For assembling stable nsp7Cnsp8 complex, purified nsp7 was incubated with nsp8 at a molar ratio of 1 1:1 for 5?h on ice. of being developed into one of the much-needed SARS-CoV-2 therapeutics. Ni-NTA (NTA) biosensors (Cat# 18-5101, ForteBio) at a concentration of 150?g/mL, resulting in a saturation response of 5C6?nm after 300?s. Subsequently, the loaded biosensors were washed for 3?min in buffer to clear up loose nonspecifically bound SARS-CoV-2 RdRp and to establish a stable baseline. For binding kinetic measurements, the association of SARS-CoV-2 RdRp and tested compounds (3.125C100?mol/L in assay buffer) was measured for 60C180?s and the dissociation of them was measured for 120?s in assay buffer. Reference wells that utilized buffer instead of tested compounds were also included to correct the baseline shift. A parallel set of Ni-NTA sensors that were incubated in buffer-only were prepared as the negative reference controls to correct the non-specific binding of the compounds to the biosensor surface. Raw kinetic data were analyzed using a double reference subtraction approach in which both the background and non-specific binding were subtracted. The binding affinity constant BL21 (DE3) cells transformed with these plasmids were cultured at 37?C in IBMX lysogeny broth (LB) media containing 100?mg/L ampicillin until IBMX the optical density at 600?nm (for 20?min, resuspended in buffer [20?mmol/L Tris-HCl (pH 8.0), 300?mmol/L NaCl, 4?mmol/L MgCl2, 2?mmol/L DTT], and lysed by sonication. Finally, the cell debris were removed through centrifugation at 10,397for 20?min. For purification of nsp12, the supernatant was first subjected to the HisTrap column (GE Healthcare) to capture the target protein, and then purified bypassing through a HiTrap Q ion-exchange column (GE Healthcare). The eluate was subjected for further purification by loading onto a Superdex 200 10/300 Increase column (GE Healthcare) in buffer containing 20?mmol/L Tris-HCl (pH 8.0), 150?mmol/L NaCl, 4?mmol/L MgCl2, 2?mmol/L DTT. The final product of nsp12 was concentrated to a minimum DCN of 5?mg/mL and stored at ?80?C for further use. For purification of nsp7, nsp8, and nsp7C6HisCnsp8, the target proteins were first purified through Ni-NTA affinity chromatography by loading onto the Histrap excel column (GE Healthcare, USA) and further purified by passing through a Hitrap Q ion-exchange column (GE Healthcare, USA). The final products were concentrated to more than 10?mg/mL and store at ?80?C. 2.6. In?vitro polymerase activity assay The measurement of SARS-CoV-2 polymerase activity was performed as previously described with slight modifications7. The activity IBMX assays were performed in the reaction buffer containing 20?mmol/L Tris-HCl (pH 8.0), 10?mmol/L KCl, 1?mmol/L DTT, and 4?mmol/L MgCl2. For assembling stable nsp7Cnsp8 complex, purified nsp7 was incubated with nsp8 at a molar ratio IBMX of 1 1:1 for 5?h on ice. Primers labelled with FAM (5?-FAM-GUCAUUCUCCUAAGAAGCUA-3?) were annealed to the complementary template (5?-CUAUCCCCAUGUGAUUUUAAUAGCUUCUUAGGAGAAUGAC-3?) with a ratio of 1 1:1 by heating at 70?C for 10?min and then cooling down to room temperature. To perform polymerase activity assay, nsp12 (1?mol/L), nsp7 (2?mol/L), and nsp8 (2?mol/L) or nsp7C6HisCnsp8 (2?mol/L) were incubated with 0.25?mol/L annealed template IBMX and 1?mmol/L NTP in reaction buffer for 30?min?at 30?C. To examine the inhibitory effect of compounds against SARS-CoV-2 polymerase activity, the selected compounds were incubated with nsp12 for at least 1?h on ice prior to performing the primer extension assay. Afterward, 1?mol/L nsp12 and 2?mol/L nsp7C6HisCnsp8 were incubated with 0.25?mol/L annealed template and 1?mmol/L NTP in the presence of compounds (2.5, 10, and 40?mol/L) in reaction buffer for 30?min?at 30?C. All samples were mixed with 2??RNA loading buffer and denatured by boiling at 100?C for 10?min. The products were separated in 20% polyacrylamide gel electrophoresis (PAGE) containing 8?mol/L urea. Images were collected with Bio-Rad ChemiDoc MP Imaging System and quantified by ImageJ software. In addition, the same samples were stained with Cybr Gold dye to mark the 20 nucleotides (nt) primer and the 40?nt template on PAGE. 2.7. Cell-based SARS-CoV-2 polymerase activity assay We established a cell-based SARS-CoV-2 RdRp reporter assay system by modifying the previously developed system40,41. The plasmids nsp12,.