Dpni

Extrachromosomal DNA was extracted from the transfected cells at 1 to 5 days post-transfection via the modified Hirt method [42 (link)], or total genomic DNA was extracted at 10 to 21 days post-transfection from U2OS cells as described previously [13 (link)]. Before Southern blot analysis, half of each extrachromosomal DNA sample or 3 μg of each total DNA sample was treated with the linearizing enzyme HindIII, and non-replicated, bacterially produced dam-methylated input DNA was fragmented with DpnI (Thermo Scientific). The DNA samples were resolved on a 0.8% (extrachromosomal DNA) or 0.6% (total genomic DNA) agarose gel using 1× Tris-acetate-EDTA (TAE) buffer. Electrophoresis was performed at 0.8 V/cm for 20 h. The separated DNA fragments were treated with 0.1 M hydrochloric acid, denatured with Sol A (0.5 M NaOH, 1.5 M NaCl), neutralized with Sol B (1 M Tris pH 7.4, 1.5 M NaCl), transferred to a Hybond-N+ membrane (Amersham Pharmacia Biotech) via the Southern blot method and hybridized with a radiolabelled HPV11-specific probe. HPV11 replication signals were detected via exposure to X-ray film.

Site directed mutagenesis (SDM) was performed in 50 µL of SDM1 (composition of all buffers is presented in Table 1) buffer. Mutated plasmids were amplified in an Eppendorf Mastercycler^® according to the program: 94 °C for 2 min, followed by 18 cycles of 94 °C for 20 s, 55 °C for 30 s, 68 °C for 7 min and final 68 °C for 10 min. The primers used for SDM are shown in Table S1. Non-mutated plasmids were removed by incubation with DpnI: 50 µL of sample was mixed with 6 µL of 10x Tango buffer and 4 µL of DpnI (10U/µL, Thermo Scientific™, Waltham, Massachusetts, USA, #ER1701), and incubated with mixing (500 rpm) for 2 h at 37 °C. The reaction was stopped by incubation at 80 °C for 20 min. Then, 8 µL of reaction mixture was added to 200 µL of competent E.coli DH5α bacteria suspension, incubated for 30 min on ice, heat shocked for 30 s at 42 °C, then 800 µL of SOC buffer was added and the bacterial suspension was incubated at 37 °C for 1 h. The bacteria were plated on LB-agar plates and incubated overnight at 37 °C. Single colonies were cultured in liquid LB overnight at 37 °C. Bacteria were then harvested and the plasmid was isolated with the use of the GeneJET Plasmid Miniprep Kit (Thermo Scientific™, Waltham, MA, USA, #K0503). All plasmids were verified by sequencing.

The single mutation H307Y was generated by PCR based site-directed mutagenesis [41 (link)], where the PCR reaction contained 10 ng of plasmid pET28_bglhi as template, 50 pmol of the primers 5’-GGCATGAACTACTACACGGCCAACTACATCAAGCAC-3’ (forward) and 5’- CGTGTAGTAGTTCATGCCGTAGAAGTCGTTGGAGCC-3’ (reverse), 200 μM dNTPs and 2.5 U of Pfu DNA polymerase (Thermo Scientific, Waltham, MA, USA) and its respective buffer. The PCR was performed with initial denaturation of the template DNA at 95°C for 3 min, followed by 30 cycles of 95°C for 1 min, 50°C for 1 min, 72ºC for 15 min and a final extension step at 72°C for 10 min. After PCR, the product was incubated with 2 U of DpnI (Thermo Scientific, Waltham, MA, USA) at 37°C for 3 h to digest the methylated template DNA. One microliter of the DpnI digested sample was used to transform E. coli DH5α. After nucleotide sequencing confirm the mutation, the plasmid (denominated pET28_307) was subsequently used to transform E. coli BL21 (DE3) for the expression of the recombinant enzyme (See the “Protein expression and purification” section).

Self-complementary pairs of primers containing the desired mutation were designed and 150 ng of the CVB3 infectious clone were used as template for amplification with Phusion High-fidelity DNA Polymerase (ThermoScientific) under the following thermal profile: initial denaturation at 98 °C for 1 min, 25 cycles of 98 °C for 1 min, 1 min at 65 °C, and 72 °C for 6 min, followed by 10 min of final extension at 72 °C. Products were digested with DpnI (ThermoScientific) at 37 °C for 2 h to remove the methylated template. Then, these reactions were transformed in NZY5α competent cells (Nzytech) and the resulting colonies were tested by Sanger sequencing for the presence of the mutation, amplified, and used for plasmid extraction by the miniprep method. Plasmids were linearized with SalI (ThermoScientific) and transcribed in vitro using the TranscriptAid T7 High Yield Transcription Kit (ThermoScientific). RNA was purified by LiCl precipitation following the manufacturer’s protocol and used to transfect 400 µl of HeLa-H1 cells (10⁶ cells/ml) by electroporation in a BIO-RAD GenePulser Xcell (240 V, 950 µF in 4-mm cuvettes).

Genomic DNA isolated from asynchronous S. acidocaldarius cells was cleaved independently with a specific REase in order to decipher the presence or the absence of m4C or m6A: BsuRI, BamHI, MboI, and DpnI (Thermo Scientific). Briefly, digestions of 250 ng of gDNA by 2.5 Units of enzyme were performed for 30 min at 37°C and loaded onto a 0.8% agarose gel at room temperature in TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.0), stained with ethidium bromide (2 μg mL⁻¹) and digitalized under UV light.