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. Author manuscript; available in PMC: 2024 Jan 1.
Published in final edited form as: Methods Mol Biol. 2023;2568:25–36. doi: 10.1007/978-1-0716-2687-0_3

Characterizing Fluorescence Properties of Turn-on RNA Aptamers

Robert J Trachman III 1,a, Katie A Link 1,a, Jay R Knutson 1, Adrian R Ferré-D’Amaré 1,*
PMCID: PMC9812286  NIHMSID: NIHMS1856750  PMID: 36227560

Abstract

Fluorescent RNA aptamers are tools for studying RNA localization and interactions in vivo. The photophysical properties of these in vitro selected RNAs should be characterized prior to cellular imaging experiments. Here, we describe the process of determining the fluorophore affinity, fluorescence enhancement, and fluorescence lifetime(s) of the Mango-III fluorescence turn-on aptamer. Parameters determined through these protocols will aid in establishing conditions for live-cell imaging.

1. Introduction

Analogous to fluorescent proteins, fluorescent RNA aptamers are genetically encodable tags useful for imaging RNA localization and interactions in live cells (1,2). Fluorescence turn-on aptamers have been in vitro selected to bind and enhance the fluorescence of small-molecule fluorophores. As such, characterization of binding affinity, fluorescence enhancement (FlE), and fluorescence lifetime are essential for successfully deploying these tags in live cells. Here we describe protocols for determining these three parameters starting with the determination of the dissociation constant (Kd), followed by FlE measurement, and fluorescence lifetime. The sequence of measurements is logical, given that the Kd is used to determine FlE, and both Kd and FlE aid in determining conditions to measure fluorescence lifetime. (see Note: 1.1) Subpopulations of chromophore with ultrafast decay processes from probe flexure that steal potential quantum yield can be deduced from changes in the radiative lifetime, defined as lifetime divided by quantum yield. Such discrepancies are indicative of quasi-static self-quenching (QSSQ) – ultrafast quenching. (3,4). In the following, we specifically describe the characterization of the Mango-III aptamer (5,6) in complex with the YO3-Biotin fluorophore (Mango-III-YO3-Biotin) (7). However, the methods presented should be useful in general for characterization of the fluorescence properties of fluorescent RNA aptamers and fluorescent proteins that bind exogenous fluorophores (8,9).

2. Materials

All solutions were prepared using purified deionized water with a resistivity of 18.2 MΩ cm (see Note 2.0). All solutions were prepared from reagents of >98% purity, and filtered through 0.22 μm membranes.

2.1. In vitro Transcription and RNA purification

  1. DreamTaq DNA polymerase (Thermo Fisher)

  2. C1000 Thermocycler (BioRad)

    10x T7 transcription buffer: 700 mM Tris-HCl pH 7.0 (Fisher), 500 mM MgCl2 (Sigma), 20 mM DTT (Sigma), 0.1% Triton-X (Sigma Aldrich)

  3. Solution of all four NTPs, (ATP, GTP, CTP, UTP, MP biomedical) each at 25 mM, pH 7.0 (neutralized with NaOH)

  4. E. coli. Inorganic pyrophosphatase (Fisher)

  5. 0.5 M EDTA pH 8.0 (pH adjusted with NaOH)

  6. 99% Formamide

  7. 10x TBE buffer: 90mM Tris-Borate (Fisher), 2 mM EDTA (Fisher)

  8. 40% polyacrylamide solution (29:1::Acrylamide:Bis-Acrylamide) (Fisher)

  9. TEMED (Fisher)

  10. Ammonium persulfate (Fisher): 30% (w/v) solution

  11. Elutrap electroelution system (Whatman)

  12. BT1 and BT2 membranes for Elutrap (Whatman)

  13. 3M KCl (Sigma)

  14. RNA buffer: 20 mM MOPS-KOH (Sigma) pH 7.0, 150 mM KCl (Sigma), 10 μM EDTA (Fisher)

  15. Amicon Ultra-15 10 kDa M.W. cutoff centrifugal filter units (Millipore)

  16. Ultrafree-MC centrifugal spin filters, 0.1 μm (Millipore)

2.2. Steady-state Fluorescence Measurements

  1. YO3-Biotin (reference 10)

  2. Cary 300 Bio UV/Vis Spectrophotometer (Varian)

  3. NanoDrop 2000 Spectrophotometer (Thermo Scientific)

  4. Fluorimeter (PTI, JYHoriba) or equivalent

  5. 0.1 cm pathlength quartz cuvette (Starna Cell Inc.)

