Notes

1. It is important to know all the DNA species and the relative population of each species present in a system to correctly interpret FRET data. Purify the desired species if necessary. For example, in Fig. 5, a nondenaturing gel image of the annealed substrate and enzyme of the DNAzyme (see Fig. 1) is shown. In the experiment, the FAM-labeled substrate is in excess. Besides the excess FAM and the desired DNAzyme complex, dimers composed of two substrates and two enzyme strands are also observed. Therefore, for FRET experiments, the desired DNAzyme complex is physically isolated from other species by cutting the band.

2. The melting property of the DNA complex should be determined. If the melting temperature is much higher than room temperature, samples can be handled at

Fig. 5. Fluorescence image of a nondenaturing gel showing the species formed by annealing the substrate and enzyme. The same gel is scanned twice by exciting at two different wavelengths. Lane 1 is loaded with the FAM-labeled substrate only, lane 2 is loaded with TMR- and Cy5-labeled enzyme only, and lane 3 is loaded with the annealed products of the substrate and the enzyme. From the gel image excited at 473 nm, three bands are observed. The lowest band is the substrate in excess. The middle band is the desired DNAzyme complex as shown in Fig. 1, whereas the higher band is the dimer formed by two substrate strands and two enzyme strands.

Fig. 5. Fluorescence image of a nondenaturing gel showing the species formed by annealing the substrate and enzyme. The same gel is scanned twice by exciting at two different wavelengths. Lane 1 is loaded with the FAM-labeled substrate only, lane 2 is loaded with TMR- and Cy5-labeled enzyme only, and lane 3 is loaded with the annealed products of the substrate and the enzyme. From the gel image excited at 473 nm, three bands are observed. The lowest band is the substrate in excess. The middle band is the desired DNAzyme complex as shown in Fig. 1, whereas the higher band is the dimer formed by two substrate strands and two enzyme strands.

room temperature. To prevent dissociation of the DNA complex, make sure that the sample is not exposed to high temperatures after nondenaturing gel purification.

3. Factors that quench fluorophores should be taken into consideration. For example, for metal-induced folding studies, it is important to titrate metal ions into singly labeled samples under the same experimental conditions. If significant quenching is observed, corresponding corrections need to be made depending on the type of quenching (see Subheading 3.3.6.). Sometimes, the bases on the backbone of single-stranded DNA can quench fluorophores. In the presence of metal ions, single-stranded DNAs tend to fold into more compact conformations, which may quench the attached fluorophores. This should be distinguished from quenching induced by metal ions. To avoid this artifact, prepare DNA as a duplex.

4. In a dual-labeled system, FRET efficiency (E) is used to assess folding of macro-molecules. According to Eq. 1, the larger the E, the shorter the distance (R), as long as R0 is kept constant. However, for a multi-fluorophore system, because the presence of additional fluorophores quenches donor fluorescence (dynamic quenching) and changes R0, E is no longer strictly dependent on R. Corrections must be made to obtain correct R values. For example, from Eqs. 15 and 16, the distance between a FRET pair is not only a function of the FRET efficiency of this pair, but also a function of FRET efficiency of the other pair(s). Therefore, for a multi-fluorophore system, R instead of Eshould be plotted to assess folding.

5. Choice of fluorophore: to acquire accurate results from multi-fluorophore FRET experiments, it is important to choose appropriate fluorophore combinations. The most important criterion is that when exciting an acceptor, fluorophores that absorb at shorter wavelength (donors) are not excited. This requires the absorption peak of each fluorophore to be well separated. On the other hand, when a donor is excited, acceptors can absorb and fluoresce, which is taken into account in the (ratio)A method (see Subheading 3.3.2.). Using the FAM-TMR pair as an example, when exciting TMR (the acceptor) at 560 nm, FAM has no absorption or fluorescence. However, when exciting FAM (the donor) at 490 nm, even though TMR has some absorption at 490 nm, the FAM-TMR is still a good FRET pair for using the (ratio)A method. For the (ratio)A method, quantum yield of acceptors is not important for the calculation. Therefore, it is suggested to design fluorophores that might have quenching or other problems as acceptors. To acquire information for all FRET pairs, efficient energy transfer should occur between any fluorophore pair. According to Eq. 2, it is important to place the pair with the smallest R0 (least spectra overlap) at the closest distance. Usually, the change of FRET efficiency is not sensitive to the change of distance when the distance exceeds 2R0. Sometimes, however, it is designed to place the two fluorophores with the smallest R0 the farthest, so that no energy transfer occurs for that pair. As a result, the calculation is simplified at the expense of losing information for one pair.

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