Name: 分離ブロッコリーRNAレポーターを用いた in vitro RNAクラスターにおける分子間RNA塩基対の可視化および定量化 (動画) | JoVE Journal
Uploaded: 2026-05-29T16:30:00.000+00:00
Duration: 10 min 52 s
Description: 学ぶ 分離ブロッコリーRNAレポーターアッセイを用いてin vitro RNAクラスターにおける分子間塩基対を可視化・定量化する方法。蛍光顕微鏡と画像解析でRNA-RNA相互作用を直接評価し、従来の化学プロービングやシミュレーションを補完する。

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During the preparation of this work, the authors used ChatGPT 5.2 to improve the readability and language of the manuscript. This research was supported by the NIGMS R35GM142737 grant awarded to T.T.

This protocol provides a step-by-step method for implementing this assay and discusses its applications. The reagents and equipment used are listed in the Table of Materials.
1. Preparation of DNA templates for in vitro RNA transcription
<ol>
	<li>Append a T7 promoter sequence to the 5′ end of each DNA template containing the split Broccoli aptamer sequences for in vitro RNA transcription. Use the split Broccoli aptamer sequences (top and bottom) alone or insert them at any position downstream of the T7 promoter, with or without an additional RNA sequence of interest. 
	NOTE: The sequences for the T7 promoter, split Broccoli reporters, and primers used to amplify the DNA templates are listed in Table 1. 
	NOTE: T7 RNA polymerase strongly prefers to initiate transcription with a “G” at the +1 position. The presence of additional Gs immediately downstream at the +2 and +3 positions can increase transcription yield19. However, when the downstream sequence begins with G, as in the top and bottom constructs that begin with two Gs, transcription can initiate at different positions20. As a result, transcripts may carry one, two, or three Gs at the 5′ end (e.g., GATCC, GGATCC, or GGGATCC), which may affect the interpretation of base pairing in the designed constructs.</li>
	<li>Use commercially synthesized gene blocks, plasmids, or DNA fragments as templates for PCR. If the original template does not contain a T7 promoter, use a forward primer with a 5′ T7 promoter overhang together with the appropriate reverse primer.</li>
	<li>Prepare a 50 µL PCR reaction in a 200 µL PCR tube containing 2.5 µL each of 10 µM forward and reverse primers, 20 ng DNA template, 25 µL 2× high-fidelity master mix, and nuclease-free water. Mix thoroughly by pipetting up and down and centrifuge at 2,000 × g for 2 s.</li>
	<li>Run the PCR reaction in a thermal cycler using the following program: 98 °C for 30 s (1 cycle); 98 °C for 10 s, optimized annealing temperature for 30 s, and 72 °C for 30 s per kilobase (35 cycles); 72 °C for 2 min (1 cycle). 
	NOTE: Determine the optimized annealing temperature using an online tool such as a Tm calculator. PCR products may be stored at 4 °C after completion.</li>
	<li>Mix 2 µL PCR product with 8 µL nuclease-free water and 2 µL 6× loading dye. Load the mixture onto a 1% agarose gel for electrophoresis. 
	NOTE: The PCR product should appear as a single band. If multiple bands are observed, excise the correct band and purify it using a gel extraction kit before proceeding.</li>
	<li>Purify the PCR product or gel-extracted DNA using a DNA purification kit and elute in 20–30 µL nuclease-free water into a 1.5 mL microcentrifuge tube.</li>
	<li>Measure DNA concentration and purity using a microvolume spectrophotometer. Ensure that A260/A280 is approximately 1.8 and A260/A230 is greater than 2.0. 