  6. KaliedaGraph (Synergy Software) or similar fitting program

2.3. Fluorescence Lifetime Measurements

  1. Thin quartz cuvettes (Precision Cells 52H, 0.700 mL capacity, 10 x 2 mm dual pathlength)

  2. Silica, colloidal, 30% (w/v) suspension in water (Sigma-Aldrich)

  3. Fluorescein or carboxyfluorescein, powder (Sigma or Molecular Probes)

  4. Rhodamine 6G, powder (Exciton)

  5. Sodium Hydroxide, pellet (Sigma-Aldrich)

  6. Ethanol, absolute (Sigma-Aldrich)

  7. Coherent Mira 900-D Ti:sapphire laser (76 MHz, fs configuration) pulse picked with electro-optic modulator (Conoptics Model 350-160) and frequency doubled with a 2mm BBO crystal (InRad)

  8. Spectra Physics Vanguard 2000-HM532 NdYVO4 (80MHz) pumping a Spectra Physics Model 3500 (4 MHz) cavity dumped dye laser

  9. JYH10 monochromator (8 nm bandwidth)

  10. Polarizer in emission path, rotatable (Thor Labs)

  11. Neutral density filters (or other attenuators), (Thor Labs)

  12. Colored glass filters (Thor Labs)

  13. Hamamatsu R3809U-50 MCP-PMT (Microchannel Plate – Photomultiplier Tube) detector

  14. 150 TCSPC (Time Correlated Single Photon Counting) module (Becker & Hickl) or “ADCAM” NIM modules for TCSPC (Ortec)

3. Methods

3.1. In vitro Transcription

  1. Perform polymerase chain reaction (PCR) by mixing DreamTaq components as described by the DreamTaq protocol (final volume, 1 mL). Add dNTP mixture to final concentration of 0.2 μM. Add forward and reverse primers, complementary to a T7 polymerase start site and the ~17 complementary nucleotides of the 3′ end respectively, to a final concentration of 2 μM (see Note 3.1a). Perform 20 cycles of PCR on a thermocycler with the annealing step at 58°C.

  2. Add 1 mL of PCR reaction product directly to the transcription reaction. Add 0.5 mL of 10x transcription buffer, 0.5 mL NTP solution, 1 unit/mL (final) inorganic pyrophosphatase, and appropriate amount of T7 RNA polymerase (see Note 3.1b). Add RNase-free H2O to a final volume of 5 mL. Place in 37°C water bath overnight (~10 hours). Add 0.5 mL of 0.5 M EDTA pH 8.0 and vortex. Add 1.5 mL of 98% formamide and place on ice.

  3. Prepare a polyacrylamide gel adequate for a single-lane comb with dimensions 3.8 x 26.7 x 0.3 cm3. To 100 g of urea add 25 mL of 10 x TBE and 94 mL of 40 % polyacrylamide:bisacrylamide (29:1). Adjust volume to 250 mL with water. Stir on magnetic hot plate until urea has dissolved (prepare the slab gel assembly while urea is dissolving), but keep mixture at room temperature or below. Add 400 μL of 30% APS and 150 μL of TEMED while stirring. Pour into slab gel casette avoiding bubbles. After polymerization is complete, remove comb and place gel on apparatus. Pre-run gel at 70 W constant power for ~ 1 hour until thermal equilibrium is reached.

  4. Heat T7 transcription reaction-EDTA-Formamide mixture to 60°C for 5 minutes and load on prewarmed gel. Electrophorese at 70 W for ~ 3 hours. Remove gel from apparatus and disassemble casette. Cover gel in film wrap and place on a fluorescent TLC plate (see Note 3.1c). Under partial darkness, briefly illuminate the gel with a handheld UV lamp (“UV-shadowing”) and excise the band containing the RNA of interest with a clean razor or scalpel.

