可点击的APEX2探针用于活细胞中增强的RNA近距离标记
Jiying Liang1, Jinghua Han1, Xutao Gao1
1Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
Analytical chemistry
|December 15, 2023
概括
研究人员开发了一种新的探针 (Ph_N3),以提高活细胞中使用APEX2近距离标签的RNA标签效率. 这种增强的方法允许敏感的转录组分析和双标记策略来研究RNA运动.
科学领域:
- 分子生物学分子生物学
- 细胞生物学 细胞生物学
- 生物化学 生物化学
背景情况:
- 在研究活细胞中的RNA局部化方面,APEX2近距离标记至关重要.
- 现有的生物-基基具有有限的标签效率,阻碍了与其他方法的兼容性.
研究的目的:
- 设计新的APEX2探头,提高反应性,水友性和脂友性.
- 为活细胞研究开发一个更有效和多功能的RNA标签策略.
- 为了启用双位点标记来跟踪RNA转位.
主要方法:
- 设计和合成的衍生物作为APEX2探针.
- 使用Ph_N3探头进行APEX2介导的近距离标记.
- 执行了用于转录组分析的高通量测序.
- 在双位置研究中采用DRAQ5的直角单点氧 (1O2) 中介标记.
主要成果:
- Ph_N3 通过两种生物化路径证明了可靠的RNA标记能力.
- APEX2/Ph_N3标签实现了对线粒体矩阵转录组分析的高灵敏度和特异性.
- 通过使用APEX2/Ph_N3和DRAQ5-1O2策略验证直角双位置标签.
结论:
- 新的Ph_N3探针显著提高了APEX2介导的RNA标记效率.
- 这种方法提供了一个敏感的工具,用于在特定的细胞区内进行转录组分析.
- 正交双标签策略为研究活细胞中的RNA动态提供了一种可行的方法.
相关概念视频
Labeling DNA Probes
8.2K
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...
8.2K
Ribosome Profiling
3.5K
Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
3.5K
Protein 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


