利用分裂西兰花RNA报告细胞在 体外 RNA簇中分子间RNA碱基配对的可视化与定量
May 29th, 2026
该方案描述了利用分裂西兰花RNA报告器进行体 外检测和定量RNA簇中分子间碱基配对的视觉测定。
我们应用分裂西兰花RNA报告测定法，在体外检测和定量RNA簇中的分子间碱基配对。化学探测和模拟表明存在RNA相互作用，但无法直接测量。该方案能够在RNA簇内进行直接、准确的评估。
在准备体外RNA转录所需的DNA模板后，用核糖核酸酶去污溶液擦拭工作表面、管架和移液器。所有移液步骤均使用无核酸酶过滤的试管。将转录试剂盒附带的反应缓冲液和核苷酸溶液，以及尿苷三磷酸氰酸5（UTP-Cy5）一起冰冻。
使用前用2000克离心机分离所有管子两秒钟。然后计算DNA中胸腺嘧啶碱基的数量。确定在保持RNA物种间标记密度一致的同时，20微升转录反应所需的UTP和UTP-Cy5体积。
接着，通过结合呈现的试剂，制备一个20微升的转录反应。通过上下移液，在2000克离心机中充分搅拌两秒。在37摄氏度下培养反应六小时后，加入套件附带的一微升脱氧核糖核酸酶，通过移液彻底混合，再次离心。
然后在37摄氏度下再孵育15分钟。将反应转移到1.5毫升的管子上。随套装附带的115微升无核酸酶水和15微升醋酸铵停止液。
混合后，将溶液以2000克离心两秒。接着，将150微升酚氯仿加入管内，轻轻混合以使两相混合。离心机在16,000克下，温度四摄氏度，持续10分钟。
然后将上层水相转移到新的管子上。向转移的溶液中加入等量氯仿，充分混合以确保相相互作用的正确。现在以16,000克离心，温度为4摄氏度，离心两分钟，再次进行相分离过程。
然后将约100至150微升的水层转移到新的管子中。加入900微升异丙醇，彻底混合。将溶液分装成不同管子后，将样品存放于零下20摄氏度的温度下过夜或最多一个月。
将RNA样品置于异丙醇中，16,000克，温度为4摄氏度，离心15分钟。小心地去除异丙醇，同时不扰动RNA沉淀。然后加入一毫升70%乙醇，用无核酸酶水制成。
离心机在16,000克下，四摄氏度，离心两分钟。去除乙醇后，将一毫升用无核酸酶水制备的70%乙醇加入管内，重复离心。在最后的乙醇洗涤过程中，使用1000微升的移液器去除大部分乙醇。
在2000克水位下短暂离心两秒，用20微升移液器去除残留乙醇。RNA沉淀干燥后，将其重新悬浮在20微升无核酸酶水中。将样品冰藏并保护其免受光照。
使用微体积分光光度计测量RNA浓度和纯度。确保吸收比在260对280纳米时约为2.0,260对230纳米时的比值大于2.0。在冰上解冻一管100毫莫拉的精液和50%聚乙二醇8,000。
结合图中组分，新制备10X RNA重折叠缓冲液。通过移液彻底混合后，在2000克的离心机中用台式迷你离心机进行两秒钟。对于共折叠条件，在200微升PCR管内，将体外转录RNA携带顶部或底部序列、RNA重折叠缓冲液和无核酸酶水结合。
按照之前的演示，通过上下移液和离心彻底混合。将样品加热到90摄氏度两分钟。立即将样品转移到室温。
避免光照，孵育一小时。在单独折叠状态下，将RNA样本放入200微升PCR管中加热，然后立即将其放入冰块中至少15分钟。在200微升PCR管中，结合体外转录RNA，携带顶部或底部序列、10倍RNA重折叠缓冲液和无核酸酶水。
在室温下孵育30分钟，避免光线遮挡。现在将包含上下序列的RNA样本合并到一个PCR管中，通过上下移液彻底混合。在2000克离心机下，用台式迷你离心机进行两秒钟。
在室温下孵育30分钟。两种情况下，添加6微升100毫莫拉精子和50%聚乙二醇8,000的预混液。混合后，在工作台式迷你离心机中以2000克离心机离心两秒。
现在将两微升1毫莫拉DFHBI-1T混合到反应中，再次离心。将反应装入玻璃腔盖玻璃中。将箱子在室温下、盖上盖子、在黑暗中孵育四小时，以减少蒸发。
将玻璃室盖玻璃装到显微镜台上。打开激光，然后用目镜定位并聚焦样本。配置显微镜在每个Z平面先用氰酸5通道成像每个感兴趣区域。
然后用绿色荧光蛋白通道成像同一感兴趣区域。所有通道的曝光时间设置为500毫秒。然后将绿色荧光蛋白通道的激光功率设置为80%，氰胺5通道设置为40%。
使用150纳米的Z步长，在室温下成像反应液滴外的覆盖滑移区域。之后，使用图像分析软件量化分子间碱基配对。在诱导RNA聚集时，两个RNA单独或共同形成簇。
与共折叠条件相比，单在上层或下层RNA中，DFHBI-1T在RNA簇中的荧光显著降低。在独立折叠条件下，归一化后的DFHBI-1T信号为0.031，与仅在顶部或底部观察到的基线水平相当。Shu-top、shu-bottom、shu-anti-top和shu-antibottom分别表现出极低的DFHBI-1T荧光。
shu-top与shu-bottom共折，或shu-antitop与shu-antibottom共折，产生的DFHBI-1T信号比单独折叠高。聚顶和聚底共折使归一化的DFHBI-1T荧光增加了5.63倍。shu-top和shu-bottom分别折叠后，仅增加了1.04倍，与基线水平相当。
Shu-top与shu-anti-bottom混合，以及shu-antitop与shu-bottom混合，在两种折叠条件下都表现出比同取向对更高的DFHBI-1T荧光。舒-顶与舒-反底共折叠，以及舒-反顶与舒-底共折叠，分别使DFHBI-1T荧光增加61.86倍和82.60倍。这些混合对的单独折叠分别带来了2.69倍和4.94倍的较小增长。
最重要的考虑是，强健的信号产生可能需要拥挤试剂，而灵敏度则依赖于分裂西兰花RNA的高效二聚化和折叠。该检测法补充了RNA拉下和凝胶电泳方法，用于研究RNA簇中分子间碱基配对。该检测方法可以检测RNA序列、解旋酶和离子对RNA簇内分子间碱基配对的影响。
Overview
This article presents a protocol for directly detecting and quantifying intermolecular base pairing in RNA clusters in vitro using a split Broccoli RNA reporter assay. The method leverages fluorescently labeled RNAs and a split aptamer system to visualize and measure RNA–RNA interactions, providing a quantitative alternative to chemical probing and computational predictions.
