Steady State FRET Assays

Steady-state fluorescence assays are performed to monitor the breakdown of FRET upon substrate cleavage as well as to measure rates of binding and dissociation of the substrate and the cleavage products. The following protocols describe in detail how to perform such assays and quantify the results.

3.3.1. Cleavage Assays

1. Prepare 150 |L substrate solution with 50 nM Cy3-Cy5 doubly labeled substrate in standard buffer (see Note 7).

2. Prepare 7 ||L aptazyme solution with 6 |M aptazyme in standard buffer. Heat at 90°C for 45 s and cool to room temperature for more than 5 min to fold RNA.

3. Prepare 7 ||L theophylline solution with 3 mM theophylline in standard buffer.

4. Incubate solutions at 25°C for 5-15 min.

5. Load 140 |L substrate solution in a clean quartz cuvet, and place it in the sample chamber of the spectrofluorometer, thermostated at 25°C.

6. Average five emission spectra between 550 and 700 nm (8 nm slit-width) at 10 nm/s acquisition rate, while exciting at 540 nm (4 nm slit-width, Fig. 2A). Adjust photomultiplier tube voltage to prevent signal saturation.

7. Average five excitation spectra between 450 and 655 nm (4 nm slit width), at 10 nm/s acquisition rate, while monitoring the emission at 665 nm (8 nm slit-width, Fig. 2B). Adjust photomultiplier tube voltage to prevent signal saturation.

8. Measure a time trace exciting Cy3 at 540 nm (4 nm slit-width), and monitoring simultaneously the emission of Cy3 (565 nm, 8 nm slit-width) and Cy5 (665 nm, 8 nm slit-width) by continuously shifting the emission monochromator between both wavelengths (Fig. 2C, bottom panel). This can be done using the "intracellular probes" function on the AMINCO-Bowman 2 spectrofluorometer. A relative FRET efficiency (Fig. 2C, top panel) is calculated as FRET = FA/(FA + FD), where FD and FA are the donor and acceptor fluorescence emissions, respectively.

9. During the initial 60 s, the relative FRET efficiency should be constant. Then, add 5 pL aptazyme solution to the quartz cuvet and mix manually with the pipet (final aptazyme concentration: 200 nM) (see Note 8). Aptazyme-substrate binding is characterized by a significant decrease in the relative FRET efficiency (Fig. 2C, top panel) as the substrate changes from a random coil conformation to the bound structure (Fig. 1). Some residual cleavage of the substrate in the absence of theophylline also causes the FRET ratio to slowly decrease after binding, with a rate constant k0. Monitor these processes in real-time for 10 min, allowing equilibrium to be reached.

10. Add 5 pL the theophylline solution to the quartz cuvet and mix manually with the pipet (final theophylline concentration: 100 pM). Monitor the theophylline induced change in relative FRET signal for 15 min. The resulting exponential decrease (Fig. 2C) is characteristic of the FRET breakdown upon substrate cleavage and product dissociation (Fig. 1).

11. Fit the measured exponential decrease to the equation y = y0 + Ae-kobst, where A is the amplitude and kobs is the pseudo-first order rate constant.

12. Repeat the experiment for final theophylline concentrations ranging from 250 nM to 10 mM. Plot obs as a function of theophylline concentration, and fit to the equation kobs = k0 + kmax [theo]/([theo] - Ktheo) to derive an apparent theophylline dissociation constant, Ktheo, and a maximum observed rate, kmax (Fig. 2D). The observed rate constant in the absence of theophylline, k0, can be held constant to the experimentally determined value (see Note 9). The binding isotherm obtained by this method provides a calibration curve to calculate the concentration of theo-phylline in an unknown solution.

A good biosensor for theophylline must be able to discriminate between the target molecule, theophylline, and the ubiquitous caffeine. Caffeine and theo-phylline only differ structurally by a single additional methyl group on the N7 of the purine ring (Fig. 1B). An important control experiment is performed using this same protocol but replacing theophylline with 1 mM caffeine. Addition of caffeine does not lead to the characteristic FRET decrease induced by theophylline (Fig. 2C).

3.3.2. Substrate and Product Binding and Dissociation Rates

Desired properties of RNA-based biosensor components include fast substrate binding, slow substrate dissociation, and fast product dissociation; this ensures that ligand-induced substrate cleavage is the rate-limiting step. The binding rates of the substrate and the product can be measured using steady-state FRET, as described in Subheading 3.3.1., and a noncleavable substrate or product analog. Here we describe detailed protocols for such measurements.

3.3.2.1. Substrate Binding Rate Constant

1. Prepare 150 |L substrate analog solution with 5 nM Cy3-Cy5 doubly labeled noncleavable substrate analog in standard buffer (see Note 7).

2. Prepare 7 ||L aptazyme solution with 1.5 ||M aptazyme in standard buffer. Heat at 90°C for 45 s and cool to room temperature for more than 5 min to fold RNA.

3. Incubate solutions at 25°C for 5-15 min.

4. Load 145 |L substrate solution in a clean quartz cuvet, and place it in the sample chamber of the spectrofluorometer, thermostated at 25°C.

5. Measure averaged emission and excitation spectra as described in Subheading 3.3.1., steps 6 and 7.

6. Measure a time trace as described in Subheading 3.3.1., step 8.

7. During the initial 60 s, the relative FRET efficiency should be constant. Then, add 5 |L aptazyme solution to the quartz cuvet and mix manually with a pipet (final aptazyme concentration: 50 nM, see Note 8). Binding of substrate to the aptazyme is characterized by an exponential decrease in the relative FRET efficiency.