	NOTE: If A260/A230 is below 2.0, perform an additional purification step.</li>
	<li>Store the DNA template at −20 °C until use.</li>
</ol>
2. Preparation of in vitro RNA transcription reaction
<ol>
	<li>Wipe the working surface, tube racks, and pipettes with RNase decontamination solution. Use RNase-free, filtered tips.</li>
	<li>Thaw reaction buffer and nucleotide solutions (ATP, CTP, GTP, UTP) supplied with the T7 transcription kit, along with UTP-Cy5, on ice. Centrifuge all tubes at 2,000 × g for 2 s prior to use. 
	NOTE: Protect UTP-Cy5 from light.</li>
	<li>Calculate the number of thymine bases in the DNA template (excluding the T7 promoter) and determine the required volumes of UTP and UTP-Cy5 for a 20 µL transcription reaction. Maintain consistent labeling density across RNA species. 
	NOTE: For example, to label an RNA containing 100 uracil bases with approximately three Cy5-labeled uracils, add 4.5 µL of 1 mM UTP-Cy5 and 1.9 µL of 75 mM UTP.</li>
	<li>Prepare a 20 µL transcription reaction by combining 2 µL each of ATP, CTP, and GTP, calculated volumes of UTP (all supplied with the kit) and UTP-Cy5, 2 µL 10× reaction buffer, 2 µL enzyme mix, 200 ng DNA template, and nuclease-free water. Mix thoroughly by pipetting up and down and centrifuge at 2,000 × g for 2 s.</li>
	<li>Incubate the reaction at 37 °C for 6 h.</li>
	<li>Add 1 µL DNase supplied with the kit. Mix thoroughly by pipetting up and down and centrifuge at 2,000 × g for 2 s.</li>
	<li>Incubate at 37 °C for an additional 15 min.</li>
	<li>Transfer the reaction (~20 µL) to a 1.5 mL tube. Add 115 µL nuclease-free water and 15 µL ammonium acetate stop solution supplied with the kit. Mix thoroughly by pipetting up and down and centrifuge at 2,000 × g for 2 s. 
	NOTE: A commercial RNA purification kit may be used instead of Steps 2.9–3.7.</li>
	<li>Add 150 µL phenol/chloroform and mix gently. 
	CAUTION: Handle phenol/chloroform in a chemical fume hood.</li>
	<li>Centrifuge at 16,000 × g for 10 min at 4 °C.</li>
	<li>Transfer the upper aqueous phase to a new tube. 
	NOTE: Avoid disturbing the interphase; the lower layer may appear blue due to unincorporated dye.</li>
	<li>Add an equal volume of chloroform and mix thoroughly. 
	CAUTION: Handle chloroform in a chemical fume hood.</li>
	<li>Centrifuge at 16,000 × g for 2 min at 4 °C.</li>
	<li>Repeat Steps 2.11–2.13.</li>
	<li>Transfer the aqueous layer (~100–150 µL) to a new tube. Add 900 µL isopropanol and mix thoroughly.</li>
	<li>Aliquot into three equal volumes in separate tubes. 
	NOTE: Each aliquot is sufficient for multiple RNA clustering reactions.</li>
	<li>Store at −20 °C overnight or up to one month.</li>
</ol>
3. Purification of in vitro transcribed RNA
<ol>
	<li>Centrifuge the RNA sample in isopropanol at 16,000 × g for 15 min at 4 °C.</li>
	<li>Carefully remove the isopropanol without disturbing the RNA pellet.</li>
	<li>Add 1 mL of 70% ethanol prepared in nuclease-free water.</li>
	<li>Centrifuge at 16,000 × g for 2 min at 4 °C.</li>
	<li>Remove the ethanol and repeat Steps 3.3–3.4.</li>
	<li>During the final ethanol wash, remove most of the ethanol using a 1000 µL pipette. Briefly centrifuge at 2,000 × g for 2 s in a benchtop minicentrifuge and remove any remaining ethanol using a 20 µL pipette.</li>
	<li>Air-dry the RNA pellet for 1–2 min at room temperature (RT).</li>
