*2.8. Single Nucleotide Incorporation Assay*

To prepare the double-stranded RNA substrate, primer (5- -AGUUGUUGAUC3- ) was 32P-labeled at the 5 position upon incubation with [γ-32P] ATP (PerkinElmer, Waltham, MA, USA) and T4 polynucleotide kinase (NEB, Ipswich, MA, USA) at 37 ◦C for 30 min. Excess [γ-32P] ATP was removed with a Bio-Spin 6 column (Bio-Rad, Hercules, CA, USA). After heat inactivation, the 5- - 32P labeled primer was combined with an excess template (5- -AUUCACUCAGAUCAACAACU-3- ). The primer and the template were annealed via step incubation at 95 ◦C (5 min), 55 ◦C (15 min), and 37 ◦C (10 min) to generate the primer/template substrate (P/T substrate), where the next correct nucleotide to be incorporated was UTP.

Single nucleotide incororation assays were modeled from previous work [19]. Briefly, ZIKV RdRP (1 μM) and P/T substrate (200 nM) were pre-incubated at 37 ◦C for 20 min. Reactions were initiated at 37 ◦C with the addition of 150 μM UTP or analog in 50 mM HEPES pH 7.5, 15% glycerol, 15 mM NaCl, 7.5 mM MnCl2, and 10 mM dithiothreitol. At specified time points, reaction aliquots were quenched with ethylenediamine tetra-acetic acid (EDTA) to a final concentration of 0.3 M.

Prior to loading equi-volume samples on a 20% polyacrylamide, 8 M urea, TBE (89 mM Tris-Borate, 2 mM EDTA) denaturing gel, the quenched samples were combined with dye (formamide, 0.01% xylene cyanol, 0.01% bromophenol blue) and 1 μL of 5 μM D10 (5- -AGTTGTTGAT-3- ) trap, followed by incubation at 95 ◦C for 5 min. The trap was used to bind to RNA template to reduce gel smearing [30]. Oligos were separated via electrophoresis at a maximum of 1900 V and 75 W for 3–6 h. Phosphorimaging (FLA 9500 imager, GE, Marlborough, MA, USA) was used to visualize the bands, and bands were quantified using ImageQuant software (GE). Plots of percent incorporation versus time were fit to a

single exponential equation using Prism (Graphpad Software, San Diego, CA, USA) to compare relative incorporation. While single-turnover conditions were used, non-physiologically relevant rates were obtained, as incorporation occurred during a steady-state time scale. This has been a common issue in the field when measuring ZIKV RdRP incorporation [19,20,31] due to measuring incorporation into a double-stranded template instead of physiologically preferred de novo RNA replication (an issue seen in HCV incorporation assays [32]), and perhaps the presence of purification tags [33]. However, since here we were interested in comparing relative incorporation efficiency rather than obtaining rate constants, we and others [19,31] have found this experimental set-up ideal and better suited to scaling up the number of analogues to be assessed.