8. Fit the measured exponential decrease to the equation y = y0 + Ae-kots', where A is the amplitude and kobs is the pseudo-first order rate constant.

9. Repeat the experiment for final aptazyme concentrations varying between 50 and 100 nM. Plot kobs as a function of aptazyme concentration, and an apparent bimo-lecular binding rate constant, kon = (1.09 ± 0.02) 107 min-1M-1, is derived as the slope (15).

3.3.2.2. Substrate Dissociation Rate Constant

Substrate dissociation rate constants are measured by first forming an aptazyme-noncleavable substrate analog complex, then "chasing" the noncleavable substrate using a large excess of unlabeled DNA analog (of the same sequence as the RNA substrate, but unlabeled) that sequesters free aptazyme liberated upon dissociation of the aptazyme-noncleavable substrate analog complex. our aptazyme-noncleavable substrate analog complex dissociates too slowly to be measured by a chase experiment at room temperature. Therefore, the substrate dissociation rate constant was measured at higher temperatures, and then extrapolated to 25°C using an Arrhenius plot.

1. Prepare 7 ||L DNA solution with 1.5 mM DNA substrate analog in standard buffer (see Note 7).

2. Assemble the ribozyme-noncleavable substrate analog complex as described in Subheading 3.3.2.1., steps 1-7, but modify step 3 to incubate solutions at 35°C for approx 15 min. Allow equilibrium to be reached.

3. Add 5 |L DNA analog solution to the quartz cuvet and mix manually with a pipet (final DNA concentration: 50 |M, see Note 8). Substrate dissociation is characterized by an exponential increase in the relative FRET efficiency.

4. Fit the measured exponential increase to the equation y = y0 + Ae-kojf, where A is the amplitude and koJJ is the dissociation rate constant.

5. Repeat the experiment at temperatures varying between 30 and 40°C. Make an Arrhenius plot by drawing ln(k0jj) as a function of 1/T (K-1). The extrapolation of the dissociation rate constant to 25°C yields an upper estimate for koff 0.21 min-1 (15).

The ratio koff/kon yields an upper estimate for the equilibrium dissociation constant KD 18.8 n M, indicating that bound substrate has a lower probability to dissociate than to be cleaved, as expected for an efficient biosensor component.

3.3.2.3. Product Dissociation Rate Constant

Fast 5' product dissociation rate constants are measured with a chase experiment at room temperature. First the aptazyme-5' product complex is formed with excess aptazyme and then the product is chased with a high concentration of DNA product analog (of the same sequence as the RNA 5' product, but unlabeled).

1. Prepare 150 |L product solution with 5 nM Cy3-labeled 5' product in standard buffer (see Note 7).

2. Prepare 7 ||L aptazyme solution with 24 |M aptazyme in standard buffer. Heat at 90°C for 45 s and cool to room temperature approx 5 min to fold RNA.

3. Prepare 7 ||L DNA solution with 1.5 mM DNA product analog in standard buffer.

4. Incubate solutions at 25°C for 1-5 min.

5. Load 140 |L product solution in a clean quartz cuvet, and place it in the sample chamber of the spectrofluorometer, thermostated at 25°C. Then, add 5 | L aptazyme solution to the quartz cuvet and mix manually with the pipet (final aptazyme concentration: 800 nM, see Note 8). Allow to equilibrate.

6. Measure a time trace exciting Cy3 at 540 nm (4 nm slit-width), and monitoring Cy3 emission at 565 nm (8 nm slit-width).

7. During the initial 60 s, the Cy3 emission should be constant. Then, add 5 | L DNA solution to the quartz cuvet and mix manually with the pipet (final product analog concentration: 50 | M). Product dissociation is characterized by an exponential decrease (quenching) in Cy3 emission.

8. Fit the measured exponential decrease to the equation y = y0 + Ae-kojf, where A is the amplitude and k0jf = 4.2 min1 is the 5' product dissociation rate constant (15).

Fig. 4. Time-resolved fluorescence resonance energy transfer (FRET) reveals slight changes in global conformation of the aptazyme-non-cleavable substrate analog complex upon theophylline addition. (A) Normalized fluorescence decay of the donor fluorophore (Cy3) in the absence (gray) and in the presence (black) of the acceptor fluorophore (Cy5). In the presence of the acceptor, the donor excitation decays more rapidly owing to FRET. (B) Resulting donor-acceptor distance distribution, P(R), in the absence (dashed line; mean distance = 37 ± 1 A, full width at half maximum = 9 ± 2 A) and in the presence (solid line; mean distance = 36 ± 1 A, full width at half maximum = 12 ± 2 A) of theophylline. The global structure of the theophylline aptamer changes only slightly in the presence of theophylline. See text for experimental details.

Fig. 4. Time-resolved fluorescence resonance energy transfer (FRET) reveals slight changes in global conformation of the aptazyme-non-cleavable substrate analog complex upon theophylline addition. (A) Normalized fluorescence decay of the donor fluorophore (Cy3) in the absence (gray) and in the presence (black) of the acceptor fluorophore (Cy5). In the presence of the acceptor, the donor excitation decays more rapidly owing to FRET. (B) Resulting donor-acceptor distance distribution, P(R), in the absence (dashed line; mean distance = 37 ± 1 A, full width at half maximum = 9 ± 2 A) and in the presence (solid line; mean distance = 36 ± 1 A, full width at half maximum = 12 ± 2 A) of theophylline. The global structure of the theophylline aptamer changes only slightly in the presence of theophylline. See text for experimental details.

The dissociation rate constant of the 3' product could not be measured because it is too fast. Such fast dissociation of both products is ideal to ensure that theophylline-induced substrate cleavage is rate-limiting for the overall reaction pathway (Fig. 1A).

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