All chemicals and biological materials were obtained from commercial suppliers. N-Hydroxy-cytidine from Cambridge Bioscience; cytidine, uridine, Lysozyme, DNase I and kanamycin were purchased from Sigma Aldrich; polymyxin B sulfate from AlfaAesar; LB agar, LB media, 2×YT media and arabinose from Formedium; Escherichia coli BL21(DE3), Q5 DNA polymerase, T4 DNA ligase and restriction enzymes from New England BioLabs; and oligonucleotides were synthesized by Integrated DNA Technologies. Construction of pET29b(+)_CD. The E. coli-optimised gene (synthesised by Integrated DNA Technologies) for cytidine deaminase (CD) from E. coli was cloned into pET29b(+) using NdeI and XhoI restriction sites to yield pET29b(+)_CD. Protein production and purification. For expression of CD and variants, chemically competent E. coli BL21 (DE3) were transformed with the relevant pET29b(+)_CD constructs. Single colonies of freshly transformed cells were cultured for 18 h in 5 mL LB medium containing 50 µg mL -1 kanamycin. Starter cultures (500 µL) were used to inoculate 50 mL 2×YT medium supplemented with 50 µg mL -1 kanamycin. Cultures were grown at 37 °C, 200 r.p.m. to an optical density at 600 nm (OD600) of around 0.5. Protein expression was induced with the addition of IPTG to a final concentration of 0.1 mM. Induced cultures were incubated for 20 h at 30 °C and the cells were subsequently collected by centrifugation (3,220 x g for 10 min). Pelleted cells were resuspended in lysis buffer (50 mM HEPES, 300 mM NaCl, pH 7.5 containing 20 mM imidazole) and lysed by sonication. Cell lysates were cleared by centrifugation (27,216 x g for 30 min) and supernatants were subjected to affinity chromatography using Ni-NTA Agarose (Qiagen). Purified protein was eluted using 50 mM HEPES, 300 mM NaCl, pH 7.5 containing 250 mM imidazole. Proteins were desalted using 10DG desalting columns (Bio-Rad) with PBS pH 7.4 and analyzed by SDS PAGE. Proteins were aliquoted, flash-frozen in liquid nitrogen and stored at -80 °C. Protein concentrations were determined by measuring the absorbance at 280 nm and assuming an extinction coefficient of 29910 M -1 cm -1 .