Ultimate 3000 uhplc

¹H NMR spectra were recorded on either a 400 MHz or 500 MHz Agilent DD2 NMR spectrometer. SEC was performed on a ThermoFisher Ultimate 3000 UHPLC equipped with two Agilent InfinityLab PolyPore columns (7.5 × 300 mm) connected in series at 60 °C. Dimethyl formamide (DMF) with 1 g/L LiBr added was used as the eluent at a flow rate of 1 mL/min. The polymer molecular weight was obtained using a T-rEX refractive index detector (Wyatt Technology) and a Dawn Heleos II (Wyatt Technology) eight angle light scattering detector. DLS including zeta potential measurements were performed using a Zetasizer Pro (Malvern Panalytical).

The dried nuclease digestion was desalted using C18 cleanup columns containing 8 mg of C-18 resin (Pierce), according the manufacturer instructions. The eluted sample was evaporated to dryness, resuspended in 10% acetonitrile in water with 0.1% formic acid, and analyzed by liquid chromatography-mass spectrometry. The LC/MS analysis was performed on a Dionex Ultimate 3000 UHPLC with an Agilent Polaris C18 100 × 2 mm column coupled to a Thermo Scientific Q-Exactive orbitrap mass spectrometer. A gradient of 0.1% formic acid in water to 0.1% formic acid in acetonitrile was used. The mass spectrometer was run with a Top-10 data-dependent acquisition in positive ion mode. The normalized collision energy was set to 27%, which resulted in robust fragmentation of the peptide bonds and consistent cleavage of the glycosidic bond in the crosslinked nucleotides, leaving a uracil modification in the fragment ions.