases by simply changing the recognition sequence found within the linker DNA. In addition, the endonuclease activity could be quantitatively analyzed by the value of the reciprocal of hydrolysis half time (t(1/2)(-1)). Furthermore, our new design could also be applied to the assay of methyltransferase activity since the methylation of DNA inhibits its cleavage by the corresponding restriction endonuclease, and thus, this new methodology can be easily adapted to high-throughput screening of methyltransferase inhibitors.

Multiplex Detection of Endonucleases by Using a Multicolor Gold Nanobeacon

“…It is anticipated that the proposed multiplex detection can be realized in an array format. Also, given the fact that DNA methylation by methyltransferase inhibits DNA cleavage by the corresponding restriction endonuclease, [24,25,28] we expect that the proposed gold nanobeacon may also be found useful in assaying methyltransferases.…”

“…[16][17][18][19][20][21][22] The use of complexes of DNA and cationic conjugated polymer as probes was recently described. [23] Colorimetric methods based on enzyme-responsive gold nanoparticle (AuNP) [24][25][26][27] and DNAzyme [28] systems have also been developed for detecting endonucleases. Although each of these techniques has its own advantages, most of them do not afford multiplex detection of endonucleases.…”

“…These were at least 100 times more sensitive than those of previously reported methods based on gel electrophoresis and chromatography, UV spectroscopy, and the general fluorescence methods. [16][17][18][19][24][25][26][27][28] These results indicated that the proposed method could be used for quantifying multiple endonucleases simultaneously with high sensitivity and specificity.…”

A novel signal-on strategy for M.SssI methyltransfease activity analysis and inhibitor screening based on photoelectrochemical immunosensor

“…There are many traditional methods for analyzing DNA MTase activity and screening inhibitor, which are focused on radioisotope labeled substrate (Bergerat et al, 1991), bisulfate treatment rolling circle amplification (Cao and Zhang, 2012), colorimetric approaches (Song et al, 2009), PCR (polymerase chain reaction) (Lyko et al, 2000), HPLC (high-performance liquid chromatography) (Lopez Torres et al, 2011), SERS (surface enhanced Raman spectroscopy) (Hu and Zhang, 2012), fluorescence (Chen and Zhao, 2013), electrochemical method (Deng et al, 2014) and so on. However, all of them have limitations, for example, radioisotope labeled substrate are harmful to biological tissue, bisulfate treatment rolling circle amplification need to convert cytosine to uracil which is time-consuming.…”

Highly sensitive fluorescence assay of DNA methyltransferase activity via methylation-sensitive cleavage coupled with nicking enzyme-assisted signalamplification

“…In recent years, a lot of efforts have been focused on DNA MTase activity assay to overcome the above drawbacks. For example, using methylation-responsive DNAgold nanoparticles (AuNPs) assembly and methylation-triggered DNAzyme-based strand displacement amplification (SDA), the colorimetric approaches have been developed to assay DNA MTase activity Liu et al, 2010;Song et al, 2009). However, in addition to relatively high detection limit, the sensing platforms also suffer from time-consuming DNA conjugation on AuNPs or polymerization process.…”