Nuclease p1

The mitochondria from Scramble and shMETTL17 RKO cells were isolated, followed by RNA extraction. A total of 400 ng mitochondrial RNA samples was digested with 1 unit of nuclease P1 (NEB, Cat#M0660S) in 30 μL NEB buffer at 37 °C for 2 h. To dephosphorylate the single nucleotides, 1 unit of rSAP (NEB, Cat#M0371S) was added and incubated at 37 °C for 2 h. The samples were then added with 200 μL nuclease-free water and centrifuged at 4 °C for 10 min. The supernatants were collected for loading into LC-MS/MS (AB SCIEX QTRAP6500+). Nucleosides were quantified using nucleoside-to-base ion mass transitions and retention time, referencing standards of m⁴C (TRC, Cat#B426568), m⁵C (TOPSCIENCE, Cat#T5912), m³C (TOPSCIENCE, Cat#T7351), m⁶₂A (MCE, Cat#HY-101984), m⁵U (TRC, Cat#389366), m⁷G (TOPSCIENCE, Cat#T7319), and m⁶A (TOPSCIENCE, Cat#T6599).

Total RNA was purified using a PolyATtract mRNA Isolation Systems kit (Promega, WI, USA) following the manufacturer's protocols. About 200 ng of mRNA was digested in 25 μl buffer with nuclease P1 (2U; M0660S, NEB, MA, USA), 2.5 mM ZnCl₂ and 25 mM NaCl at 42°C for 2 h. Then alkaline phosphatase (0.5 U; Thermo Fisher, MA, USA) and NH₄HCO₃ (1 M, 3 ml) were added and incubated at 37°C for 2 h. Next, the sample was diluted 5-fold and filtered (pore size 0.22 mm; MilliporeSigma, MA, USA). A total of 5 μl of the solution was injected into an LC-MS/MS instrument. The total amount of m⁶A in mRNA was assessed using an Agilent 1290 Infinity II-6470 (Agilent Technology, CA, USA) system as previously reported (15 (link)). The concentration of m⁶A and A was calculated according to the standard curve, and then m⁶A/A (%) was obtained.

Triphosphorylated RNA (pppG-RNA₁₂ or pppA-RNA₁₂, 10 μM) were capped using the vaccinia virus capping enzyme (New England Biolabs) in the presence of 2 μCi α-³²P-GTP (Perkin Elmer) to yield radiolabeled G*pppN capped RNA (where * represents the radioactive α-³²P). Duplicate reactions were run in the presence of 0.1 mM AdoMet, for creation of N7-guanine methylated cap controls (^mG*ppppN). Additional reactions were additionally prepared using pppN_m-RNA₁₂ (pre-methylated at the 2′O position), in the presence or absence of AdoMet, for generation of G*pppN_m and ^mG*pppN_m cap controls. Capped RNA was incubated with 1 μM (final concentration) of the purified WB MTase enzyme in a final reaction containing 40 mM Tris, pH 8, 40 μM AdoMet, and 1 μM capped RNA. RNA was digested with Nuclease P1 (New England Biolabs) for 3–4 h at 37°C, and loaded onto polyethylenimine cellulose TLC sheets (Macherey Nagel), along with cap controls. Samples were separated using 0.65 M LiCl as mobile phase. TLC sheets were exposed and visualized using using an Amersham Typhoon BiomolecularImager (GE Healthcare).

The global DNA methylation levels were determined by measuring the 5-methyl-cytosine level with a Global DNA Methylation competitive enzyme-linked immunosorbent assay (ELISA) kit from Cell Biolabs (San Diego, CA, USA). Briefly, genomic DNA was extracted from cell samples using the Wizard® Genomic DNA purification kit (Promega, Madison, WI, USA). A total of 1 μg genomic DNA (0.1 μg/μL concentration) was denatured into single-stranded DNA by heating the samples at 95° C for 10 mins, followed by quick cooling on ice. Next, 1 μL of nuclease P1 (New England Biolabs, Ipswich, MA, USA) and an appropriate amount of nuclease P1 reaction buffer (New England Biolabs, Ipswich, MA, USA) were added to single-stranded DNA samples, and the whole mixture was incubated at 95° C for 1 hr to digest DNA into nucleosides. After that, the samples were treated with calf intestinal alkaline phosphatase (Takara Bio Inc., Shiga, Japan) for 1 hr at 37° C. Further steps were conducted according to the manufacturer’s recommendation. Sample absorbance was measured at 450 nm using a microplate reader (DTX880, Beckman Coulter, Inc., California, USA).