	<li>Resuspend the RNA pellet in 20 µL nuclease-free water. Keep the sample on ice and protect it from light.</li>
	<li>Measure RNA concentration and purity using a microvolume spectrophotometer. Ensure that A260/A280 is approximately 2.0 and A260/A230 is greater than 2.0.</li>
</ol>
4. Preparation of in vitro RNA clustering reaction
NOTE: In this protocol, induction of RNA clustering requires spermine and PEG 8000, based on previous studies2,4,5. Specifically, a 100 mM spermine tetrahydrochloride stock solution prepared in nuclease-free water was aliquoted into multiple 1.5 mL microcentrifuge tubes and stored at −20 °C.
<ol>
	<li>Thaw a tube of 100 mM spermine solution and 50% PEG 8000 on ice. 
	NOTE: After opening, 50% PEG 8000 should be used for no longer than two months due to potential degradation.</li>
	<li>To prepare 500 µL freshly made 10× RNA refolding buffer, combine 50 µL 2M KCl, 50 µL 1M MgCl2, 50 µL 1M Tris (pH 7.0), and 350 µL nuclease-free water. Mix thoroughly by pipetting up and down. Centrifuge at 2,000 × g for 2 s in a benchtop minicentrifuge. 
	NOTE: The refolding buffer should be prepared on the day of the RNA clustering experiment. 
	NOTE: Steps 4.3-4.4 depend on the experimental conditions and may be adjusted as needed. Two example conditions for RNA clustering used in this study are described below. The co-folding condition is adapted from a method used to anneal antisense oligonucleotides to RNA2. In this method, RNAs are mixed in a salt buffer and heated together to promote intermolecular base pairing between RNAs carrying complementary sequences. In contrast, for the separate folding condition, each RNA is folded individually prior to mixing. This condition is intended to approximate a cellular scenario in which RNAs are folded prior to interacting.</li>
	<li>Co-folding 
	NOTE: This condition promotes intermolecular base pairing between RNAs containing the top and bottom sequences. It serves as a positive control for reporter-driven intermolecular base pairing under the RNA clustering condition.
	<ol>
		<li>In a 200 µL PCR tube, combine 16 pmol of each in vitro transcribed RNA carrying either top or bottom sequence, 2 µL of 10× refolding RNA buffer, and nuclease-free water to a final volume of 14 µL. Mix thoroughly by pipetting up and down. Centrifuge at 2,000 × g for 2 s in a benchtop minicentrifuge. 
		NOTE: To calculate the RNA mass corresponding to 16 pmol.</li>
		<li>Heat the RNA sample at 90 °C for 2 min.</li>
		<li>Immediately transfer the sample to RT and protect it from light. Incubate the reaction at RT for 1 h.</li>
	</ol>
	</li>
	<li>Separate folding
	<ol>
		<li>Heat the RNA sample at 90 °C for 2 min and then immediately place it on ice for at least 15 min.</li>
		<li>In a 200 µL PCR tube, combine 16 pmol of in vitro transcribed RNA carrying either top or bottom sequence, 1 µL of 10× refolding RNA buffer, and nuclease-free water to a final volume of 7 µL. 
		NOTE: To calculate the RNA mass corresponding to 16 pmol.</li>
		<li>Incubate the reaction at RT for 30 min, protected from light.</li>
		<li>Combine the RNA samples containing the top and bottom sequences into a single PCR tube. Mix thoroughly by pipetting up and down. Centrifuge at 2,000 × g for 2 s in a benchtop minicentrifuge. Incubate at RT for 30 min.</li>
	</ol>
	</li>
