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. 2021 Jul 2;478(13):2425–2443. doi: 10.1042/BCJ20210200

Figure 3. Optimisation of assay conditions for HTS.

Figure 3.

(A–C) Strand displacement assay using insect cell expressed (Sf) nsp12-HF/7L8 at the indicated concentrations alone (A) or after pre-incubating at 22°C for 30 min with Ec nsp7 and Ec nsp8 (ratio 1 : 3 : 6) (B) or with Sf 7H8 (ratio 1 : 6) (C). As control, a condition without NTPs using 200 nM RdRp was included. (D) Optimisation experiment to decide on RdRp concentration using HTS conditions (100 nM strand displacement substrate, 300 µM NTPs). Sf nsp12-HF/7L8 was preincubated at 22°C for 30 min with Sf 7H8 at either 1 : 3 or 1 : 5 ratio as specified and tested in the strand displacement assay at the indicated concentrations. Under these conditions, the unquenched Cy3 template alone had an average fluorescence value of 50 × 103 a.u. (E) Reaction velocities extracted from the curves shown in (D).