HEPES (Sigma-Aldrich, 99.5% purity, copper ≤ 5 ppm); MgCl₂.H₂O (Sigma-Aldrich, 99% purity, copper ≤ 5 ppm); LiOH·H₂O (Sigma-Aldrich, ≥ 99% purity, copper ≤ 5 ppm); sodium ascorbate (Sigma-Aldrich, 98% purity); NaCl (Sigma-Aldrich, 99% purity); KCl (ACP Chemicals, 99%); sodium acetate (Bio Basic, 99% purity); 5-hexyn-1-ol (Sigma-Aldrich, 96% purity); propargyl alcohol (Sigma-Aldrich, 99% purity); 3-azido-7-hydroxycoumarin (AK Scientific 98% purity); azide-PEG3-biotin conjugate (Sigma-Aldrich, purity not specified by the manufacturer); NaOH (Sigma-Aldrich, 99% purity); CuSO₄ (Fisher Scientific, 99% purity); Azido-PEG2-NHS ester (BroadPharm, 98% purity); Fluorescein-NHS ester (BroadPharm, 95% purity); AFDye 546 (Click Chemistry Tools, ≥95%); Lysozyme, from chicken egg white (Sigma-Aldrich, ≥ 98% purity); Nuclease P1 (NEB, 100,000 Units/mL).

Mass spectrometry was carried out in a Bruker maXis Impact Quadrupole Time-of-Flight LC/MS System. For unconjugated lysozyme-(N₃)_n, 1 mg/ml solution was prepared in ddH2O, and loaded directly. For ≡-CLICK-17/lysozyme-(N₃)_n conjugates, the DNA was first cleaved by Nuclease P1 (New England Biolabs, MA, USA) to leave one guanosine (the 5′-most deoxyguanosine of the CLICK-17 sequence) per conjugated DNA still attached to lysozyme.
For mass-spectrometry, in cis conjugation of ≡-CLICK-17 and lysozyme-(N₃)_n, 2 μM ≡-CLICK-17 DNA, 2 μM lysozyme-(N₃)_n, were reacted together at 22°C for 1 h in R Buffer supplemented with 4 μM CuSO₄ and 100 μM sodium ascorbate. The reaction mixture was passed through Microcon-10K centrifugal filter to desalt. The concentrated solution was diluted with ddH₂O, supplemented with 1/10 volume of 10× Nuclease P1 reaction buffer, and 0.1 μl Nuclease P1 per 50 μl reaction. Digestion was carried out at 37°C for 15 min, and the reaction quenched by heating at 75°C for 10 min. The resulting solution was cleaned up by passing through a Microcon-10K centrifugal filter. After adjusting the sample volume to make the concentration of protein ∼1 mg/ml, samples were analyzed by mass spectrometry.

RNA and DNA libraries were prepared from each of the selected samples according to the manufacturer’s instructions with the following exceptions. First, an enzymatic fragmentation was used for the preparation of DNA libraries instead of ultrasonication. The nucleic acid sample (up to 1 μg DNA, as measured by NanoPhotometer) was cut with the Nuclease P1 (New England BioLabs, Ipswich, MA, USA) as described previously [32 (link)], then cleaned with 1.8× AmPure magnetic beads. After the analysis of the fragment size distribution by the 5200 Fragment Analyzer System (Agilent Technologies, USA), if necessary, the sample was additionally cut for 5 min with the NEBNext dsDNA Fragmentase (New England BioLabs, Ipswich, MA, USA). The fragmented DNA (20–100 ng) was then subjected to the library preparation, starting from the end repair and A-tailing step. Second, instead of bead-based normalization, all prepared libraries were measured with the Qubit 4.0 fluorometer and the 5200 Fragment Analyzer System and pooled at equimolar concentrations for sequencing on the Illumina NextSeq 550 instrument.