	<li>Add 6 µL of a 1:2 (v/v) premix of 100 mM spermine and 50% PEG 8000. Mix thoroughly by pipetting up and down. Centrifuge at 2,000 × g for 2 s in a benchtop minicentrifuge. 
	NOTE: 50% PEG 8000 is highly viscous. Pipette slowly and carefully.</li>
	<li>Add 2 µL of 1 mM DFHBI-1T to the reaction. Mix thoroughly by pipetting up and down. Centrifuge at 2,000 × g for 2 s in a benchtop minicentrifuge. The reaction should appear yellow-green. 
	NOTE: To prepare a 20 mM DFHBI-1T stock solution from 1 mg of DFHBI-1T (MW: 320.21) lyophilized dye, add 156 µL of molecular biology-grade anhydrous DMSO. Aliquot the stock in small amounts in multiple microcentrifuge tubes and store in -20 °C. Avoid repetitive freeze-thaw cycles for the dye. Prepare a freshly diluted 1 mM DFHBI-1T working solution by diluting the 20 mM stock in nuclease-free water and storing it on ice.</li>
	<li>Load the reaction into a glass chambered coverglass.</li>
	<li>Incubate the chamber at RT for 4 h in the dark with the cover on to minimize evaporation.</li>
</ol>
5. Preparing for imaging
<ol>
	<li>Acquire three-dimensional images using a structured illumination or confocal microscope equipped with appropriate lasers and objectives. 
	NOTE: Confocal microscopy is required to minimize out-of-focus light.</li>
	<li>Configure the microscope to image each region of interest (ROI) with the Cy5 channel first at each z-plane, followed by imaging the same ROI using the GFP channel.</li>
	<li>Set exposure time to 500 ms for all channels. Set laser power to 80% for GFP and 40% for Cy5.</li>
	<li>Image a coverslip region outside the reaction droplet at RT using a z-step size of 150 nm.</li>
</ol>
6. Quantification of intermolecular base pairing
<ol>
	<li>Import images into image analysis software21. Identify GFP (DFHBI-1T) and Cy5 (RNA) channels.</li>
	<li>Draw a 5 × 5 pixel ROI within an RNA cluster and measure integrated density for both channels at the same z-plane. Select 5–8 ROIs from different RNA clusters in each image. 
	NOTE: Adjust ROI size depending on camera setup, but maintain consistency across experiments.</li>
	<li>Select 5–8 ROIs outside the reaction droplet for background measurement and calculate the average integrated density for both channels.</li>
	<li>Calculate normalized DFHBI-1T intensity for each ROI using: 
	<img alt="Integrated density equations, fluorescence analysis, comparing GFP and Cy5 in ROI and background." src="/files/ftp_upload/70886/70886eq1.jpg" style="margin: auto;" data-altupdatedat="1787025648000" data-alt="Equation 1"></li>
</ol>
7. Common problems and troubleshooting
<ol>
	<li>If no RNA pellet is observed after centrifugation, consider RNA degradation, insufficient transcription time, inactive polymerase, residual salts, or low RNA concentration. Use RNase-free reagents, extend transcription time, use fresh enzyme, and purify templates.</li>
	<li>If no Cy5-labeled RNA clusters are observed, consider RNA degradation or degraded PEG 8000 or UTP-Cy5. Use fresh reagents and RNase-free conditions.</li>
	<li>If no DFHBI-1T signal is observed under co-folding conditions, consider poor dye quality, lack of Broccoli structure formation, or truncated transcripts. Use fresh dye, ensure proper buffer composition, and verify transcript size by gel analysis.</li>
	<li>If fluorescence signal bleaches, reduce laser power and exposure time, minimize imaging steps, and protect samples from light during incubation.</li>
</ol>

