Fluorescent SAM analogues for methyltransferase based DNA labeling

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Gel based restriction assay
The compatibly of the various synthesized cofactors with DNA methyltransferases was examined through a gel based restriction assay. The principle of this assay takes advantage of the methylationsensitive restriction enzymes. Briefly, if the DNA methyltransferase is compatible with the synthetic cofactor, the successful labeling will block the subsequent digestion of DNA by a restriction enzyme that recognizes the same sequence. In case of digestion, the DNA is cut into smaller fragments which will migrate faster through the gel. Thus, the digestion patterns from electrophoresis gels indicate the compatibility between the enzymes and cofactors of interest.
When a non-digested plasmid is loaded into an agarose gel and exposed to the applied voltage, naturally three bands can be expected corresponding to the three plasmid conformations (from high to low: nicked, linear and supercoiled DNA). Any band lower than the natural conformations can be considered as the result of plasmid digestion, indicating incomplete protection.

Counting assay
Fluorescence counting assay was performed as described in literature 10 in order to assess the DNA labeling efficiency. Briefly, 10 samples of labeled pUC19 DNA were prepared using the direct cofactors 5a-5j. Next, for each sample, the labeled molecules were deposited on a poly-L-lysine (PLL) (0.01% w/v in H 2 O) coated coverslip and visualized using fluorescent microscopy. After long excitation the reporter fluorophores will be photobleached. Each bleaching step can be counted, yielding the number of dyes bound to a single DNA molecule. Up to 20000 DNA molecules per sample were measured and the average dye content was calculated. These averages are presented in Fig. S10 for all 10 samples.

M.TaqI directed labeling using a rhodamine B-tagged SAM analogue
A similar labeling protocol 11 was used as described by Bouwens et al. In brief, Lambda DNA (Thermo Scientific) was enzymatically labeled using 35 µM of the fluorescent analogue 4 or 5a and 0.14 mg/ml M.TaqI methyltransferase enzyme (recognition sequence 5'-TCGA-3'), in a final concentration of 50 ng/µl. The reaction was carried out at 60°C for 2 hours in a custom labeling buffer. Next, proteinase k was added and reacted for 1 hour at 50°C. Finally, the reaction product was purified using CHROMA SPIN+TE-1000 columns (Clontech, Takara Bio).
Purified DNA was dissolved in 50 mM MES (pH 5.6), and deposited in stretched conformation by mechanically dragging a 2 µl droplet over the surface of a Zeonex-coated coverslip at a speed of 4.4 mm/min, as described earlier 12 . Stretched samples were vacuum dried overnight prior to imaging.
Imaging was performed with a Zeiss SIM Elyra microscope with a Zeiss Plan-APOCHROMAT 63× oil immersion objective (numerical aperture 1.4) and an EMCCD camera. For each field of view, 25 frames were recorded for 5 SIM modulation angles and 5 phases/angle. The illumination patterns for SR-SIM were created by a grating with a period of 34 µm. A wide-field image was calculated by averaging over the 25 frames. SR-SIM reconstruction was done with the open-source fairSIM plugin for ImageJ. DNA fragments were segmented manually on the SR-SIM images using ImageJ. For each imaged DNA fragment, both widefield and SR-SIM signals were extracted.                ) of purified 2b. Blue peak correspond to cofactor 2b, red peak corresponds to the starting material 1b which has formed due to degradation of the cofactor during evaporation.   ) of purified 5a. Blue peak correspond to cofactor 5a, red peak corresponds to the starting material S5 which has formed due to degradation of the cofactor during evaporation. ) of purified 5b. Blue peaks correspond to cofactor 5b, red peak corresponds to the starting material S6 which has formed due to degradation of the cofactor during evaporation. ) of purified 5c. Blue peak correspond to cofactor 5c, red peak corresponds to the starting material S7 which has formed due to degradation of the cofactor during evaporation. ) of purified 5d. Blue peak correspond to cofactor 5d, red peak corresponds to the starting material S8 which has formed due to degradation of the cofactor during evaporation. ) of purified 5e. Blue peak correspond to cofactor 5e, red peak corresponds to the starting material S9 which has formed due to degradation of the cofactor during evaporation. ) of purified 5f. Blue peak correspond to cofactor 5f, red peak corresponds to the starting material S10 which has formed due to degradation of the cofactor during evaporation. ) of purified 5g. Blue peak correspond to cofactor 5g, red peak corresponds to the starting material S11 which has formed due to degradation of the cofactor during evaporation. ) of purified 5h. Blue peak correspond to cofactor 5h, red peak corresponds to the starting material S12 which has formed due to degradation of the cofactor during evaporation. ) of purified 5i. Blue peak correspond to cofactor 5i, red peak corresponds to the starting material S13 which has formed due to degradation of the cofactor during evaporation. ) of purified 5j. Blue peak correspond to cofactor 5j, red peak corresponds to the starting material S14 which has formed due to degradation of the cofactor during evaporation. to the respective starting material S5 through hydrolysis of the amino acid.