  5. Elute the RNA from the gel using an Elutrap electroelution system. Place the RNA gel slices between the BT1 (macromolecule impermeable) and BT2 (macromolecule permeable) membranes in the gel cartridge. Fill chambers and submarine apparatus with 1xTBE and run Elutrap overnight at 100 V. Remove RNA/TBE solution from eluate chamber. Increase voltage to 200 V and run for an additional 2 hours. Remove RNA/TBE solution and pool with first eluate. UV-shadowing of gel slices as in step 4 will confirm complete elution of RNA.

  6. Exchange RNA into RNA buffer using a 15 mL Amicon Ultra-15 10 kDa M.W. cutoff centrifugal filter unit. Place RNA solution from the Elutrap (~4 mL) into the centrifugal filter and add KCl to a final concertation of 1M. Centrifuge at 5000 x g until volume is reduced to ~0.25 mL. Add H2O to 15 mL final volume and centrifuge at 5000 x g until volume is reduced to ~0.25 mL. Add RNA buffer to 15 mL final and centrifuge at 5000 x g until volume is reduced to ~0.25 mL (repeat two times for a total of three buffer exchanges). Filter the RNA solution with a 0.1 μm Ultrafree-MC centrifugal spin filter.

  7. Determine the concentration of the RNA using a UV spectrophotometer. Dilute the RNA in water and measure the absorbance at 260 nm and 280 nm. Measure the 260/280 nm ratio to help assess the purity of the sample. Determine the concentration of RNA using the Beer-Lambert-Bouguer law. (see Note 3.1d)

3.2.1. Binding Affinity Measurement by Steady-state Fluorescence

  1. Make an ~0.5 μM solution of YO3-Biotin in RNA buffer and add to 1 cm path length quartz cuvette. Perform an absorbance scan from 240 nm – 680 nm on Cary 300 Bio spectrophotometer. Plot data to determine maximum absorbance wavelength (Absmax).

  2. Add the fluorophore to the RNA buffer so that the final concentration is no greater than the Kd (see Note 3.2b). Add RNA aptamer to a final concentration of ~100 nM and place in 0.1 cm quartz cuvette. Exciting at Absmax, perform a fluorescence emission scan from 590 nm (10 nm longer wavelength than Absmax) to 660 nm, with 1 nm step size and 2 nm bandwidth. Plot data to determine maximum emission wavelength (Emmax).

  3. Determine fluorescence excitation maximum (Exmax) by measuring fluorescence at Emmax while scanning excitation wavelengths over 200 nm shorter wavelength range (410 nm - 610 nm). Plot data to determine Exmax.

  4. Using a NanoDrop Spectrophotometer, make three solutions of the RNA aptamer in RNA buffer ranging in concentration from 20 nM to 2 μM. Anneal the RNA by heating to 95°C for 3 minutes followed by cooling to room temperature in a sample holder on the bench (see Note 3.2.1a).

  5. Add 120 μl of the fluorophore solution assembled in step 3.2.2 and record an initial fluorescence reading by exciting at Exmax and measuring Emmax.

  6. Starting with 20 nM RNA aptamer solution, add 1 μl of RNA solution to the cuvette (mix thoroughly; see Note 3.2.1b) and record fluorescence reading. Repeat with incrementally increasing volume of RNA aptamer added by 1 μl (i.e. 1 μl, 2 μl, 3 μl, etc.) for 5 cycles. After 5 cycles, perform 5 cycles of same titration series with 200 nM stock RNA aptamer solution, followed by the 2 μM. The final volume in the cuvette will be 153 μl.

  7. Correct each fluorescence reading for dilution of the fluorophore. Multiply the fluorescence reading by the ratio of the current volume / initial volume.

  8. Plot the dilution correct fluorescence emission reading (ordinates) vs log10 concentration of RNA aptamer (abcissa). The data points should conform to a sigmoidal shape with two well-defined baselines. If not, adjust concentration of RNA titrants appropriately and repeat titration.

