Ultrasensitive fluorescence detection of multiple DNA methyltransferases based on DNA walkers and hyperbranched rolling circle amplification.

The accurate and ultrasensitive detection of multiple methyltransferases was in great request for clinical diagnosis and epigenetic therapy. Here, a novel fluorescence assay was proposed for ultrasensitive CpG methyltransferase (M.SssI) and DNA adenine methyltransferase (Dam) activity detection based on hyperbranched rolling circle amplification (HRCA) and DNA walkers. The biosensor showed an extremely high sensitivity due to the dual-amplification strategy of HRCA and DNA walker. The LOD of the biosensor for M.SssI and Dam methyltransferase was estimated at 0.0004 U/mL and 0.001 U/mL, respectively. Without the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was cleaved by HpaII endonuclease, generating a DNA fragment (T-DNA) and inducing the DNA walker-HRCA reaction. Since the HRCA products contained numerous double-strand DNA (dsDNA), SYBR Green I could be embedded in the dsDNA, leading to a high fluorescent signal. In the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was methylated and the HpaII endonuclease-catalyzed stem of hairpin HM dissociation was hindered, leading to no DNA fragment (T-DNA) present. Hence, the DNA walker-HRCA reaction was not initiated and the fluorescent signal of SYBR Green I remained at a low level. Similarly, DNA adenine methyltransferase (Dam) and its inhibitors could also be detected by redesigning hairpin HD with the Dam recognition sequences. Furthermore, the sensing system was applied to analyze the endogenic Dam methyltransferase in the real samples such as E. coli cell lysate.

Mapping Retinoic Acid-Dependant 5mC Derivatives in Mouse Embryonic Fibroblasts.

Methylase-assisted bisulfite sequencing (MAB-seq) is a derivatization technique to evaluate the presence of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) at base-pair resolution. Although MAB-seq was originally designed to study these metabolites under steady-state conditions, we have developed an alternative protocol to evaluate the dynamics of 5-fC/5-caC accumulation in response to agonists, such as all-trans retinoic acid (ATRA). In addition, this protocol utilizes a lower quantity of the M.SssI enzyme without compromising methylation efficiency and requires less bench time. Herein, we describe the use of MAB-seq assay to evaluate the generation of 5-fC/5-caC in response to ATRA in mouse embryonic fibroblasts, using the hypermethylated in cancer 1 (Hic1) locus as a model system.