Figure 5.

Genotyping SNPs. (a) Positive identification of any of four SNP targets (X^A, X^C, X^G, X^U) by a mixture of four HCR systems labeled with spectrally distinct fluorophores. Native PAGE: green channel, H^U labeled with Alexa488; red channel, H^G labeled with Cy3; blue channel, H^C labeled with Cy5; yellow channel, H^A labeled with Alexa750. (b) In isolation, H^U is not selective for cognate target X^A (forming Watson–Crick pair U·A) over off-target X^G (forming nearly isoenergetic wobble pair U·G). The selectivity of H^U is restored by competitive inhibition from H^C, which is selective for X^G. Targets at 2 μM, scRNAs for systems H^U and H^A at 1 μM, scRNAs for systems H^C and H^G at 2 μM to shift the selectivity window earlier in time. Reactions run for 1 h in 1× PKR at 37 °C. (c) Target sequences depicted 5′ to 3′ with nucleotide variants in orange. For each target, the input domain of the cognate detecting scRNA is depicted with the location of the output domain indicated by an ellipsis. See Supporting Information Table S3 for scRNA sequences and label locations; see Supporting Information Section S4 for additional data.