From the HPLC run the Uridine signal is measured at 260 nm and the S4U signal is measured at 330 nm. To then calculate the S4U incorporation rate, first determine the area under the curves for Uridine and S4U and determine the extinction coefficients for each nucleotide from the HPLC analysis.
For the calculation, divide the area under the curve by the extinction coefficients for each nucleoside: S4U (at 330 nM) and Uridine (U) at 260 nM, and multiply these together to give the incorporation rate as the percentage of Uridine substituted by S4U.
% U substituted by S4U = (Area4SUArea4SU,330 nm/ε4SUε4SU,330 nm) × (εUεU,260 nm/AreaUAreaU,260 nm) × 100 Anchor