HpaII-assisted and linear amplification-enhanced isothermal exponential amplification fluorescent strategy for rapid...

The detection of methyltransferase (MTase) activity is of great significance in methylation-related disease diagnosis and drug screening. Herein, a HpaII-assisted and linear amplification-enhanced exponential amplification strategy is proposed for sensitive and label-free detection of M.SssI MTase activity. The P1 probe contains self-complementary sequence 5'-CTAGCCGGCTAG-3' at 3'-terminal. After denaturation and annealing, P1 probes hybridize with itself to generate P1 duplexes. M.SssI MTase induces methylation of cytosine at 5'-CG-3' in P1 duplexes, and thus, HpaII fails to cleave at 5'-CCGG-3' due to methylation sensitivity, leaving P1 duplex intact. Then, these intact P1 duplexes are extended along 3'-terminal through Vent (exo^-) DNA polymerase to generate dsDNA, which is recognized and nicked at the recognition sites by Nt.BstNBI, releasing two copies of primer X. Primer X hybridizes with X' at the amplification template T1 (X'-Y'-X') and then serves as primers to trigger the exponential amplification reaction (EXPAR). The point of inflection (POI) values of real-time fluorescence curves is linearly correlated with the logarithm of M.SssI MTase concentration in the range of 0.125 [Formula: see text] 8 U mL^-1 with a low detection limit of 0.034 U mL^-1. In the absence of M.SssI, P1 duplexes are cut by HpaII and separated into ssDNA under the executed temperature of EXPAR and thus unable to trigger the amplification. The strategy provides good selectivity against other types of MTases and protein and is able to detect M.SssI activity in human serum. Furthermore, the analytical method has the generality and can be extended to the analysis of other types of DNA MTases.