MST measurements for the relative stability of four unmodified and 11 modified RNA nucleotides in an R•D-D triple helix. Schematic depicting R•D-D triple helix with the varying position (A) Z•A-T and (E) Z•G-C in red. The putative Watson–Crick and Hoogsteen interactions are represented by a solid line (|) and a dot (•), respectively. The asterisk (*) denotes the location of Cy-5. Representative MST traces for the R•D-D triple helix containing (B) m5U•A-T base triple and (F) m5C•G-C base triple at Z•X-Y position. Traces go from light gray to black as the concentration of the RNA third strand is increased. Red bar shows the 10–11 sec time frame where all data are averaged. Representative binding curve generated using MST data points for R•D-D triple helix containing the (C) m5U•A-T and (G) m5C•G-C base triples. Please note that data points were collected up to at least 50,000 nM of the third strand RNA but are not shown in these plots for better visualization of the measurements near the KD, MST (see Supplemental Fig. S2 for plot containing all data points). The relative stability of each (D) Z•A-T base triple and (H) Z•G-C base triple is shown as bar plots. The relative stability was calculated as (D) KD, MST(U•A-T)/KD, MST(Z•A-T) and (H) KD, MST(C•G-C)/KD, MST(Z•G-C). Each bar color represents a specific RNA nucleobase: pink for C, blue for U, green for A, and orange for G. Reported KD, MST values and relative stability values are an average of at least three independent replicates and the associated standard deviation.
