Visualization and Quantification of Intermolecular RNA Base Pairing in in vitro RNA Clusters Using Split Broccoli RNA Reporters

We apply the split broccoli RNA reporter assay to detect and quantify intermolecular base pairing in RNA clusters in vitro. Chemical probing and simulations suggest RNA interactions but cannot directly measure them. This protocol enables direct, accurate assessment within RNA clusters.

After preparing the DNA templates for in vitro RNA transcription, wipe the working surface, tube racks, and pipettes with ribonuclease decontamination solution. Use ribonuclease-free filtered tips for all pipetting steps. Thaw the reaction buffer and nucleotide solutions supplied with the transcription kit along with uridine triphosphate Cyanine 5, or UTP-Cy5, on ice.

Centrifuge all tubes at 2, 000g for two seconds before use. Then calculate the number of thymine bases in the DNA. Determine the required volumes of UTP and UTP-Cy5 for a 20 microliter transcription reaction while maintaining consistent labeling density across RNA species.

Next, prepare a 20 microliter transcription reaction by combining the presented reagents. Mix thoroughly by pipetting up and down and centrifuge at 2, 000g for two seconds. After incubating the reaction at 37 degrees Celsius for six hours, add one microliter of deoxyribonuclease supplied with the kit, mix thoroughly by pipetting, and centrifuge again.

Then incubate at 37 degrees Celsius for an additional 15 minutes. Transfer the reaction to a 1.5 milliliter tube. Add 115 microliters of nuclease-free water and 15 microliters of ammonium acetate stop solution supplied with the kit.

After mixing, centrifuge the solution at 2, 000g for two seconds. Next, add 150 microliters of phenol chloroform to the tube and mix gently to combine the phases. Centrifuge at 16, 000g for 10 minutes at four degrees Celsius.

Then transfer the upper aqueous phase to a new tube. Add an equal volume of chloroform to the transferred solution and mix thoroughly to ensure proper phase interaction. Now centrifuge at 16, 000g for two minutes at four degrees Celsius and repeat the phase separation process once again.

Then transfer the aqueous layer of approximately 100 to 150 microliters to a new tube. Add 900 microliters of isopropanol and mix thoroughly. After aliquoting the solution in separate tubes, store the samples at minus 20 degrees Celsius overnight or up to one month.

Centrifuge the RNA sample in isopropanol at 16, 000g for 15 minutes at four degrees Celsius. Carefully remove the isopropanol without disturbing the RNA pellet. Then add one milliliter of 70%ethanol prepared in nuclease-free water to the pellet.

Centrifuge at 16, 000g for two minutes at four degrees Celsius. After removing the ethanol, add one milliliter of 70%ethanol prepared in nuclease-free water to the tube and repeat centrifugation. During the final ethanol wash, remove most of the ethanol using a 1, 000 microliter pipette.

Briefly centrifuge at 2, 000g for two seconds in a benchtop mini centrifuge and remove any remaining ethanol using a 20 microliter pipette. Once the RNA pellet is dried, resuspend it in 20 microliters of nuclease-free water. Keep the sample on ice and protect it from light.

Using a micro volume spectrophotometer, measure RNA concentration and purity. Ensure that the absorbance ratio at 260 over 280 nanometers is approximately 2.0 and the ratio at 260 over 230 nanometers is greater than 2.0. Thaw a tube of 100 millimolar spermine solution and 50%polyethylene glycol 8, 000 on ice.

Freshly prepare 10X RNA refolding buffer by combining the components shown. After mixing the components thoroughly by pipetting, centrifuge at 2, 000g for two seconds in a benchtop mini centrifuge. For the co-folding condition, in a 200 microliter PCR tube, combine in vitro transcribed RNA carrying either top or bottom sequence, RNA refolding buffer, and nuclease-free water.

Mix thoroughly by pipetting up and down and centrifuge as demonstrated earlier. Heat the sample at 90 degrees Celsius for two minutes. Immediately transfer the sample to room temperature.

Protect from light and incubate for one hour. For the separate folding condition, heat the RNA sample in a 200 microliter PCR tube and then immediately place it on ice for at least 15 minutes. In a 200 microliter PCR tube, combine in vitro transcribed RNA carrying either top or bottom sequence, 10 times RNA refolding buffer, and nuclease-free water.

Incubate the reaction at room temperature for 30 minutes, protected from light. Now combine the RNA samples containing the top and bottom sequences into a single PCR tube and mix thoroughly by pipetting up and down. Centrifuge at 2, 000g for two seconds in a benchtop mini centrifuge.

Incubate at room temperature for 30 minutes. For both conditions, add six microliters of a one to two premix of 100 millimolar spermine and 50%polyethylene glycol 8, 000. After mixing the content, centrifuge at 2, 000g for two seconds in a benchtop mini centrifuge.

Now mix two microliters of one millimolar DFHBI-1T to the reaction and centrifuge again. Load the reaction into a glass chambered cover glass. Incubate the chamber at room temperature for four hours in the dark with the cover on to minimize evaporation.

Load the glass chambered cover glass onto the microscope stage. Turn on the laser, then use the eye pieces to locate and focus on the sample. Configure the microscope to image each region of interest with the cyanine 5 channel first at each Z plane.

Then image the same region of interest using the green fluorescent protein channel. Set the exposure time to 500 milliseconds for all channels. Then set the laser power to 80%for the green fluorescent protein channel and 40%for the cyanine 5 channel.

Using a Z step size of 150 nanometers, image a cover slip region outside the reaction droplet at room temperature. Afterwards, quantify intermolecular base pairing using image analysis software. Upon induction of RNA clustering, both RNAs formed clusters either individually or together.

DFHBI-1T fluorescence within RNA clusters was substantially lower for top or bottom RNAs alone compared to the co-folding condition. In the separate folding condition, the normalized DFHBI-1T signal was 0.031, comparable to baseline levels observed in top or bottom alone. Shu-top, shu-bottom, shu-anti-top, and shu-anti-bottom individually exhibited minimal DFHBI-1T fluorescence.

Co-folding of shu-top with shu-bottom or shu-anti-top with shu-anti-bottom produced a higher DFHBI-1T signal than separate folding. Co-folding of shu-top and shu-bottom resulted in a 5.63 fold increase in normalized DFHBI-1T fluorescence. Separate folding of shu-top and shu-bottom showed only a 1.04 fold increase comparable to baseline levels.

Shu-top mixed with shu-anti-bottom and shu-anti-top mixed with shu-bottom exhibited higher DFHBI-1T fluorescence than same-orientation pairs under both folding conditions. Co-folding of shu-top with shu-anti-bottom and shu-anti-top with shu-bottom resulted in 61.86 and 82.60 fold increases in DFHBI-1T fluorescence respectively. Separate folding of these mixed pairs yielded smaller increases of 2.69 and 4.94 fold respectively.

The most important consideration is that robust signal generation may require crowding reagents while sensitivity depends on efficient dimerization and folding of split broccoli RNAs. This assay complements RNA pull down and gel electrophoresis approaches for studying intermolecular base pairing in RNA clusters. This assay can examine the effects of RNA sequences, helicases, and ions on intermolecular base pairing within RNA clusters.