Sep 02, 2021
Assessment of the RNA Chaperone Activity with a Molecular Beacon
Book Chapter
- Pilar Menendez-Gil1,
- Carlos J. Caballero1,
- Cristina Solano2,
- Alejandro Toledo-Arana1
- 1Instituto de Agrobiotecnología, IDAB, CSIC-UPNA-Gobierno de Navarra, Pamplona, Navarra, Spain;
- 2Navarrabiomed-Universidad Pública de Navarra (UPNA)-Complejo Hospitalario de Navarra (CHN), IDISNA, Pamplona, Navarra, Spain
- Springer Nature Books
Protocol Citation: Pilar Menendez-Gil, Carlos J. Caballero, Cristina Solano, Alejandro Toledo-Arana 2021. Assessment of the RNA Chaperone Activity with a Molecular Beacon. protocols.io https://dx.doi.org/10.17504/protocols.io.bnikmccw
Manuscript citation:
Menendez-Gil P., Caballero C.J., Solano C., Toledo-Arana A. (2020) Fluorescent Molecular Beacons Mimicking RNA Secondary Structures to Study RNA Chaperone Activity. In: Heise T. (eds) RNA Chaperones. Methods in Molecular Biology, vol 2106. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0231-7_3
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 16, 2020
Last Modified: September 02, 2021
Protocol Integer ID: 43308
Keywords: RNA, Chaperone, RNA-binding protein, Hairpin, Stem loop, Molecular beacon, Fluorescein, Quencher, FAM,
Abstract
This is part 3.2 of the "Fluorescent Molecular Beacons Mimicking RNA Secondary Structures to Study RNA Chaperone Activity" collection of protocols.
Collection Abstract: Molecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5' and 3' ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from Staphylococcus aureus. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases.
Materials
Materials
Note
Prepare all solutions using ultrapure water (prepared by purifying deionized water to reach a sensitivity of at least 18 MΩ at 25 °C ) and analytical grade reagents for use in molecular biology. Store solutions atRoom temperature unless stated otherwise. Follow safety and waste disposal regulations when handling harmful products accordingly.
- Molecular beacon mimicking the hairpin structure under study (see Note 3).
- Spectrophotometer equipped with a UV lamp (e.g., NanoDrop).
- Recombinant RNA chaperone CspA (or protein of interest).
- Bovine Serum Albumin (BSA) dissolved in CspA storage buffer at the same concentration as the protein of interest (see Note 4).
- TE buffer: 10 millimolar (mM) Tris–HCl 7.5 and 1 millimolar (mM) EDTA .
- 96-well PCR plates suitable for the Real-Time PCR System available. Plate is sealed with optically clear adhesive film (see Note 5).
- AriaMx Real-Time PCR System (Agilent Technologies) or any other equivalent thermal cycler including an optical system able to excite the FAM fluorophore and register the fluorescence emission at different temperature incubation times.
- CspA storage buffer (see item 17 in part 3.1, Materials for section "Purification of Recombinant CspA from Total Protein Crude Extracts").
- 10 X reaction buffer : 100 millimolar (mM) MTris–HCl 7.5 , 300 millimolar (mM) KCl , 200 millimolar (mM) NH4Cl, 15 millimolar (mM) DTT , 50 millimolar (mM) MgCl2 (see Note 6).
- 4 U/μl Ribolock (see Note 7).
- Proteinase K (Sigma) stock solution. Dissolved Proteinase K powder in water to a final concentration of 20 mg/mL (see Note 8). Store at -20 °C .
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Molecular Beacon Design
Molecular Beacon Design
Note
The success of this assay lies in an adequate MB design, which is based on two main principles:
(1) the presence of an RNA structure targeted by the RNA chaperone under study.
(2) fluorescence quenching exerted by a quencher dye (e.g., BHQ_1) on a fluorophore (e.g., FAM), which occurs when both molecules are in close proximity to one another.
