5 methyluridine

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).

The enzymatic reaction was carried out using 99% pure uridine, citidine, adenosine, guanosine, inosine, xanthosine, 2′–deoxyuridine, thymidine, vidarabine or 5–methyluridine from Sigma–Aldrich (Burlington, MA, USA) and water purified using a Milli-Q unit (Merck, Darmstadt, Germany). The reaction was carried out as follows: the desired stock concentration of nucleoside was prepared by weighing it and dissolving it in Tris–HCl buffer pH 7.5, then 500 μL of said nucleoside at the required concentration (diluted with water when needed) was added to plastic test tubes (total volume 1.5 mL), and five replicates were made for each final concentration of nucleoside. The number of points on the curve (i.e., the number of nucleoside samples) depends on the further accuracy of plotting the reaction rate versus substrate concentration curve; in this work, it was 15 points for each substrate where applicable. Next, 5 μL of purified RihC solution at a concentration of approximately 200 μg mL⁻¹ was added to each sample (the final concentration in the solution is 2 μg mL⁻¹) and stirred. At certain time intervals, the enzymatic reaction was stopped by adding 5 µL of concentrated HCl (time differed for different nucleosides). The prepared samples were then used for analysis.