Direct and Accurate Detection of DNA Methylation Positions using Biological Nanopore

Abstract

We describe a method of detecting 5-methylcytosine (5mC) positions using nanopore measurement. The determination of 5mC in the genome is promising to be used as epigenetic detection. In the general method of the 5mC determination, bisulfite conversion, it requires multiple procedures and the incomplete conversion sometimes occurs to reduce the reliability of the epigenetic analysis. In the previous study, Wang et al. detected the 5mC existence using nanopore measurement with tetramethylammonium salt1. However, this method is hard to detect the position of 5mC. Here, we report a simple method of determining a 5mC position using α-hemolysin (αHL) nanopore without bisulfite conversion, chemical modification, or enzyme amplification. αHL reconstituted in the lipid bilayers made a nanopore for capturing the target molecules under an applied voltage. A probe DNA hybridizes to a DNA which has a 5mC, and then the complementary DNA is captured and passed through in the nanopore with unzipping strands electrophoretically. The αHL-filling electrolyte record the ion current change in a single-molecular level triggered by unzipping the double-stranded (dsDNA). To determine the 5mC-position information, we analyzed the unzipping current data including the unzipping time and blocking ratio using the bootstrap method2. The bootstrap method is based on the resampling of the original random sample drawn from a population. The bootstrapped data offered the estimation of the unknown distribution, which was shown by plotting the blocking ratio against unzipping time. As a result, the scatter plot graph showed the possibility to distinguish between the 5mC positions at the single nucleotide level using nanopore sensing technology. Our method offers a simple and rapid tool for a 5mC-position determination as a nanopore epigenetic detection. Reference 1. Wang, Y. et al., Scientific Reports (2017). 2. Liu, P. et al., Small Methods (2020).