Additionally, the selected RNA structure must keep the quencher close enough to the fluorophore at the working temperature (switch OFF). MB mimicking hairpin-like structures have been shown to comply these criteria before [7–9]. Likewise, alternative MB conformations can be tested according to the characteristics of the protein of interest. Having decided the MB configuration, the synthesis of the labelled probe may be ordered to any oligonucleotide supplier (see Note 18). Based on the binding capacity of the CspA protein and the stability and cost of the probe, we decided to use single-stranded DNA instead of RNA oligonucleotides (see Note 19).
Testing the Effectiveness of the Designed MB and Setup of the Working Conditions
Testing the Effectiveness of the Designed MB and Setup of the Working Conditions
20m
20m
Note
Before assessing if the RNA chaperone is able to unwind the MB structure, the effectiveness of the designed MB must be tested following two main criteria. On the one hand, no fluorescence emission should be detected when the MB is in an OFF state. On the other hand, maximum fluorescence levels should be registered when the MB is completely unfolded (ON state). In other words, a MB design will be appropriate when a large ON/OFF fluorescence ratio is detected. Incubating different MB concentrations at increasing temperatures (that denature the oligonucleotide and open the structure leading to FAM fluorescence emission) helps determining both the background fluorescence (see Note 20) and the lowest quantity of oligonucleotide needed to obtain good fluorescence levels when the MB is in an ON state (see Note 21). To test if the MB design was successful, proceed as follows:
Dissolve the MB in TE buffer to obtain a concentration of 100 micromolar (µM) , following the manufacturer recommendations (see Note 22). Concentration of the MB should be corroborated with a spectrophotometer (e.g., NanoDrop).
Program the AriaMx thermal cycler to incubate the MB samples as follows: 37 °C , 00:05:00 ; 45 °C , 00:05:00 ; 55 °C , 00:05:00 ; and 65 °C during 00:05:00 (see Note 23).
Note
Register the emission of FAM fluorescence every minute.
20m
Make serial dilutions of the MB in an optical 96-well plate as indicated in Table 1, which shows mixtures of the components to analyze different concentrations of the MB. Triplicates are highly recommended.
| MB concentration (pmol) | |||||||||
| 0 | 0.5 | 1 | 2 | 5 | 10 | 15 | 20 | ||
| MB 10 μM | - | - | - | - | 0.5 | 1 | 1.5 | 2 | |
| MB 1 μM | - | 0.5 | 1 | 2 | - | - | - | - | |
| CspA storage buffer b | 12.5 | 12 | 11.5 | 10.5 | 12 | 11.5 | 11 | 10.5 | |
| 10X reaction buffer | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | |
| Ultrapure water | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
| Final volume | 25 μL | ||||||||
Table 1
Preparation of dilution mixes for testing MB effectivenessa
aVolumes of each reactive are indicated in μL
bSince the CspA protein is diluted in CspA storage buffer, the MB effectiveness test is performed including this buffer
Seal the plate with an optically clear adhesive film (see Note 5) and load it into the thermal cycler. Start the incubation program.
Once the incubation time is finished, plot the obtained fluorescence signals in function of the MB concentration at the different temperatures. If replicates are used, plot the means of the fluorescence signals. The instrument background signal should be previously subtracted. Figure 4 shows an example of the results obtained with the MB designed for the analysis of S. aureus CspA activity [9] (Fig. 4). In this example, when the MB was incubated at 55 °C and 65 °C , fluorescence emission was registered, indicating that the MB was in an ON state. These fluorescence levels were directly proportional to the MB concentration. In contrast, when the MB was incubated at 37 °C and 45 °C , the fluorescence values were close to those of the background confirming that the MB was in an OFF configuration. This experiment validated the functionality of the designed MB (see Note 24).
Fig. 4 Test of the molecular beacon functionality. Different concentrations of the MB mimicking the hairpin structure located at the 5'UTR of the cspA mRNA were incubated at different temperatures and fluorescence emission was registered. The experiment was carried out using the AriaMx thermal cycler
Determination of the RNA Chaperone Activity Using the Designed MB
Determination of the RNA Chaperone Activity Using the Designed MB
1h 55m
1h 55m
Based on the data obtained from the MB effectiveness test, select the lowest MB concentration that gives a good ratio between the fluorescence and background signals (see Note 21).