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R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+metabolite+dynamics+in+living+cells+using+a+Spinach-based+riboswitch.">Imaging metabolite dynamics in living cells using a Spinach-based riboswitch.</a> Proc Natl Acad Sci U S A. 112, E2756-E2765 (2015).</li><li>Zinskie, J. A., Roig, M., Janetopoulos, C., Myers, K. A., Bruist, M. F. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+of+small+nucleolar+RNA+tagged+with+the+broccoli+aptamer+in+yeast.">Live-cell imaging of small nucleolar RNA tagged with the broccoli aptamer in yeast.</a> FEMS Yeast Res. 18, (2018).</li></ol>

The authors declare no competing interests.

In this study, the split Broccoli aptamer system18 was adapted for direct visualization and quantification of intermolecular base pairing under in vitro RNA clustering conditions. This approach enables assessment of intermolecular base pairing across different in vitro conditions and complements downstream biochemical assays, such as RNA pull-down experiments using base-pairing crosslinkers15,16,17&#160;and gel electrophoresis4,6,7. Unlike these biochemical methods, which lack the spatial resolution to distinguish base pairing within RNA clusters from that occurring outside them, this approach enables direct spatial visualization of these interactions within clusters. Specifically, it allows monitoring of base pairing between top and bottom RNAs, or RNAs carrying these reporters, under defined RNA clustering conditions. In addition, it provides a real-time, quantitative readout of base pairing between reporter RNAs without requiring crosslinking or RNA extraction, thereby minimizing sample loss and buffer incompatibility.
Using fluorescence microscopy, intermolecular base pairing between the split Broccoli aptamer sequences was quantitatively measured through DFHBI-1T signal, demonstrating compatibility of DFHBI-1T with the in vitro RNA clustering protocol. The extent of intermolecular base pairing varied depending on RNA folding conditions, as observed under co-folding and separate folding conditions (Figure 1F and Figure 2Di&#8211;2Dii). These findings indicate that RNA folding conditions critically influence intermolecular RNA interactions and should be carefully considered when interpreting experimental results.
This assay also provides a platform to evaluate whether sequences flanking the top and bottom reporters enhance intermolecular base pairing, as demonstrated using shu. DFHBI-1T fluorescence was higher for shu-top with shuanti-bottom and shuanti-top with shu-bottom than for shu-top with shu-bottom or shuanti-top with shuanti-bottom, indicating that intermolecular interactions involving shu and shuanti further enhance the Broccoli signal. These observations suggest that the DFHBI-1T fluorescence signal obtained from the co-folding of the top and bottom reporters alone does not represent the upper limit of the assay.
DFHBI-1T fluorescence measured in these experiments spanned a broad dynamic range, from a 1.04-fold increase (shu-top with shu-bottom under separate folding) to an 82.60-fold increase (shuanti-top with shu-bottom under co-folding conditions). This dynamic range enables the detection of differences in intermolecular RNA interactions among RNAs carrying these reporters.
Several steps in this protocol are critical for the successful detection of intermolecular RNA base pairing between split Broccoli top and bottom RNAs in RNA clusters. Fresh aliquots of PEG 8000 should be used to reliably induce RNA clustering, and repeated freeze&#8211;thaw cycles should be minimized due to reagent instability. DFHBI-1T should be aliquoted and stored at &#8722;20 &#176;C to preserve fluorescence properties. In vitro transcription products should be analyzed on a denaturing RNA gel prior to clustering to confirm the correct transcript size. In addition, maintaining RNase-free conditions, including a clean working environment and imaging chamber, is essential because RNA droplets are incubated at room temperature for extended periods prior to imaging.
One limitation of this assay is that DFHBI-1T specifically reports intermolecular base pairing mediated by the top and bottom sequences. Other intermolecular base pairing events occurring in different RNA regions cannot be detected. In addition, the Broccoli structure formed by base pairing between the top and bottom strands is thermodynamically stable26 and may not fully reflect weaker or transient interactions, such as those formed by scattered, surface-exposed bases, as observed in Drosophila germ granule mRNA clusters4.
Furthermore, intermolecular RNA&#8211;RNA interactions can be promiscuous and non-specific, and sequences flanking the top and bottom reporters may influence DFHBI-1T fluorescence. These flanking regions may directly interact with the reporter sequences, inhibiting Broccoli formation or altering RNA structure, thereby affecting interactions between RNAs carrying the reporters. Accordingly, the assay reflects the combined effects of RNA structure, top&#8211;bottom base pairing, interactions among flanking sequences, and interactions between flanking sequences and the reporters.
This assay provides opportunities for future investigation. For example, altering RNA sequences flanking the reporters, introducing RNA helicases or chaperones, or modifying salt conditions may influence intermolecular base pairing in RNA clusters. Such effects can be assessed by measuring the DFHBI-1T signal under co-folding and separate folding conditions. Given that similar RNA aptamers have been used in cells, bacteria, and yeast26,27,28,29,30, this approach may be extended to in cellulo systems or model organisms to examine intermolecular base pairing in mRNA clusters.

