A single natural RNA modification can destabilize a U•A-T-rich RNA•DNA-DNA triple helix

EMSA measurements of the relative stability of 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 the 5'-[³²P]-radiolabel. Representative gel image for R•D-D triple helix containing the (B) m⁵U•A-T base triple and (F) m⁵C•G-C base triple, showing a shift from dsDNA (D-D) to triple helix (R•D-D) as increasing amounts of RNA are added. The binding curve generated from the (C) m⁵U•A-T and (G) m⁵C•G-C gel data points. The relative stability of each R•D-D triple helix with (D) Z•A-T base triple and (H) Z•G-C base triple is shown as bar plots. The relative stability was calculated as K_{D, EMSA}(U•A-T)/K_{D, EMSA}(Z•A-T) in panel D and as K_{D, EMSA}(C•G-C)/K_{D, EMSA}(Z•G-C) in panel H. Each bar color represents a specific RNA nucleobase: pink for C, blue for U, green for A, and orange for G. Reported K_{D, EMSA} values and relative stability values are an average of at least three independent replicates, and error bars represent standard deviation.