Incorporation of 2′-F,Me-UTP, 3′-F-dTTP, TFV-DP,
and 3′-N3-dTTP by SARS-CoV-2 RdRp to terminate the
polymerase reaction. The sequences of the primer and template used
for these extension reactions, which are at the 3′ end of the
SARS-CoV-2 genome, are shown at the top of the figure. Polymerase
extension reactions were performed by incubating (a) 2′-F,Me-UTP,
(b) 3′-F-dTTP, (c) UTP + TFV-DP, and (d) 3′-N3-dTTP with preassembled SARS-CoV-2 polymerase (nsp12, nsp7, and nsp8),
the indicated RNA template and primer, and the appropriate reaction
buffer, followed by detection of reaction products by MALDI-TOF MS.
The accuracy for m/z determination
is ±10 Da.