TITLE: Assessment of the RNA Chaperone Activity with a Molecular Beacon
AUTHORS: Pilar Menendez-Gil, Carlos J. Caballero, Cristina Solano, Alejandro Toledo-Arana

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is part 3.2 of the "Fluorescent Molecular Beacons Mimicking RNA Secondary Structures to Study RNA Chaperone Activity" collection of protocols.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract: </span><span>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 </span><span style = "font-style:italic;">Staphylococcus aureus</span><span>. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases.</span></div></div>

[STEPS]
?. [Molecular Beacon Design]
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]
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:
?. [Testing the Effectiveness of the Designed MB and Setup of the Working Conditions]
Dissolve the MB in TE buffer to obtain a concentration of , following the manufacturer recommendations (see Note 22). Concentration of the MB should be corroborated with a spectrophotometer (e.g., NanoDrop).
?. [Testing the Effectiveness of the Designed MB and Setup of the Working Conditions]
Program the AriaMx thermal cycler to incubate the MB samples as follows: , ; , ; , ; and  during  (see Note 23).
37 °C
45 °C
55 °C
65 °C
Register the emission of FAM fluorescence every minute.
?. [Testing the Effectiveness of the Designed MB and Setup of the Working Conditions]
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.  ABCDEFGHI12MB concentration (pmol)300.51251015204MB 10 μM----0.511.525MB 1 μM-0.512----6CspA storage buffer b12.51211.510.51211.51110.5710X reaction buffer2.52.52.52.52.52.52.52.58Ultrapure water10101010101010109Final volume25 μL
ABCDEFGHI12MB concentration (pmol)300.51251015204MB 10 μM----0.511.525MB 1 μM-0.512----6CspA storage buffer b12.51211.510.51211.51110.5710X reaction buffer2.52.52.52.52.52.52.52.58Ultrapure water10101010101010109Final volume25 μL
?. [Testing the Effectiveness of the Designed MB and Setup of the Working Conditions]
Seal the plate with an optically clear adhesive film (see Note 5) and load it into the thermal cycler. Start the incubation program.
?. [Testing the Effectiveness of the Designed MB and Setup of the Working Conditions]
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  and , 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  and , 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).
55 °C
65 °C
37 °C
45 °C
?. [Determination of the RNA Chaperone Activity Using the Designed MB]
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).
?. [Determination of the RNA Chaperone Activity Using the Designed MB]
Program the AriaMx thermal cycler to incubate the MB samples as follows: , ; PAUSE, , ; PAUSE, , ; , ; STOP (Table 2).   ABCDEFGHI1Samples b2123456783MB tube labelling-+++++++4CspA tube labelling--+++---5BSA tube labelling-----+++6Water (Vf: 100 μL)39383838383838387CspA storage buffer50503015-3015-810X reaction buffer10101010101010109MB 1 μM-111111110Ribolock 4 U/μL111111111112Seal the plate with adhesive film13Incubate 37 °C—5 min14Register fluorescence emission every minute15PAUSE incubation programc1617CspA stock (~200 μM)--203550---18BSA stock (~200 μM)-----2025501920Re-seal the plate with adhesive film21Incubate 37 °C—15 min22Register fluorescence emission every minute23PAUSE incubation program2425Proteinase K 20 mg/mL10101010101010102627Re-seal the plate with adhesive film28Incubate 37 °C—30 min29Incubate 65 °C—10 min30Register fluorescence emission every minute31Collect the fluorescence data from AriaMx thermal cycler32Plot the data accordinglyPlot the data accordinglyPlot the data accordinglyPlot the data accordinglyPlot the data accordinglyPlot the data accordinglyPlot the data accordinglyPlot the data accordingly
37 °C
37 °C
37 °C
65 °C
Register the fluorescence emission every minute (see Note 25).
ABCDEFGHI1Samples b2123456783MB tube labelling-+++++++4CspA tube labelling--+++---5BSA tube labelling-----+++6Water (Vf: 100 μL)39383838383838387CspA storage buffer50503015-3015-810X reaction buffer10101010101010109MB 1 μM-111111110Ribolock 4 U/μL111111111112Seal the plate with adhesive film13Incubate 37 °C—5 min14Register fluorescence emission every minute15PAUSE incubation programc1617CspA stock (~200 μM)--203550---18BSA stock (~200 μM)-----2025501920Re-seal the plate with adhesive film21Incubate 37 °C—15 min22Register fluorescence emission every minute23PAUSE incubation program2425Proteinase K 20 mg/mL10101010101010102627Re-seal the plate with adhesive film28Incubate 37 °C—30 min29Incubate 65 °C—10 min30Register fluorescence emission every minute31Collect the fluorescence data from AriaMx thermal cycler32Plot the data accordinglyPlot the data accordinglyPlot the data accordinglyPlot the data accordinglyPlot the data accordinglyPlot the data accordinglyPlot the data accordinglyPlot the data accordingly
?. [Determination of the RNA Chaperone Activity Using the Designed MB]
Prepare an optical 96-well plate including the reaction mixes as indicated in Table 2 (see Note 26).
Note that the CspA and BSA proteins should be added later.
?. [Determination of the RNA Chaperone Activity Using the Designed MB]
Seal the plate with adhesive film (see Note 5) and load it into the thermal cycler. Start the incubation program.
?. [Determination of the RNA Chaperone Activity Using the Designed MB]
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.
?. [Determination of the RNA Chaperone Activity Using the Designed MB]
Reintroduce the plate into the thermal cycler and continue the incubation at  during .
37 °C
Register the fluorescence emission every minute.
?. [Determination of the RNA Chaperone Activity Using the Designed MB]
During the second incubation pause, pull out the plate, remove the adhesive film and add . Re-seal the plate with a new adhesive film. This step must be performed swiftly.
[proteinase K (20 mg/mL)]
?. [Determination of the RNA Chaperone Activity Using the Designed MB]
Reintroduce the plate into the thermal cycler and continue the incubation for  at  and then increase the temperature up to during .
37 °C
65 °C
Register fluorescence emission every minute.
?. [Determination of the RNA Chaperone Activity Using the Designed MB]
Once the incubation program is finished, collect the result data sheet.
?. [Determination of the RNA Chaperone Activity Using the Designed MB]
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  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).
65 °C