Extended Data Fig. 10: Design, characterization, and binding measurements for the 88 nt synthetic and SARS-CoV-2 virus extracted targets.
From: Heteromultivalency enables enhanced detection of nucleic acid mutations
a, Scheme showing the 88 nt original and omicron target sequences, as well as the mutations unique to the omicron target and the primer locations. The 9S1-9S2 n = 2 bead is complementary to the Q498R and Y505H mutations (yellow) while the n = 1 bead is complementary to Q498R, N501Y, and Y505H. Therefore, the n = 2 bead binds the original target with two mismatches, whereas the n = 1 bead binds with three mutations. Primer sequences were designed to not overlap with any mutations, including the Q493R and G496S mutations. b, PAGE gel comparing synthetic 88 nt original and omicron targets to virus extracted original and omicron targets. The left image (DNA) shows ethidium bromide fluorescence, middle image (Atto647N) shows Atto647N fluorescence, and the right image (Merged) shows the two channels merged. c,d, Representative flow cytometry histograms for the n = 1 and 9S1-9S2 n = 2 beads binding 10 nM virus extracted original and omicron targets (c) and 10 nM synthetic 88 nt original and omicron targets (d). Moreover, to assess the impact of target concentration on heteroMV binding specificity, synthetic versions of the same targets were tested at concentrations ranging from 1 to 100 nM. e–i, Measured median fluorescence intensity values for the n = 1 and 9S1-9S2 n = 2 beads binding 1 nM (e), 5 nM (f), 10 nM (g), 25 nM (h), and 100 nM (i) of the synthetic 88 nt original and omicron targets. j, Measured discrimination factors corresponding to the data shown in e-i (P vales: 1 nM = 0.3583, 5 nM = 0.0907, 10 nM = 0.1717, 25 nM = 0.1071, 100 nM = 0.0224). n = 2 beads yielded increased average DF values at each concentration tested, showing that heteroMV beads can be used to improve specificity in a range of target concentrations. Error bars represent standard error of the mean from n = 3 distinct samples. Values were compared using two-sided paired student t tests (nsP > 0.05, *P < 0.05).
