Figure 1: Time-resolved FRET measurements showing a decrease in the fluorescence intensity of the acceptor upon addition of the mir-21 key. The fluorescence intensity of the donor increases when the key is added. The sample was excited at 530 nm and fluorescence was measured at 560 nm (donor) and 665 nm (acceptor).
Figure 2: The black and red curves (excitation at 530 nm) show the fluorescence spectra before and after addition of the mir-21 key, respectively. The blue curves (excitation at 600 nm) show the spectra of the directly excited acceptor, which is shown as a reference to the calculations of the transfer efficiencies and as an indication of the normalization.
Figure 3: Fluorescence spectra before and after addition of the key, of a structure containing only one fluorophore (Cy3). The spectra shows a small decrease in the intensity upon addition of the key, corresponding to a dilution of the sample.
Figure 4: The fluorophores are attached to the 3’ (a) and 5’ (b) ends of a staple strand via linkers that are bound to the 3’-OH and 5’-phosphate groups, respectively. The internally modified staples have fluorophores attached through a linker bound to the methyl group of thymine (c).
Figure 5: Absorption measurements of the four FRET staples. The FRET staples were examined to investigate the fluorophore labeling efficiency.
The concentration of both the DNA and fluorophores in the sample was calculated by measuring the absorption and using Lambert-Beers law. The calculations were done using the wavelengths 260nm for DNA, 550nm for Cy3 and 649nm for Cy5. The extinction coefficients for the DNA were calculated using the calculator on RiboTasks webpage. The sequences used can be found in the Supplementary. The extinction coefficients for the fluorophores were data from previous work. The cuvette used had a path length of 3mm. The concentration of the fluorophore are higher than that of DNA in all four samples, which can be interpreted as there being a surplus of fluorophores in each sample. It is therefore expected that all of the FRET staples to have a fluorophore attached. It is also noted that the difference in concentration indicate a lack of purification after labeling of the staples, which is not a problem because the assembled structure is purified before the FRET measurements.