Nowadays colorimetric assay has been applied in detecting DN
Nowadays, colorimetric assay has been applied in detecting DNA MTase activity. Li et al. used DNA modified gold nanoparticles (AuNPs) coupled with enzyme-linkage reactions to detect the activity of methylases . In a previous study, our group presented a label-free colorimetric method, using unmodified Au nanorods with enzyme-linkage reaction for the assay of DNA methylation . The methods applying AuNPs with enzyme linkage reactions have emerged as the most important colorimetric reporters owing to their high extinction coefficients and strong size-dependent surface plasmon resonance (SPR) properties . The color of the AuNPs solution can be changed from red to purple or pale, in response to the SPR Glycopyrrolate of dispersed and aggregated nanoparticles.
Although AuNPs based methods have accomplished substantial progress, relatively poor sensitivity is one of the common limitations, which is mainly due to the shortage of signal amplification . It is extremely important to develop amplification strategies for AuNPs-based methods to address this drawback. Up until now, several signal amplification strategies have been reported including loop-mediated isothermal amplification (LAMP) , rolling circle amplification (RCA) , exponential amplification reaction (EXPAR) . Motoi and coworkers designed a fluorescent-amplified adenosine aptasensors by toehold mediated click chemical ligation DNA strand displacement system . A new approach of reverse DNA strand displacement by using functional nucleic acids as toeholds was reported by Yang’s group . Among these methods, strand displacement amplification (SDA) has attracted increasing attention due to its excellent property of homogeneous and lable-free. DNA displacement reactions between double-stranded (ds)-DNA with strands of unequal length and single-stranded (ss)-oligonucleotides using a toehold structure, as a trigger point, enables DNA rehybridization in a fast “base-by-base” programmable controlled manner , . This method, SDA, has the advantage that the route of strand displacement can be easily predicted due to the fact that the displacement is always known at the commencement. Furthermore, the reacting oligonucleotides sequence is known, considering all mismatches, such as deletions, insertions, or point mutations occurring during strand exchange, making it possible to forecast the kinetic, thermodynamic behavior and outcome of the displacement reaction . Therefore, the SDA method has potention applications in many fields. Nevertheless, there are only a few amplified strategies available for development of colorimetric methods based on AuNPs.
Here we take advantage of strand displacement to design colorimetric SDA biosensor for CpG methyltransferase (M.SssI) detection. The M.SssI catalyzes the transferring of methyl to C-5 position of cytosine in the CpG region of double-stranded DNA from SAM and the HpaII restriction endonuclease can identify the duplex symmetrical sequence of 5′-CCGG-3′ and catalyze the digestion of double-stranded DNA between the unmethylated cytosines. According to the procedure, HpaII endonuclease catalyzed cleavage will be blocked once the CpG dinucleotide site in the 5′-CCGG-3′ sequence is methylated . In this work, the amplification is triggered by the HpaII digestion products region I hybridization with a hairpin structure DNA, and then region I is replaced by DNA probes. With the use of AuNPs-based colorimetric assay, the developed strategy can create a convenient platform for visualized detection of enzyme activity as well as the screening of the inhibitors of M.SssI with high sensitivity and selectivity.
Results and discussion
Acknowledgements We greatly appreciate the supports of the National Natural Science Foundation of China (21265012and 21365015), the Program for New Century Excellent Talents in University (NCET-11-1002 and NCET-13-0848) and the Department of Education (Jiangxi Province, China, Grant No. GJJ13109).