  9. Using KaliedaGraph or a similar program capable of performing non-linear least-suqare analysis, fit the data to the Hill equation (11,12) allowing the Kd and ligand stoichiometry to float. If the concentration of fluorophore at the mid-point of the titration is equal to or greater than the Kd, reduce the concentration of the fluorophore.

3.2.2. Fluorescence Enhancement Measurement by Steady-state Fluorescence

  1. Determine the background signal of the fluorimeter (FlBK) by placing 120 μl of RNA buffer in 0.1 cm pathlength quartz cuvette and measure Emmax.

  2. Add YO3-Biotin dissolved in RNA buffer to a final concentration of approximately 500 nM and measure fluorescence at EmMax. Fluorescence signal should be at least double the background signal. If not, add additional YO3-Biotin to cuvette.

  3. Using the binding isotherm from 3.2.1., estimate the amount of RNA aptamer required to bind 99.99% of all YO3-Biotin in solution. Add prefolded RNA aptamer to the cuvette and take reading at EmMax. Make a correction for the dilution of YO3-Biotin fluorophore by multiplying EmMax by the quotient of the final volume and initial volume. Add additional amount of RNA aptamer to ensure that no free YO3-Biotin is in solution.

3.3. Fluorescence Lifetime Measurement

  1. Initialize instruments and lasers and allow them to come to thermal equilibrium to ensure maximum stability. Tune laser wavelength(s) to the excitation band of the fluorophore-Aptamer complex. (see Note 3.3.1) The Coherent Mira system is used for excitation bands below 490 nm and the Spectra Physics laser system is used for excitation bands between 560-590 nm.

  2. The near-IR fs Mira is tunable from 700-980 nm. To produce a wavelength that overlaps with the excitation band of the fluorophore-Aptamer complex, tune the Mira to double the desired wavelength. Place a beta barium borate (BBO) nonlinear doubling crystal in the optical path after the laser to double the frequency of the light. (see Note 3.3.2) To double the frequency of the light the BBO should be oriented with its principal plane perpendicular to the polarization of the laser. Rotate the crystal until the doubled (second harmonic) beam is visible on fluorescent paper, after filtering the combination of IR and UV beams through a low-pass color filter. With the BBO, the operating wavelengths of the Mira ranges from ~350-490 nm. (see Note 3.3.3) Always wear laser eye protection that attenuates beams of both wavelengths to final powers below .25 mW, in case a direct reflection of either beam to the eye accidentally occurs.

  3. The Mira repetition rate is too fast to collect the entire lifetime of the fluorophore. Use an electro-optic modulator (EOM) to pulse pick and provide sufficient time between pulses to collect the entire (e.g., lifetime x 10) decay response. (see Note 3.3.4)

  4. Prepare a light scattering solution from the colloidal silica stock by diluting the stock 30 % (w/v) suspension with pure deionized water (1:3). Add diluted suspension to one of the thin quartz cuvettes.

  5. Prepare a slightly basic (pH ~8) aqueous solution of NaOH (<0.1 M) and fluorescein (~1 μM). Add ~500 μL to the 10 x 2.0 mm dual pathlength cuvette. Take the fluorescence measurement at or near the peak emission (512 nm). If the detector counting rate is saturated (e.g., >3% of picked laser repetition), dilute further until the detector is no longer saturated. (see Note 3.3.5)

  6. Prepare a dilute (~1 μM) rhodamine 6G solution in ethanol and add ~500 μL to the 10 x 2.0 mm dual pathlength cuvette. Take the fluorescence measurement at or near the peak emission (~620 nm). If the detector rate is likely to ‘pile up’ the paralyzable detection counter (>3% of picked/dumped repetition), dilute further until the detector is no longer saturated.

  7. Prepare room-temperature equilibrated samples of fluorophore bound turn-on RNA aptamers in separate cuvettes and cover with a light blocking material until ready to use. (see Note 3.3.6)

  8. Measure the instrument response function (IRF) with the colloidal silica suspension. Put the colloidal silica cuvette in the sample holder. Attenuate the laser before the cuvette with neutral density filters (>100x) so that the detector is not saturating. In our configuration, both laser sources are vertically polarized so we set the polarizer after the sample and before the detector (emission polarizer) to 0 degrees (vertical) as Rayleigh scattering is parallel to excitation. Take the transient of the silica suspension using the same excitation wavelengths used for the RNA aptamer sample (Rayleigh scattering, excitation=emission).