In vitro transcribed RNAs can self-assemble into visible clusters upon the addition of salts and molecular crowding reagents1,2,3,4,5. Notably, these RNA clusters can form in the absence of other cellular components, including proteins, indicating that under specific conditions, intermolecular RNA&#8211;RNA interactions are sufficient to drive RNA condensation in vitro.
Among the various types of intermolecular RNA&#8211;RNA interactions, intermolecular base pairing has received substantial attention in the condensate field1,2,4,6,7,8,9,10. These interactions can be driven by exposed complementary sequences (&#8220;zipcodes&#8221;) between transcripts, as demonstrated by co-clustering of BNI1 and SPA2 mRNAs in the filamentous fungus Ashbya gossypii2 or oligomerization of oskar mRNA in Drosophila melanogaster 6,7. Alternatively, intermolecular base pairing can be promoted by remodeling of RNA secondary structures, exposing complementary sequences that are otherwise structurally embedded. This mechanism has been observed in repeat RNAs in silico9 and guanidine riboswitches in vitro10. Both exposed complementary sequences and RNA structural remodeling can be measured using chemical probing11,12 or simulation approaches4,9,13,14. However, methods that directly visualize intermolecular base pairing in in vitro RNA clustering assays remain limited.
RNA pull-down assays using base-pairing crosslinkers15,16,17and gel electrophoresis4,6,7are commonly used to study intermolecular RNA base pairing in vitro. However, these methods lack the spatial resolution to distinguish base pairing within clusters from that outside them. Moreover, isolating RNA clusters for downstream analysis is technically challenging, as it requires preservation of cluster stability while ensuring buffer compatibility with subsequent assays.
A visual assay based on split Broccoli RNA reporters18 conjugated to fluorescently labeled RNAs of interest has previously been employed to enable direct detection of intermolecular base pairing during RNA condensation in vitro4. In this context, the split Broccoli system reports on the likelihood of intermolecular interactions between reporters or between RNAs carrying them under defined in vitro clustering conditions. Thus, the assay probes how sequence context and environmental factors influence the propensity for intermolecular base pairing within an RNA cluster-like environment.
The split Broccoli system consists of two complementary RNA sequences, top and bottom, that dimerize to reconstitute the Broccoli RNA aptamer (Figures 1A&#8211;C)18. Upon dimerization, the aptamer binds the fluorophore DFHBI-1T, resulting in green fluorescence (Figures 1A&#8211;C)18. Using this system, the extent of intermolecular base pairing between reporter complementary sequences within RNA clusters is shown to depend on RNA folding. By comparing the ratio of DFHBI-1T fluorescence to RNA levels across different experimental conditions, the influence of in vitro conditions on reporter-mediated intermolecular base pairing within RNA clusters can be quantitatively assessed.
This assay complements RNA pull-down and gel electrophoresis approaches for studying intermolecular base pairing in RNA clusters. However, robust signal detection may require molecular crowding reagents, and sensitivity is limited by the efficiency of dimerization and folding of the split Broccoli top and bottom RNAs.

