PMC

The Probe Synthesis of Near-Infrared Rhodol Dyes (Probes A and B) and Their Structural Changes in Trifluoracetic Acid Treatment or under UV Radiation and Heat Treatment

NAB2 configuration. (A) NAB2 was heat denatured in water and renatured in high-concentration MgCl₂ buffer. Then, NAB2 was digested with the dsRNA-specific RNase III and RNase V1 and single-strand RNA-specific RNase T2. Urea and dyes were added, and the samples were run on a 6% polyacrylamide-urea slab gel under denaturing conditions. Lane 1, NAB2; lane 2, NAB2 digested with RNase III; lane 3, NAB2 digested with RNase T2; lane 4, NAB2 digested with RNase V1; lanes L, RNA ladder (numbers on the left right are in bases). NAB2 is sensitive to the two dsRNA-specific RNases. (B) Electron microscopy analysis of NAB2. Platinum-shadowed nucleic acid molecules were observed in a transmission electron microscope. Analyses revealed a linear molecule without bulges or forks.

Validation of labeling efficiency. (A) Effect of DMSO. Five synthetic miRNAs containing different sequences were either directly labeled (blue) at 0%, 15%, or 25% DMSO or heated prior to labeling (red) at 0%, 15%, 25%, or 30% DMSO. At 25% DMSO, high labeling yields were observed independent of heat denaturation. Fifty-five separate reactions are shown. (B) Evaluation of dye bias. Box plots of labeling efficiencies of 53 different synthetic miRNAs labeled individually with Cy5-pCp and Cy3-pCp are shown. Results from 220 miRNA ligations are shown (see Supplement 1 for all detailed ligation data). The mean yield and standard error was 80±2 for Cy3 and 81±1 for Cy5. ANOVA p value of the Cy3 and Cy5 labeling efficiency distributions was 0.70, showing no difference between the labeling efficiencies of the two dyes. (C) Evaluation of sequence bias. Box plots of ligation efficiencies of the 220 reactions from B categorized according to 3′ nucleotide are shown. The mean percent yields of the labeled product and the standard errors were 82 ± 3 for A, 86 ± 2 for C, 78 ± 2 for G, and 78 ± 2 for U. ANOVA p value distinguishing the four distributions was 0.015, indicating that the labeling bias, while slight, is statistically significant. Ligations in A and B with low yields (<60%) were not reproducible, as subsequent ligations generally resulted in significantly higher yields (>80%). The reasons for these occasional instances of low yields are unclear. Infrequency and lack of reproducibility suggest such low yields are atypical experimental variations. High yields (>90%) were very reproducible.