3.3. Single-Molecule FRET-Based DNA Methylation Assay

The single-molecule FRET technique has been reviewed in numerous papers [76,77,78] and the setup used in our assay was also described previously (we used 532 nm CW laser (Coherent, Inc., Santa Clara, CA, USA), the power of which was ~3 mW at the laser. The beam was shone at the sample obliquely through the objective lens (CFI 60× Apochromatic TIRF, Nikon, Tokyo, Japan) for TIRF imaging. The donor and acceptor fluorescent signals were directed to the EMCCD (iXON Ultra897, Andor, Belfast, Northern Ireland, UK)) [74]. The experimental procedure and details are described as follows. First, the sample chamber was coated with 1 mg/mL biotinylated BSA and then 0.2 mg/mL NeutrAvidin. The sample solution was introduced to the chamber and incubated for the immobilization of the DNA tethers for 10 min. The chamber was then washed with a 4× chamber volume of buffer solution containing various concentrations of magnesium ions ([Mg²⁺]) and incubated for 30 min. Within 30 min, the B-Z transition normally reached equilibrium. For fluorescence measurements, the chamber was washed with oxygen scavenging solution (2 mM Trolox, 2.6 mM PCA (Protocatechuaic acid, Sigma-Aldrich, St. Louis, MO, USA), 0.4 unit/mL of rPCO (Protocatechuate 3,4-dioxygenase, OYC Japan, Tokyo, Japan)) supplemented with 5~500 mM MgCl₂.

The experimental method is depicted schematically in Figure 4B. In our TIRF-based smFRET assay, the core in the B-state and Z-state exhibited the FRET efficiency (E_FRET) of ~0.5 and ~0.15, respectively. In the assays to determine the degree of methylation or to compare the levels of methylation, we used [Mg²⁺] = 50 mM. The rationale for this choice is that at this concentration, cores with different degrees of methylation exhibit distinctive relative populations of B- and Z-states. In comparison with this reference information, we can determine the degree of methylation from the tested samples, measuring the activity of DMT and the inhibitory effect of dietary compounds on DMT.