<table><tbody><tr><td>1M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Millipore Sigma</td><td>M1028</td><td>Used for preparation of 10&amp;times; RNA refolding buffer.</td></tr><tr><td>2M KCl</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AM9640G</td><td>Used for preparation of 10&amp;times; RNA refolding buffer.</td></tr><tr><td>50% PEG 8000</td><td>NEB</td><td>M0204S</td><td>Component of T4 RNA ligase kit; used as a molecular crowding reagent to induce RNA clustering.&amp;nbsp;</td></tr><tr><td>Absolute ethanol, 200 proof</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>T038181000CS</td><td>Used for preparation of RNA wash buffer.</td></tr><tr><td>Acid phenol:chloroform, pH 4.5 (with IAA, 125:24:1)</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AM9722</td><td>Used for RNA purification.</td></tr><tr><td>Aminoallyl-UTP-Cy5</td><td>Jena Bioscience</td><td>NU-821-CY5</td><td>Used for RNA labeling during in vitro transcription.</td></tr><tr><td>Chambered coverglass</td><td>Thermo Fisher Scientific</td><td>155409PK</td><td>Imaging chamber for RNA clustering reactions.</td></tr><tr><td>Chloroform</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>C298-500</td><td>Used for RNA purification.</td></tr><tr><td>DFHBI-1T</td><td>Lucerna</td><td>410</td><td>Used as a fluorophore for detection of Broccoli RNA aptamer.</td></tr><tr><td>DMSO</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>D12345</td><td>Anhydrous; molecular biology grade; used for DFHBI-1T preparation.&amp;nbsp;</td></tr><tr><td>DNA Clean &amp;amp; Concentrator Kit</td><td>Zymo</td><td>D4014</td><td>Used for cleanup of DNA products prior to T7 transcription.</td></tr><tr><td>DNA Gel Extraction Kit</td><td>NEB</td><td>T1020L</td><td>Used for gel extraction.</td></tr><tr><td>Dry bath</td><td>Benchmark Scientific</td><td>BSH1001</td><td>Used for heating RNA samples in microcentrifuge tubes.&amp;nbsp;</td></tr><tr><td>FIJI/ImageJ</td><td>NIH (National Institutes of Health)</td><td>N/A</td><td>Open-source image analysis software; used for quantification of fluorescence intensity and ROI analysis.</td></tr><tr><td>Gel electrophoresis system&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>B2-BP</td><td>Used for running DNA gel electrophoresis.</td></tr><tr><td>Gel loading dye, purple (6&amp;times;)</td><td>NEB</td><td>B7024S</td><td>Used as a loading dye for DNA gel electrophoresis.</td></tr><tr><td>Instant structured illumination microscope</td><td>VisiTech international</td><td>VT-iSIM</td><td>A confocal microscope capable of imaging GFP and Cy5 fluorescence.&amp;nbsp;</td></tr><tr><td>Isopropanol</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>327272500</td><td>Used for RNA purification.</td></tr><tr><td>MEGAscript T7 Transcription Kit</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AM1334</td><td>Used for in vitro T7 transcription. The kit includes nucleotide solutions (ATP, CTP, GTP, UTP) and DNase.</td></tr><tr><td>Microcentrifuge tubes</td><td>Genesee Scientific</td><td>22-284</td><td>1.5mL tube; used for storing DNA templates for in vitro transcription, RNA products, and aliquots of spermine and DFHBI-1T.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Mini centrifuge</td><td>Benchmark Scientific</td><td>Z763845</td><td>Used for brief centrifugation.</td></tr><tr><td>Nanodrop One Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>13-400-518</td><td>Microvolume spectrophotometer for measuring DNA and RNA concentration and quality.</td></tr><tr><td>Nuclease-free water&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AM9937</td><td>Used for resuspension of RNA pellets, preparation of RNA wash and refolding buffers, and dilution of spermine and DFHBI-1T.</td></tr><tr><td>Online molar mass calculator</td><td>New England Biolabs (NEB)</td><td>N/A</td><td>Online tool used to calculate RNA mass from molar quantity (e.g., pmol).</td></tr><tr><td>Polypropylene PCR tubes</td><td>Corning</td><td>3745</td><td>200 &amp;mu;L PCR tubes used for PCR, in vitro transcription, and RNA clustering reactions</td></tr><tr><td>PowerPac Basic Power Supply</td><td>Bio-rad</td><td>1645050</td><td>Used for running DNA gel electrophoresis.</td></tr><tr><td>Proflex PCR thermal cycler</td><td>Thermo Fisher Scientific</td><td>44-840-73</td><td>Used for PCR, in vitro transcription and heating RNA samples in PCR tubes.</td></tr><tr><td>Q5 High-Fidelity 2&amp;times; Master Mix</td><td>NEB</td><td>M0492S</td><td>Used as 2&amp;times; high-fidelity master mix.</td></tr><tr><td>Refrigerated centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>5424R</td><td>Used for RNA purification and pelleting.&amp;nbsp;</td></tr><tr><td>RNase Decontamination Solution</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AM9782</td><td>Used for cleaning lab benches and surfaces to minimize RNase contamination.</td></tr><tr><td>Spermine&amp;nbsp;tetrahydrochloride</td><td>Sigma-Aldrich</td><td>S1141-1G</td><td>Used for inducing RNA clustering.</td></tr><tr><td>Tris Base</td><td>Millipore Sigma</td><td>T1503-1KG</td><td>Used for preparation of Tris buffer (pH 7.0).</td></tr><tr><td>Ultrapure agarose</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>16500500</td><td>Used for DNA gel electrophoresis.&amp;nbsp;</td></tr></tbody></table>

