5 methylcytidine

Guanosine
(Guo), cytidine (Cyt), adenosine (Ado), uridine (Urd), 5-methylcytidine
(5-me-Cyt), 5-methyluridine (5-me-Urd), N⁶-methyladenosine (N⁶-me-Ado), 2′-deoxyguanosine (dG), 2′-deoxycytidine
(dC), 2′-deoxyadenosine (dA), thymidine (dT), 5-methyl-2′-deoxycytidine
(5-me-dC), N⁶-methyl-2′-deoxyadenosine
(N⁶-me-dA), 8-oxo-7,8-dihydro-2′-deoxyguanosine
(8-oxo-dG), nucleosides test mix [containing cytidine (Cyt), guanosine
(Guo), adenosine (Ado), uridine (Urd), inosine (Ino), 5-methylcytidine
(5-me-Cyt), 2′-O-methylcytidine (2-O-me-Cyt), 3-methylcytidine methosulfate (3-me-Cyt), 7-methylguanosine
(7-me-Guo), 1-methyladenosine (1-me-Ado), 5-methyluridine (5-me-Urd),
β-pseudouridine (β-Urd), 2-thiocytidine dihhydrate (ThioC)],
DNA from calf thymus (ctDNA) sodium salt, nuclease P₁ from Penicillium citrinum (NP1), phosphodiesterase I from Crotalus adamanteus (snake) venom (SVPDE), alkaline
phosphatase from Escherichia coli (AKP),
ammonium acetate, ammonium bicarbonate, tris(hydroxymethyl)aminomethane
(Tris-buffer, pH 7.4), zinc chloride, and formic acid were obtained
from Sigma-Aldrich (St. Louis, MO). Chelex-100 resin was purchased
from Bio-Rad (Solna, Sweden). All the solvents used were of HPLC grade.
Experiments containing nucleic acids were carried out in DNA LoBind
tubes, 1.5 mL (Eppendorf).

Synthetic modified nucleosides for preparation of calibration solutions were purchased from: Sigma-Aldrich, Munich, Germany: cytidine (C), uridine (U), guanosine (G), adenosine (A), 5-methylcytidine (m⁵C), 2′-O-methylcytidine (Cm), 4-thiouridine (s⁴U), 5-methyl-2-thiouridine (m⁵s²U), 7-methylguanosine (m⁷G), 2′-O-methyladenosine (Am), 1-methyladenosine (m¹A). 6-Dimethyladenosine (m⁶₂A) and inosine (I); Berry&Associates, Dexter, MI, USA: 5-methyluridine (m⁵U), pseudouridine (Ψ), 2′-O-methyluridine (Um) and 2′-O-methylguanosine (Gm). 6-Methyladenosine (m⁶A) was a gift from Glenn Björk. Each nucleoside powder was weighed into a clean tube (5–10 mg per nucleoside) and dissolved in pure water to reach a final concentration of 10 mM. The nucleosides were then mixed in a 100 μM solution (major nucleosides final concentration in the mix: 1000 μM). This calibration mix stock solution was then used to prepare 15 calibration solutions in the range of 1 pM (1 amol/μl) to 10 μM (100 pmol/μl). Two sets of these calibration solutions were prepared, either containing 10% of the prepared ¹³C SIL-IS or not. The solutions were then analyzed as described and the measurements repeated after 1, 2, 4, 10 and 12 weeks to assess the methods reproducibility. In between measurements, the calibration solutions were stored at −20°C.

Binding measurements were performed on a MicroCal PEAQ ITC (Malvern) at 25 °C. UHRF1 MBP–SRA was dialyzed overnight at 4 °C in 25 mm HEPES, 100 mm NaCl, and 1 mm DTT. The next morning, 5-methylcytidine (Sigma, M4254) or annealed He5mC (sense, CCATG(5mC)GCTGAC; antisense, GTCAGCGCATGG), was resuspended in the same dialysis buffer as the SRA domain. UHRF1 SRA (35 μm) was loaded into the cell, and 5-methylcytidine (1 mm) or He5mC (430 μm) was loaded into the syringe. Nineteen injections (2 μl each, separated by 150 s) were performed after an initial injection of 0.4 μl.