Figure 1.

Substrate properties of 6Az-dTTP and DBCO-dTTP in primer elongation catalyzed by HIV-1 RT. All samples contained an 18 nt FAM-labeled primer/template complex (Scheme 1C) and 50 nM HIV RT in reaction buffer (50 mM Tris pH 8.0, 100 mM KCl, 8 mM MgCl₂, 100 mg/mL BSA, and 2 mM DTT). The samples contained 5 mM dTTP, 6Az-dTTP, DBCO-dTTP, and 5 mM dVTP (V is either A, C, or G) added in different orders as indicated (e.g., +₁ means first added). All samples were incubated at 37 °C for 20–30 min at each stage of dNTP addition as described in the Supporting Information followed by analysis by electrophoresis in 15% polyacrylamide gel containing 7 M urea. The product of click reaction is indicated by green arrows (lanes 8 and 9). The primer elongation products with detached DBCO groups are indicated by blue arrows in lanes 6, 10, and 11.