  9. To set up the sample area for standards and fluorophore samples, set the emission polarizer to the ‘magic angle’ (54.7°). This removes effects from rotational motion of the fluorophore. Use a blocking colored-glass filter (one that absorbs or reflects undesired excitation light but passes longer emission wavelengths) after the sample to prefilter fluorescence light passing through the monochromator to the PMT.

  10. Take a fluorescence decay of the standards, rhodamine 6G when employing Spectra Physics dye laser (560-590 nm) and fluorescein when employing Coherent Mira (<490 nm) at the same wavelengths that will be used for the fluorophores. Excellent signal to noise ratios (SNR) are achieved when the peak counts in the decay exceed 10,000 and the total counts exceed a million. Alternative standard dyes can be used if their excitation and emission profiles are similar to fluorophores of interest.

  11. Take fluorescence decay of RNA fluorophore complex(es) in question. For good SNR, the acquisition time typically targets >5,000 photons/time channel at the peak.

3.4. Fluorescence Lifetime Analysis

  1. Using the colloidal silica scattering solution, determine the ‘lamp’ (IRF) profile. After loading the lamp profile, the standard (in our cases fluorescein or rhodamine 6G) should be analyzed with iterative reconvolution to optimize both the “color shift Q” and the choices for time channels where the fitting should begin and end. “Q” compensates for the small, typically <50 ps, difference in transit times between excitation and emission generated signals in the photon detector (transit time vs. wavelength shift).

  2. Analyze data from fluorophore samples using reconvolution and nonlinear least squares optimization with Decay Fit (or similar) fitting program.

  3. Using the optimized fixed parameters gleaned from standards (e.g., Q-shift), fit the data for the sample to obtain minimal fit “cost parameters”. A good fit will typically have a reduced χ2 (see Note 3.4.1) value below 1.5 and ideally close to 1.

  4. Once a good fit (a reduced χ2 close to 1) has been found using the least number of fitting parameters necessary, find the 90% confidence limits. In this case we will assume that two lifetimes (τ1 and τ2) were needed to get a good fit. To find the 90% confidence limits of τ1, adjust τ1 in the fit by fixing its value to push reduced χ2 ± 10% (see Note 3.4.2) while other τ (lifetimes) and α (amplitudes) remain free. The change in τ 1 required to surpass this limit will be the boundary for the 90% confidence limit. Repeat for τ 2 (and any additional τ if more than 2 were needed).

Fig 1.

Fig 1.

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Steady-state fluorescence characterization of Mango-III-YO3-Biotin. (a) Binding isotherm for Mango-III-YO3-Biotin. Fluorescence measurements were performed at 20°C at an excitation wavelength of 580 nm and an emission wavelength of 618 nm. Acquisitions were obtained with a 2 nm bandwidth and averaged for 2 s. (b)

Fig 2.

Fig 2.

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Fluorescence lifetime measurement. (a) Fluorescence lifetime decay (red points) with fit (dark blue) and lamp (light blue). (b) autocorrelation (c) weighted residuals. The weighted residuals and their autocorrelation are used in addition to the reduced χ2 to determine when the best possible fit is reached.

Acknowledgments

This work was supported by the intramural research program of the National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health. R.J.T. is the recipient of a K22 Career Transition Award from NHLBI.

Footnotes

Note 1.0

Subpopulations of chromophore with ultrafast decay processes from probe flexure that steal potential quantum yield can be deduced from changes in the “radiative lifetime”, defined as lifetime divided by quantum yield. Such discrepancies reveal “QSSQ” – ultrafast quenching.

Note 2.0.

