The assay was designed as previously described17 (link), with some adjustments. In short, we
digested our synthesized pLCMV-NanoLuc plasmid with NheI and PflFI to remove the
NanoLuc start codon region. We then ligated in annealed oligos containing a G-G
mismatch in the third position of the NanoLuc start codon; this was referred to
as our “Mismatch plasmid.” A set of annealed oligos with a WT
start codon was also ligated in to be used as a normalization control. The
annealed oligos also contained a disrupted HpaI restriction site, allowing us to
specifically eliminate any uncut parental pLCMV-NanoLuc plasmid (which contains
an intact HpaI site directly upstream of start codon) following plasmid
ligation. Validation of the MMR activity assay was done using A549 cells
transfected with MSH2 and MSH6 siRNA. For the comparison of A549 and H441 cells,
the cells were infected at MOI=10, transfected with 0.5 μg of the WT or
Mismatch plasmid at 24 hpi, and then analyzed at 16 h post-transfection. To
measure NanoLuc expression, cells were lysed with Luciferase Cell Lysis Buffer
(NEB) and then evaluated using the Nano-Glo Luciferase Assay Reagent Kit
(Promega).
digested our synthesized pLCMV-NanoLuc plasmid with NheI and PflFI to remove the
NanoLuc start codon region. We then ligated in annealed oligos containing a G-G
mismatch in the third position of the NanoLuc start codon; this was referred to
as our “Mismatch plasmid.” A set of annealed oligos with a WT
start codon was also ligated in to be used as a normalization control. The
annealed oligos also contained a disrupted HpaI restriction site, allowing us to
specifically eliminate any uncut parental pLCMV-NanoLuc plasmid (which contains
an intact HpaI site directly upstream of start codon) following plasmid
ligation. Validation of the MMR activity assay was done using A549 cells
transfected with MSH2 and MSH6 siRNA. For the comparison of A549 and H441 cells,
the cells were infected at MOI=10, transfected with 0.5 μg of the WT or
Mismatch plasmid at 24 hpi, and then analyzed at 16 h post-transfection. To
measure NanoLuc expression, cells were lysed with Luciferase Cell Lysis Buffer
(NEB) and then evaluated using the Nano-Glo Luciferase Assay Reagent Kit
(Promega).