Program the AriaMx thermal cycler to incubate the MB samples as follows: 37 °C , 00:05:00 ; PAUSE, 37 °C , 00:15:00 ; PAUSE, 37 °C , 00:30:00 ; 65 °C , 00:10:00 ; STOP (Table 2).
Note
Register the fluorescence emission every minute (see Note 25).
| Samples b | |||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | ||
| MB tube labelling | - | + | + | + | + | + | + | + | |
| CspA tube labelling | - | - | + | + | + | - | - | - | |
| BSA tube labelling | - | - | - | - | - | + | + | + | |
| Water (Vf: 100 μL) | 39 | 38 | 38 | 38 | 38 | 38 | 38 | 38 | |
| CspA storage buffer | 50 | 50 | 30 | 15 | - | 30 | 15 | - | |
| 10X reaction buffer | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
| MB 1 μM | - | 1 | 1 | 1 | 1 | 1 | 1 | 1 | |
| Ribolock 4 U/μL | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | |
| Seal the plate with adhesive film | |||||||||
| Incubate 37 °C—5 min | |||||||||
| Register fluorescence emission every minute | |||||||||
| PAUSE incubation programc | |||||||||
| CspA stock (~200 μM) | - | - | 20 | 35 | 50 | - | - | - | |
| BSA stock (~200 μM) | - | - | - | - | - | 20 | 25 | 50 | |
| Re-seal the plate with adhesive film | |||||||||
| Incubate 37 °C—15 min | |||||||||
| Register fluorescence emission every minute | |||||||||
| PAUSE incubation program | |||||||||
| Proteinase K 20 mg/mL | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
| Re-seal the plate with adhesive film | |||||||||
| Incubate 37 °C—30 min | |||||||||
| Incubate 65 °C—10 min | |||||||||
| Register fluorescence emission every minute | |||||||||
| Collect the fluorescence data from AriaMx thermal cycler | |||||||||
| Plot the data accordingly | |||||||||
Table 2
Determination of RNA chaperone activity: preparation of reaction mixesa
aVolumes of each reactive are indicated in μL
bReplicates of samples should be included
cIf the thermal cycler software allows it, the entire incubation protocol can be pre-programed including the corresponding PAUSE times
1h
Prepare an optical 96-well plate including the reaction mixes as indicated in Table 2 (see Note 26).
Note
Note that the CspA and BSA proteins should be added later.
Seal the plate with adhesive film (see Note 5) and load it into the thermal cycler. Start the incubation program.
At the first pause of the incubation program, pull out the 96-well plate from the thermal cycler, remove the adhesive film and add the appropriate quantity of CspA and BSA. Re-seal the plate with a new adhesive film. This step must be performed swiftly.
Reintroduce the plate into the thermal cycler and continue the incubation at 37 °C during 00:15:00 .
Note
Register the fluorescence emission every minute.
15m
During the second incubation pause, pull out the plate, remove the adhesive film and add 10 µL proteinase K (20 mg/mL) . Re-seal the plate with a new adhesive film. This step must be performed swiftly.
Reintroduce the plate into the thermal cycler and continue the incubation for 00:30:00 at 37 °C and then increase the temperature up to65 °C during 00:10:00 .
Note
Register fluorescence emission every minute.
40m
Once the incubation program is finished, collect the result data sheet.
Plot the obtained data subtracting the background fluorescence levels. If the experiments work as expected, fluorescence emission should be registered after addition of the RNA chaperone. This fluorescence should disappear after treatment with Proteinase K, showing the specificity of the reaction. Finally, increasing the temperature at 65 °C should lead to maximum levels of fluorescence, indicating that the MB remains functional through the course of the experiment. Logically, in the negative controls, no fluorescence emission should be detected until the last step, when temperature is raised (e.g., see ref. 9).