在活哺乳动物细胞中的成像和跟踪RNA通过光光近亲性标记
Alexandria L Quillin1, Diane B Karloff1,2, Tewoderos M Ayele2
1Department of Chemistry, Washington University, St. Louis, Missouri 63130, United States.
ACS chemical biology
|February 15, 2025
概括
这项研究引入了一种新的光亲和标记方法,用于强大的细胞RNA跟踪. 通过紫外线的共价附着提高了成像灵敏度,并使RNA动态的精确可视化,克服了非共价方法的局限性.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 细胞生物学 细胞生物学
背景情况:
- 基于aptamer的RNA标签为基于蛋白质的方法提供了一个基因编码的替代方案.
- 由于染料洗和解离,非共价的阿巴-素相互作用限制了RNA成像的稳定性和跟踪准确性.
- 现有的方法难以在RNA分子之间进行染料转移,从而损害了动态研究.
研究的目的:
- 开发一种共价RNA标记策略,以克服基于非共价APTAMER系统的局限性.
- 为了实现强大的,空间时间控制的RNA成像和跟踪在活细胞内.
- 为了提高研究RNA动态的灵敏度和时间分辨率.
主要方法:
- 用可光激活的迪亚齐林组对阿帕特默配体的功能化,用于UV诱导的共价RNA附着.
- 将光亲和链接器纳入马拉绿色中,并将其吸收体融合到向RNA (snRNA和mRNA) 中.
- 活细胞成像实验,以评估标记效率,灵敏度和RNA动态.
主要成果:
- 在紫外线辐射下对活细胞RNA成像的感觉得到改善.
- 通过共价标签成功可视化了几分钟的时间尺度上的RNA动态.
- 展示了共价链接的稳定性,防止染料转移,并使精确的RNA跟踪成为可能.
结论:
- 光亲和标签为共价RNA标签提供了强大的方法,增强了成像能力.
- 这种方法使得RNA局部化和动态的精确,时间解析的研究成为可能.
- 该方法对细胞RNA行为和相互作用的多样化研究具有前景.
更多相关视频
10:59Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy
Published on: October 18, 2024
518
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
相关概念视频
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
03:31Labeling DNA Probes
8.1K
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
8.1K