PMC

A) Relative fluorescence yield from SPN2A and 2xSPN2A on a molar basis, B) Binding curves for SPN2A and 2xSPN2A in Buffer S, C) Binding curves for SPN2A and 2xSPN2A in Buffer IC, D) K_ds of DFHBI and PFP-DFHBI for SPN2A and 2xSPN2A in buffers IC and S.

A) Fluorescence spectrum of MGA upon binding 10 µM MG, λ^ex = 630 nm. Dashed line, fluorescence of control RNA with 10 µM MG, B) Relative fluorescence yield from 10 µM MG interacting with MGA, 4xMGA and 8xMGA on a molar basis. Aptamer concentrations were 0.8, 0.2, and 0.08 µM respectively. C) Binding of MG to MGA, 4xMGA and 8xMGA (each normalized to the maximum fluorescence). D) K_ds of MGA (1) 4xMGA (2) and 8xMGA (8) for MG.

A) Chemical structures. B) The ligands were measured for their affinity to the Spinach aptamer by the increase in fluorescence as a function of the ligand concentration in buffer S. C) Fluorescence of DFHBI versus PFP-DFHBI under UV illumination with 254nm UV lamp. D) K_ds of DFHBI and PFP-DFHBI determined as in B in buffer S (black bars) or buffer IC (grey bars). Error bars are the standard deviations of two or more independent replicates under the same conditions.

A) Images of CHO cells transiently transfected with plasmids from which either 4xMGA or control RNA was expressed. The cells were imaged with MG 9 h after transfection, B) Time course of the increase in fluorescence background after the addition of 10 µM MG to the cell medium, C) Mv1Lu cells were transiently transfected with plasmids from which control or SPN1A RNA were expressed from the CMV promoter. All cells were cotransfected with a plasmid from which DsRed was expressed from the CMV promoter. DsRed was used as an internal control to verify transfection of the cells and transcription of RNA from the plasmids. Cells were imaged 24h after transfection.

A) Yeast cells expressing control RNA, SPN1A or 2xSPN2A were incubated at 30°C with DFHBI or PFP-DFHBI and fluorescence and DIC images taken after 60 min, B) The fluorescence images in A, and others from the same experiment, were quantified for fluorescence per cell using ImageJ. The results are shown for each ligand normalized to the average fluorescence per cell of the control RNA-expressing cells. Images of field as higher resolution are shown in . C) Representative images of cells (63x objective with 3.5X zoom in) from the experiment in A to demonstrate that the high background signal is not due to vacuolar uptake of the ligands. D) Images of yeast cells expressing GAL1 promoter-driven 6XPDC IMAGEtags taken 99 min after the promoter was induced by the addition of 2% galactose to cells that had previously been grown for 12 h in 2% raffinose to reach 0.7 OD⁶⁰⁰. The images for Cy3-PDC only, Cy5-PDC only, or Cy3-PDC, Cy5-PDC are of cells that have been incubated with either one or the other PDC ligand or the ligand pair (Cy3-PDC, Cy5-PDC) that interacts in FRET. Images were taken in the Cy3, Cy5 or FRET channels. The DIC is of the field shown for the ligand pair and is representative of all fields in this experiment. E) FRET efficiencies from the experiment in D determined from images taken each 1 or 2 min after induction were quantified for 8 cells expressing control RNA and for 12 cells expressing 6xPDC IMAGEtags and averaged for each group. The average FRET/cell of the IMAGEtag expressing group was divided by the average FRET/cell of the control RNA expressing group and plotted as a function of time after the addition of galactose. F) The chemical structure of Cy3-PDC. Cy5-PDC is identical in structure with the exception that Cy5 replaces Cy3.