To minimize ribonuclease (RNase) contamination, deionized water can be treated with diethylpyrocarbonate (DEPC). To 4 liters of deionized water, add 1 ml of DEPC. Close the container and shake vigorously. Autoclave for 30’. DEPC is a toxic, alkylating agent. Suitable precautions (personal protective equipment, fume hood) should be observed. Autoclaving destroys residual DEPC, converting it into ethanol and carbon dioxide.

Note 3.1a

In vitro transcription using T7 RNA polymerase often results in non-encoded nucleotide addition to the 3′ end of the transcript. By replacing the first two nucleotides of the reverse PCR primer with their 2’-methoxy analogs, incorporation of additional nucleotides to the 3′ end of the transcript can be reduced. Nucleotide addition can be further reduced by adding DMSO to the transcription reaction (13).

Note 3.1b

T7 RNA polymerase can be obtained from many sources including in-house expression and purification. As each preparation of this enzyme will vary in activity, care should be taken to ensure that the appropriate amount is used that maximizes RNA yield.

Note 3.1c

Fluorescent thin layer chromatography (TLC) plates can be used to detect nucleic acids, which absorb UV light and thus produce a shadow upon illumination. Care should be taken to limit the time in which RNA is exposed to a UV lamp so as to minimize photodamage (14).

Note 3.1d

Determining accurate RNA concentrations is critically dependent on the use of an accurate extinction coefficient. RNA extinction coefficients can be estimated from the RNA sequence; however, this may result in substantial error. A more accurate approach requires empirically determining the extinction coefficient through RNA hydrolysis. Using this approach, the absorbance is measured for a purified RNA and its products of complete alkaline hydrolysis (15). Using the known sequence and extinction coefficients of each nucleotide, the extinction coefficientof the intact RNA can be determined.

Note 3.2.1a

Optimal annealing temperatures and times vary between RNA species. Annealing protocols should be tested prior to experimentation by varying temperature and time assessing folding efficiency by native polyacrylamide gel electrophoresis or size-exclusion chromatography.

Note 3.2.1b

Steady-state thermodynamic parameters can only be determined under reversible binding conditions (at equilibrium). It is important to ensure that the fluorescence signal is stable and that the system has equilibrated prior to taking a measurement. A fluorescence time-course experiment prior to titration should be used to ensure equilibrium (16).

Note 3.3.1:

A fixed diode laser can be used in lieu of a tunable laser if the laser emission of the diode laser overlaps with the excitation band of the fluorophore. The peak emission of the laser does not have to be exactly matched to the fluorophore’s excitation maximum—there just needs to be overlap between the laser emission and excitation band of the fluorophore.

Note 3.3.2

There are many nonlinear crystals in addition to a BBO crystal. A BBO crystal was chosen because of their high damage threshold, their effective range (400-800 nm), and because they are widely available. Some other crystals that may work better for other wavelengths can be found in sources such as (17).

Note 3.3.3:

This reduced repetition rate can alternatively be achieved by cavity dumping a laser with an acousto-optic deflector.

Note 3.3.4:

A precise total volume of solution/suspension in the cuvettes is not critical; rather the solution/suspension must fill the cuvette enough so that the laser is below the meniscus and hitting neither the meniscus nor the bottom of the cuvette.

Note 3.3.5:

RNA must be well in excess over the fluorophore so as to reduce the lifetime fraction of unbound fluorophore state. The excess fraction of RNA required to minimize the unbound fluorophore can be calculated from a binding isotherm.

Note 3.3.6:

Other standards may be used. To choose another standard that is applicable, use a dye that is excited and emits at wavelengths similar to the fluorophore of interest.

Note 3.3.7:

If the detector is saturated, attenuate the signal with neutral density filters until the detector is no longer saturated.

Note 3.4.1:

Reduced χ2 is the sum over all points of actual squared error divided by the expected squared error, then reduced (divided) by the “degrees of freedom” (number of points-number of parameters-1).

3.4.2

The choice of χ2 ± 10% as the F-test limits was driven by using a 90% confidence interval with between 100-1000 channels to fit. More information about calculating confidence intervals can be found (18,19).

Note 3.2b

Given the Kd is unknown, a low concentration of fluorophore that will result in a reasonably large signal when saturated with the fluorescent RNA-aptamer should be used.

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