Materials and Reagents

1.5 ml Eppendorf tube
Semi-micro quartz cell 108F-QS (Sigma-Aldrich, Hellma Analytics, catalog number: HL108-F-10-40 )
1 mm quartz cell 350 ml (Sigma-Aldrich, Hellma Analytics, catalog number: HL110-F-1-40 )
Cacodylic Acid (Sigma-Aldrich, catalog number: C0125-5G ). Store at 4 °C
KOH in pellets, BioXtra (Sigma-Aldrich, catalog number: P5958 )
RNase-free water (Sigma-Aldrich, catalog number: W4502 )
RNA oligonucleotide with 5’-FAM and 3’-TAMRA fluorescent probes (5’-FAM-AAG AGA GCU UAA UUG UCA GUU UAU UCU CUG-3’-TAMRA in case of the PIF7 hairpin); the RNA sequence should cover the entire predicted hairpin-forming sequence, but should not exceed more than 3 nucleotides beyond this sequence on either side to ensure efficient FRET (IBA Gmbh, HPLC Purified). Store at 4 °C
Cacodylate buffer (see Recipes)
1 M KOH (see Recipes)

Equipment

2 L glass beaker
Stirring bar
SevenCompact pH meter S220 (Mettler Toledo, model: 30019029 )
-80 °C freezer
Varian Cary Eclipse Fluorescence Spectrometer fitted with a Varian temperature control unit (Agilent, Varian, Cary Eclipse Fluorimiter)
ThermoMixer C (Eppendorf, model: EP5382000015 ) fitted with SmartBlock 1.5 ml (Eppendorf, model: EP5361000031 )
Applied Photophysics Chirascan (CD) fitted with temperature control unit (Applied Photophysics, Chirascan^TM)

Software

Cary Eclipse Kinetics software (Agilent, https://www.agilent.com/en/product/molecular-spectroscopy/fluorescence-spectroscopy/fluorescence-software/cary-eclipse-software)
GraphPad Prism 8 (GraphPad Software, https://www.graphpad.com/scientific-software/prism/)

Procedure

60 mM Cacodylate Buffer Preparation
Notes:
1. It is important to wear appropriate PPE whilst performing buffer preparation, including nitrile gloves, corrosive resistant lab coat and safety goggles.
2. Cacodylic Acids contains Arsenic and therefore is toxic and should be disposed in dedicated heavy metal waste collection.
1. Weight 8.280 g of Cacodylic Acid in a 2 L glass beaker and dissolve it in 500 ml of RNase free water, ensuring all white powder is fully solubilized. If needed, use additional water up to a total volume of 700 ml to ensure full solubilization of the Cacodylic Acid.
2. Measure the pH of the Cacodylic Acid solution generated in Step A1 with any standard laboratory pH-meter, ensuring that calibration of the pH-meter has been performed on the same day. The measured pH of the starting solution is expected to be ~4.0.
3. Prepare a 1 M solution of potassium hydroxide (KOH) by dissolving 5.61 g of KOH in 100 ml of RNase free water. Given the highly exothermic reaction caused by KOH, dissolving in water is recommended to perform in an ice bath.
4. With a glass pipette add 1 M KOH solution dropwise to the Cacodylic Acid solution while stirring the solution with a stirring bar over a magnetic stirring plate. Measure the pH after each drop addition until it stabilizes at 7.4, which is the final pH required for both CD and FRET experiments.
5. Add RNase-free water to the Cacodylate buffer (pH 7.4) to a total volume of 1 L.
6. Place the solution at 4 °C for long term storage. Buffer storage at 4 °C should not exceed 3 months. After this time, preparation of fresh buffer is strongly recommended.
RNA Annealling
Note: It is important to anneal the RNA sequence the day before the experiment is performed to allow sufficient time for the correct RNA structure to be properly folded.
1. Resuspend the dually labelled RNA oligonucleotide in the appropriate volume of RNase-free water to obtain a stock solution with the final concentration of 100 μM. To achieve this, add a volume of water that is 10× with respect to the total numbers of RNA nanomoles. For example, for 5 nmol of RNA add 50 μl of RNase free water. Store this solution at -80 °C for long term storage.
2. Dilute RNA as follows:
  1. For FRET measurements, dilute 1 μl of the 100 μM RNA stock solution in 499 μl (200 nM final concentration) of 60 mM Cacodylate Buffer (pH 7.4) in a 1.5 ml Eppendorf tube.
  2. For CD measurements, dilute 10 μl of the 100 μM RNA stock solution in 190 μl (5 μM final concentration) of 60 mM Cacodylate Buffer (pH 7.4) in a 1.5 ml Eppendorf tube.
3. Anneal the RNA solution prepared in Step B2 by heating the Eppendorf tube containing the solution at 90 °C for 10 min using the Eppendorf Thermomixer equipped with the 1.5 ml SmartBlock. Prepare an ice box while the RNA solution is heating and immediately place the RNA-solution in ice after the 10 min heating cycle is completed.
4. Store the RNA-solution at 4 °C overnight before performing any FRET or CD measurements.
FRET protocol
In this experiment it is possible to gain insight into the distance between the 5’ and the 3’ ends of the RNA sequence using FRET. To achieve this, excite the FAM fluorophore (placed at the 5’- end of the sequence) at 488 nm and record the emission between 500 and 700 nm, covering the emission peaks of both the FAM and the TAMRA (placed at the 3’ end of the sequence) fluorophore.
1. Transfer 500 μl of the RNA solution prepared in Step B2a in the Semi-micro quartz cell 108F-QS cuvette and insert it in the Cary Eclipse Fluorimiter pre-equilibrated at the temperature of 17 °C.
2. After equilibrating the cuvette in the fluorimeter for 10 min record the fluorescence emission using the following parameters:
  Excitation wavelength 488 nm;
  Emission range 500-700 nm;
  Excitation slit 5 nm;
  Emission slit 5 nm;
  Detector Voltage High.
3. Increase the temperature of the Cary Variant heating block at 27 °C and once the temperature has been reached equilibrate the sample for at least 10 min.
4. Record the fluorescence emission using the following parameters:
  Excitation wavelength 488 nm;
  Emission range 500-700 nm;
  Excitation slit 5 nm;
  Emission slit 5 nm;
  Detector Voltage High.
5. Repeat Steps C2 to C4 as appropriate. Due to the RNA’s relatively low stability this cycle can be repeated for a maximum of 12-14 times. Representative spectra are displayed in Figure 1A.
  
