FLEth RNA间隙探测器是小干扰RNA的方便报告器
Ingrid M van der Wiel1, Jenny Cheng, Roger Koukiekolo
1Steacie Institute for Molecular Sciences, National Research Council, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6.
Journal of the American Chemical Society
|July 9, 2009
概括
一个新的光探测器FLEth检测双重短干扰RNA (siRNA) 并监测蛋白质-RNA相互作用. 这种探测器提供了关于siRNA结合动态和细胞定位的见解,有助于RNA沉默研究.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 在RNA治疗方面,RNA疗法.
背景情况:
- 短干扰RNA (siRNA) 对于RNA干扰 (RNAi) 和基因沉默至关重要.
- 开发监测siRNA行为和相互作用的工具对于理解RNAi机制和优化治疗应用至关重要.
- 追踪siRNA-蛋白相互作用的现有方法可能很复杂,缺乏实时监测能力.
研究的目的:
- 开发和描述一种基于光的新型探针 (FLEth),用于检测双重siRNA.
- 调查FLEth在报告蛋白质-RNA相互作用方面的实用性,特别是与病毒RNA沉默抑制剂.
- 探索FLEth在细胞环境中的行为及其跟踪siRNA传递的潜力.
主要方法:
- 合成一种与光素部分 (FLEth) 相关联的光素衍生物.
- 光谱学,包括光共振能量转移 (FRET),用于研究FLEth-siRNA复合体的形成和解离.
- 结合试验以确定FLEth与siRNA的解离常数 (Kd).
- 与siRNA,FLEth和p19蛋白进行共同化实验,以评估蛋白质-RNA相互作用.
- 使用脂质体和光显微镜在Huh 7.5细胞中的细胞吸收研究.
主要成果:
- FLEth作为双重siRNA的灵敏光基探针,在复杂化时显示FRET信号.
- 至少有两个FLEth分子与21核酸复合siRNA结合,并报告了解离常数.
- 当FLEth与卡纳的意大利环点病毒蛋白p19相互作用时,可以报告未结合siRNA的部分,而不会直接结合siRNA-p19复合体.
- FLEth在脂质体输送系统内结合siRNA,但在进入细胞时分离,局部化到细胞核中.
结论:
- 在体外检测双重siRNA和监测蛋白质-RNA相互作用时,FLEth是一个有价值的工具.
- 探测器的行为提供了关于siRNA结合动态的见解,其解离表明它不会干扰核心RNA沉默机制.
- FLEth能够报告未结合的siRNA及其细胞定位的能力为研究siRNA在细胞内的传递和功能提供了潜力.
相关概念视频
01:23RNA Interference
RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
01:23RNA Interference
RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
02:15Experimental RNAi
RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
02:31In-situ Hybridization
In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...
02:30Small interfering RNAs (siRNA)
Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
02:30siRNA - Small Interfering RNAs
Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...