通过单细胞检测 in vivo 活动中跨剪接的 ribozyme 的活性
Sumitaka Hasegawa1, Jung W Choi, Jianghong Rao
1Department of Radiology & Bio-X Program, Molecular Imaging Program at Stanford, Stanford University, 300 Pasteur Drive, Stanford, California 94305-5344, USA.
Journal of the American Chemical Society
|June 10, 2004
概括
研究人员开发了一种新的方法来跟踪使用β-lactamase的活细胞中的Tetrahymena ribozyme活性. 这种敏感的技术可以有效地选 ribozyme 变体,从而改善 RNA 编辑.
科学领域:
- 分子生物学分子生物学
- 生物化学 生化学
- 细胞生物学 细胞生物学
背景情况:
- 铁胺跨拼接 рибо酶编辑RNA,但需要提高治疗应用的效率.
- 目前评估 ribozyme 活性的方法在灵敏度和细胞兼容性方面是有限的.
研究的目的:
- 开发一种灵敏的单细胞方法,用于监测活哺乳动物细胞中Tetrahymena ribozyme转接合活性.
- 为了使 ribozyme 库能够进行高通量选,以寻找改进的变体.
主要方法:
- 使用细菌β-乳酸酶作为Tetrahymena ribozyme转接的记者.
- 采用光显微镜和流细胞计,用于单细胞分析.
- 证明与活细胞测试的兼容性.
主要成果:
- 在单活哺乳动物细胞中成功报告了tetrahymena ribozyme的转接活动.
- 基于酶的检测方法被证明是高度敏感的.
- 该方法与活细胞流细胞计相兼容.
结论:
- 已经建立了一种新的,敏感的和细胞兼容的方法来检测Tetrahymena ribozyme的跨拼接活性.
- 这种方法有助于系统的,基于细胞的选 ribozyme 库.
- 开发的方法对于识别潜在治疗用途的增强型转接合的 ribozyme 变体至关重要.
相关概念视频
RNA Splicing
Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
Ribozymes
The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
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Ribosome Profiling
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 helps...
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Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
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