Key Study Components
Area of Science
- Molecular biology
- RNA biochemistry
- Fluorescence microscopy
Background
- RNA clustering is driven by multivalent intermolecular RNA–RNA interactions.
- Traditional methods like chemical probing and computational simulations infer but do not directly measure these interactions.
- Direct assessment of intermolecular base pairing within RNA clusters has been limited.
- The split Broccoli RNA aptamer system enables fluorescence-based detection of RNA dimerization.
Purpose of Study
- To develop and demonstrate a visual assay for direct detection of intermolecular base pairing in RNA clusters.
- To quantify how in vitro clustering conditions affect RNA–RNA interactions.
- To provide a complementary approach to existing RNA interaction assays.
Methods Used
- Preparation of DNA templates and in vitro transcription of RNAs labeled with Cy5.
- Purification of transcribed RNAs using phenol-chloroform extraction and ethanol precipitation.
- Folding of RNAs under co-folding and separate folding conditions with crowding reagents (spermine, PEG 8000).
- Assembly of split Broccoli RNA reporters and addition of DFHBI-1T fluorophore.
- Fluorescence imaging using confocal microscopy and quantitative image analysis.
Main Results
- DFHBI-1T fluorescence was substantially higher in co-folded RNA samples compared to separately folded or single RNA controls.
- Co-folding of complementary split Broccoli RNAs resulted in up to 82.6-fold increases in fluorescence, indicating robust intermolecular base pairing.
- Mixed orientation pairs (shu-top with shu-anti-bottom, etc.) showed the highest fluorescence increases upon co-folding.
- Robust signal generation depended on crowding reagents and efficient RNA dimerization.
Conclusions
- The split Broccoli RNA reporter assay enables direct, quantitative assessment of intermolecular base pairing in RNA clusters.
- This method complements traditional RNA interaction assays such as pull-downs and gel electrophoresis.
- The assay can be used to study the effects of RNA sequence, helicases, and ions on RNA–RNA interactions in vitro.
Frequently Asked Questions
Executive Industry Relevance
Direct quantification of intermolecular RNA base pairing in vitro addresses a critical gap in early discovery, enabling mechanistic de-risking of RNA-driven clustering phenomena. The split Broccoli RNA reporter assay provides a quantitative, fluorescence-based readout for RNA–RNA interactions, supporting predictive confidence in target validation and assay development. This capability enhances portfolio triage by clarifying the molecular basis of RNA clustering under defined conditions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct interrogation of RNA–RNA interaction hypotheses within clustered assemblies.
- Supports mechanistic de-risking by distinguishing specific intermolecular base pairing from background association.
- Provides quantitative data to inform target validation and early-stage portfolio decisions.
Screening & Assay Development
- Facilitates preparation of validated RNA cluster systems for downstream screening workflows.
- Delivers standardized, reproducible fluorescence outputs for assay benchmarking.
- Enables reliable evaluation of sequence, helicase, or ionic effects on RNA–RNA interactions.
Translational & Preclinical Research
- Aligns with disease-relevant RNA clustering models when mechanistic continuity is required.
- Supports risk-adjusted advancement by providing quantitative, comparative readouts across experimental conditions.
- Offers predictive de-risking for RNA-targeted therapeutic strategies.
Pipeline & Workflow Integration
This assay integrates into the discovery continuum from hypothesis-driven target validation through assay development and preclinical mechanistic studies.
- Discovery Biology: Quantifies direct intermolecular base pairing, clarifying RNA clustering mechanisms.
- Screening: Provides reproducible, quantitative fluorescence outputs for comparative analysis.
- Analytics: Enables statistical comparison of folding conditions and sequence variants using normalized fluorescence measurements.
- Translational Research: Supports continuity by modeling RNA–RNA interactions relevant to disease or therapeutic context.
- Enterprise Reuse: Functions as a reusable platform for diverse RNA interaction studies across programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in RNA clustering studies.
- Operational Value: Standardizes detection and quantification of RNA–RNA interactions for scalable workflows.
- Strategic Value: Informs go/no-go decisions and reduces late-stage biological risk for RNA-targeted assets.
- Portfolio Impact: Enables risk-adjusted prioritization based on direct, quantitative mechanistic data.
Implementation Considerations
- Requires expertise in RNA biochemistry and fluorescence microscopy.
- Needs access to confocal imaging and quantitative image analysis infrastructure.
- Demands rigorous cross-team standardization of RNA preparation and folding protocols.
- May require adaptation for different RNA sequences or clustering models.
- Sensitivity depends on efficient dimerization and folding of split Broccoli RNAs and use of crowding reagents.
