Assessment of the RNA Chaperone Activity with a Molecular Beacon

Dissolve the MB in TE buffer to obtain a concentration of 100micromolar (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, 0h 5m 0s; 45°C, 0h 5m 0s; 55°C, 0h 5m 0s; and 65°C during 0h 5m 0s ( see Note 23 ).

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.

A	B	C	D	E	F	G	H	I
	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 1Preparation of dilution mixes for testing MB effectivenessa aVolumes of each reactive are indicated in μLbSince 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 ).

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, 0h 5m 0s; PAUSE, 37°C, 0h 15m 0s; PAUSE, 37°C, 0h 30m 0s; 65°C, 0h 10m 0s; STOP (Table 2).

A	B	C	D	E	F	G	H	I
	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	Plot the data accordingly	Plot the data accordingly	Plot the data accordingly	Plot the data accordingly	Plot the data accordingly	Plot the data accordingly	Plot the data accordingly

Table 2Determination of RNA chaperone activity: preparation of reaction mixesaaVolumes of each reactive are indicated in μLbReplicates of samples should be includedcIf the thermal cycler software allows it, the entire incubation protocol can be pre-programed including the corresponding PAUSE times

Prepare an optical 96-well plate including the reaction mixes as indicated in Table 2 ( see Note 26 ).

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 0h 15m 0s.

During the second incubation pause, pull out the plate, remove the adhesive film and add 10µL. 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 0h 30m 0s at 37°C and then increase the temperature up to65°C during 0h 10m 0s.

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).