Neu a

Nanoparticles were incubated with neuraminidase A (Neu A, New England biolabs, Inc., Beverly, MA, USA), neuraminidase S (Neu S, New England biolabs, Inc.), or PNGase F (New England biolabs, Inc.) at 37 °C for 18 h. Proteins were transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Waltham, MA, USA). After blocking with 5% (w/v) bovine serum albumin in Tris-buffered saline with 0.05% TWEEN 20 (TBST) for 1 h at 25 °C, membranes were probed with monoclonal mouse anti-flag (1:1000, Sigma-Aldrich, St. Louis, MO, USA) antibodies, biotinylated Sambucus nigra agglutinin (SNA, Vector Laboratories, Burlingame, CA, USA), or biotinylated Maackia amurensis lectin-I (MAL-I, Vector Laboratories, Burlingame, CA, USA) as a primary staining, and then with horseradish peroxidase-conjugated goat anti-mouse IgG (Bioss Antibodies, Woburn, MA, USA) and streptavidin at 1:5000 dilution in TBST. Membranes were developed using ECL substrate (Thermo Fisher Scientific, Waltham, MA, USA) and signals were detected with Fusion Solo X (Vilber, Paris, France).

NMR was performed by the Complex Carbohydrate Research Centre (Athens, Georgia). A solution was prepared in a 5-mm NMR tube with 320 μl of 1.78 mg/ml 4MU-α-NeuAc in D₂O (2 mm final concentration after addition of enzyme), 60 μl of 200 mm sodium phosphate in D₂O, pH 7.4 (20 mm final concentration after addition of enzyme), and 190 μl of D₂O. The solution was mixed, and the NMR tube placed into a 600-MHz Inova NMR spectrometer (Agilent Technologies, Santa Clara, CA) at 25 °C. A one-dimensional proton NMR experiment with water presaturation (using the two-step purge option) was recorded as the t = 0 measurement. Then 30 units of ORF12p–His sialidase or 240 units of NeuA (New England Biolabs) was added, the NMR tube was inverted several times to ensure good mixing, and the tube was replaced into the NMR spectrometer. One-dimensional proton spectra with water presaturation were recorded at time intervals with 32 transients each. Chemical shifts were referenced relative to the residual HDO peak, set at 4.78 ppm.