Extended Data Fig. 5. The KRAS Gl2D-targeting XNAzyme performs G12D-specific mRNA knockdown inside PANC-l cells.

Example droplet digital RT-qPCR (ddPCR) multiplex assays (see Supplementary Table 3) of (a) total KRAS mRNA (channel 1, blue) and reference EIF2B2 mRNA (channel 2, green), and (b) KRAS alleles G12D (c.35G>A) mRNA (channel 1, blue) and wild-type (wt) mRNA (channel 2, green), in RKO cells (homozygous for either G12D or wt KRAS) or PANC-1 cells (heterozygous for G12D and wt KRAS). Together, these results show that the baseline level of total KRAS expression in PANC-1 cells was ~3-fold higher than in RKO KRAS^G12D/G12D, with the G12D allele mRNA transcribed at approximately twice the level of the wild-type KRAS mRNA. (c) Dose response curves showing (bottom graph) total KRAS mRNA levels (relative to reference EIF2B2 mRNA and normalised to cells transfected with buffer alone (yellow circles)) and (top graph) ratio of G12D : wt KRAS mRNA, in PANC-1 cells 3 h after transfection with XNAzyme “FR6_1_KRas12B” (Fz12B)(red circles and dashed line) or the catalytically inactive mutant “FR6_1_KRas12Bi” (Fz12Bi)(black triangles and dashed line) at the concentrations shown. Each datum represents an independent biological replicate and error bars show ddPCR total error. (d) Chart showing proposed interpretation of data from (c); levels of G12D (blue bars) and wt (green bars) KRAS mRNA are equally reduced by RNase H-mediated knockdown (represented as dashed blue and green bars) induced by both the active and inactive XNAzymes (although to a greater degree by the inactive XNAzyme). In the case of the inactive XNAzyme, due to the unequal baseline transcription of G12D and wt KRAS mRNA, this causes an increase in the ratio of G12D : wt KRAS mRNA. With the active XNAzyme - in addition to this effect - allele-specific XNAzyme-mediated knockdown (represented as dashed purple bar) reduces the level of G12D, but not wt KRAS mRNA, maintaining (or reducing) of the baseline ratio of G12D: wt KRAS mRNA.