  Figure 1. Temperature-dependent hairpin dynamics measured as changes in FRET efficiency. A 5’-FAM and 3’-TAMRA fluorescently labelled 32 nt RNA oligonucleotide harbouring the hairpin sequence was used for the experiment. Emission spectra between 500 and 700 nm (A) were recorded upon excitation at 488 nm over multiple shifts between 17 and 27 °C; peak emissions at 520 and 578 nm were used to calculate FRET efficiencies (B). FRET efficiency is calculated using the following equation: [Fa/(Fa + Fd)] × 100, where Fa is the emission of the acceptor (FAM) and Fd is the emission intensity of the donor (TAMRA).
CD measurement
In this experiment the overall secondary structure adopted by the RNA under different conditions will provide a characteristic CD signature that can be monitored in real time. Spectra with a maximum at ~270 nm and a minimum at ~210 nm are indicative of classical RNA hairpin structures.
1. Transfer 200 μl of the RNA prepared in Step B2b into a 1 mm quartz cell and place it in the Chirascan^TM instrument pre-equilibrated at the temperature of 17 °C.
2. After equilibrating the cuvette in the Circular Dichroism machine for at least 10 min record the CD intensity (reported as molar ellipticity θ) between 300 and 180 nm using the following parameters:
  Record 1 point per nm;
  Bandwidth 0.5 nm;
  Time per point 1 s.
3. Acquire the CD spectra at 17 °C for 3 times consecutively.
4. Raise the Chirascan^TM to 27 °C and equilibrate the sample at this temperature for at least 10 min.
5. Record the CD intensity between 300 and 180 nm using the following parameters:
  Record 1 point per nm;
  Bandwidth 0.5 nm;
  Time per point 1 s.
6. Acquire the CD spectra at 27 °C for 3 times consecutively.
7. Repeat Steps D1 to D6 with 200 μl of 60 nM Cacodylate Buffer to record a baseline.

Data analysis

For FRET measurements, divide the recorded emission at 578 nm (TAMRA emission peak) by the sum of the recorded emission at 578 nm and 520 nm (FAM emission peak) for each temperature (Figure 1B); this ratio is inversely correlated with the distance between the two fluorophores (i.e., the ends of the RNA oligonucleotide). A reduction in FRET efficiency thus reflects a (partial) opening or destabilisation of the hairpin structure.
For CD spectroscopy, average the three replicate measurements for each temperature to reduce background noise and subtract the baseline recorded at the respective temperature. Plot the baseline-corrected CD intensity versus wavelength and overlay the two spectra obtained in Steps D3 and D6 to visualize structural differences between the RNA hairpin structures at 17 and 27 °C (Figure 2). Hairpin-type characteristic signals display a detectable maximum at ~270 nm and a minimum ~210 nm, with a more pronounced peak ~210 nm suggesting an increased stability of the hairpin structure.

Figure 2. Temperature-dependent hairpin dynamics observed as changes in CD spectra. CD spectra of the hairpin-containing RNA oligonucleotide were recorded at 17 and 27 °C.

Notes

We recommend that RNase-free labware be used throughout the protocol.
The assay temperatures of 17 and 27 °C used in this protocol may be adjusted to the experimenter’s temperature of interest.

Recipes

Cacodylate buffer
60 nM Cacodylic Acid in RNase-free water, pH 7.4 adjusted with 1 M KOH; store at 4 °C (see Section A for details on buffer preparation)
1 M KOH
In RNase-free water

Acknowledgments

M.B. was supported by an EMBO Long-term Fellowship (ALTF 4815), M.D.A and his group are supported by a BBSRC David Phillips Fellowship (BB/R011605/1). B.Y.W.C. and her group are supported by grants from the Wellcome Trust (096082), MRC (MR/R021821/1) and the Royal Society (RGS\R2\192222).

This work is based on the original research paper “An RNA thermoswitch regulates daytime growth in Arabidopsis” by Chung et al. (2020).

Competing interests

We declare that there is no conflict of interest.

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