visualization and quantification of intermolecular rna base pairing in in vitro rna clusters using split broccoli rna reporters

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

RNA clustering, driven by multivalent intermolecular RNA&#8211;RNA interactions, can occur in vitro in the absence of proteins. While chemical probing and computational simulations have been used to predict these interactions, methods for directly assessing intermolecular base pairing in RNA clusters remain limited. This study presents a visual assay based on split Broccoli RNA reporters conjugated to fluorescently labeled RNAs of interest. The assay enables the detection and quantification of intermolecular base pairing in RNA clusters. The split Broccoli system contains complementary reporter sequences that dimerize to form the Broccoli RNA aptamer. The resulting dimerized RNA structure binds DFHBI-1T, a fluorophore that emits green fluorescence upon binding. Upon RNA clustering, the appearance of green fluorescence reports base pairing mediated by the complementary reporter sequences among the RNAs of interest. Measurement of DFHBI-1T fluorescence enables quantitative assessment of how in vitro RNA clustering conditions influence intermolecular base pairing between these reporter sequences.

This protocol describes a visual assay using split Broccoli RNA reporters to detect and quantify intermolecular base pairing in RNA clusters in vitro.

RNA clustering, driven by multivalent intermolecular RNA&#8211;RNA interactions, can occur in vitro in the absence of proteins. While chemical probing and ...

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.

visualization-quantification-intermolecular-rna-base-pairing-in-vitro

Research

JoVE Journal

Biology

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Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

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Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

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Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

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Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

The protocol presents two methods to determine the kinetics of the fluorogenic RNA aptamers Spinach2 and Broccoli. The first method describes how to measure fluorogenic aptamer kinetics in vitro with a plate reader, while the second method details the measurement of fluorogenic aptamer kinetics in cells by flow cytometry.

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

We established a Duplex in situ hybridization assay to map insulin receptor isoforms IR-A and IR-B in the murine choroid plexus, overcoming limitations of traditional methods. This high-resolution approach reveals isoform-specific expression, offering new insights into brain insulin signaling and potential isoform-targeted therapies for neurological disorders.

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RNA-Associated Chromatin DNA-DNA Interaction Method

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学ぶ 分離ブロッコリーRNAレポーターアッセイを用いてin vitro RNAクラスターにおける分子間塩基対を可視化・定量化する方法。蛍光顕微鏡と画像解析でRNA-RNA相互作用を直接評価し、従来の化学プロービングやシミュレーションを補完する。

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

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