长RNA的光终端标记和封装用于单分子FRET-TIRF显微镜
Esra Ahunbay1, Besim Fazliji1, Besa Maloku1
1Department of Chemistry, University of Zurich.
Journal of visualized experiments : JoVE
|November 4, 2024
概括
我们开发了一种新的RNA双端标记方法,使用单分子Förster共振能量转移 (smFRET) 来研究RNA的动态. 这种技术可以在没有人工修改的情况下精确观察RNA构造变化.
科学领域:
- 生物化学和分子生物学
- 生物物理学的生物物理.
- 核酸化学的核酸化学
背景情况:
- 单分子弗斯特共振能量转换 (smFRET) 对于观察动态生物分子构造变化至关重要.
- 监测RNA动态需要精确的标记策略,避免人工修改.
- 现有的方法可能不适用于所有RNA序列和大小.
研究的目的:
- 开发一种适合smFRET研究的RNA多功能和直接终端标记方法.
- 为了使RNA动态的观察能够独立于序列或大小.
- 适应现有的化学反应,使其在核酸研究中广泛适用.
主要方法:
- 开发了一种双端标签策略,针对RNA的5'-酸盐 (通过EDC/NHS的碳胺激活) 和3'-酸盐 (通过周期酸氧化).
- 使用商用化学品进行标签,避免需要定制的探针合成.
- 在脂囊中封装的双端标记单个RNA分子用于全内部反射光 (TIRF) 显微镜.
主要成果:
- 通过FRET一对光体成功实现了RNA末端的共价标记.
- 展示了一种适应各种RNA分子的方法,无论大小或序列如何.
- 建立了一个单分子成像方法,通过将标记的RNA封装在囊泡中,用于TIRF显微镜.
结论:
- 开发的双端标记方法为使用smFRET研究RNA动态提供了可概括的方法.
- 这种技术允许对RNA行为进行详细,动态的观察,而无需引入人工修改.
- 5'-EDC/NHS激活策略对用5'-酸盐标记核酸具有更广泛的影响.
更多相关视频
07:51Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments
Published on: December 21, 2017
8.2K
10:19Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin
Published on: January 24, 2025
388
相关概念视频
01:05Total Internal Reflection Fluorescence Microscopy
5.7K
Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
5.7K
01:19Protein Dynamics in Living Cells
2.1K
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
2.1K