Kinetex evo c18 column

Dynamic light scattering (DLS) and ζ-potential measurements were carried out in a Zetasizer Nano ZS (Malvern Instruments Ltd.) stabilised at 37 °C. DLS was read at 173° (backscattering) for 60 s in triplicate and ζ-potentials were recorded 30 times at 140 V. TEM images of PIC nanoparticles were acquired on a JEM-1200EX (JEOL USA Inc.). PIC particle size was measured from TEM micrographs using ImageJ software (version 1.48v) and measuring each nanoparticle twice: both in their longest and shortest diameters. Reverse phase HPLC analysis was run through a Kinetex® C18-EVO column (Phenomenex®): 5 μm, 100 Å, 250 × 4.60 mm, stabilised at 35 °C and fitted to a SPD-M20A UV–vis detector (Shimadzu Co.) monitoring absorbance at 210 nm. A Loctite® LED flood array (Henkel Ltd.) operating at 405 nm was used to sterilise the samples studied in the bacterial growth experiment. A FLUOstar Omega (BMG Labtech GmbH.) microplate reader was used to incubate and measure the optical density at 600 nm (OD₆₀₀) in the bacterial growth experiments. Pictures of agar plates were taken on a ChemiDoc™ MP imaging system (Bio-Rad laboratories Inc.).

Nucleosides and nucleotides were quantified by reversed-phase ion-pairing HPLC. Typically, 10 μL of diluted sample containing ∼1 mM of analyte were loaded on a Kinetex C18 EVO column (Phenomenex, Aschaffenburg, Germany; 5 μm, 100 Å, 150 × 4.6 mm). Analytes were separated in 15-min long isocratic runs using 20 mM phosphate buffer, pH 5.9, containing 40 mM tetra-n-butylammonium bromide. The flow rate was 0.25 mL/min and the temperature set to 35 °C. Eluting compounds were detected at 260 nm. A 15-min HPLC trace is shown in Supplementary Fig. 40. Typical retention times were as follows: Ψ (7.1 min), uracil (7.4 min), U (8.7 min), and ΨMP (13.5 min).
To analyze the reaction mixture of conversion of ΨMP to ΨTP, the HPLC method was adapted slightly. A Kinetex C18 column (Phenomenex; 5 μm, 100 Å, 50 × 4.6 mm) was used. The eluting buffer from above additionally contained 12.5% (by volume) acetonitrile. A 5-min long isocratic run at 2.0 mL/min flow rate was used. Under these conditions, the retention times were as follows (Supplementary Fig. 41): Ura (0.3 min), ΨMP (0.4 min), ΨDP (1.1 min), ADP (1.1 min), ΨTP (2.7 min), and ATP (4 min).

dNTP and rNTP concentration (pmol/million cells) for activated CD4⁺T cells were collected from a previous study having 4 replicates, and the precalculated average was used to represent dNTP/rNTP ratios (41 (link)). The nucleotide samples were extracted based on the established protocol (42 (link)) with some modifications. To prepare the nucleotide samples, 2 × 10⁶ cells of HEK293T or hESC-h9 were counted and centrifugated to obtain a cell pellet. The pellet was washed with PBS and then vortexing was performed for 2 min with 200 μl of cold 65% methanol for cell lysis. The cell mixture was incubated at 95°C for 3 min and then incubated on ice for 1 min to complete the cell lysis. By centrifugation at 14 000 rpm for 3 min, the supernatant containing nucleotides was isolated. To quantify the intracellular dNTPs and rNTPs, an ion pair chromatography-tandem mass spectrometry method (43 (link)) was applied, with modifications. Chromatographic separation and detection were performed on a Vanquish Flex system (Thermo Fisher Scientific) coupled with a TSQ Quantiva triple quadrupole mass spectrometer (Thermo Fisher Scientific). Analytes were separated using a Kinetex EVO-C18 column (100 × 2.1 mm, 2.6 μm) (Phenomenex) at a flow rate of 250 μl/min. Pmol/million cells were calculated for rNTPs and dNTPs for all four replicates to calculate the ratios.