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技术文章 / article 您的位置:网站首页 > 技术文章 > 荧光蛋白的简述

荧光蛋白的简述

发布时间: 2024-06-13  点击次数: 68次

 

荧光蛋白广泛用于报告基因表达,蛋白质动态变化和代谢活动。与蛋白质类似,RNA在细胞中位置分布,行为和功能极其复杂。鉴于此,作为荧光蛋白的模拟物,荧光RNA(FR)被用来进行RNA的研究。

Fluorescent proteins are widely used in as reporters of gene expression, protein dynamics and metabolic activities. Similar to proteins, RNAs have highly complex distributions, behaviors, and functions in cells. To this end, Fluorescent RNAs (FRs) are mimicary of fluorescent proteins for RNA studies.


尽管目前已开发出几个可用的荧光RNA,但是,这其中多数都不够稳定,无法用于追踪和定量活细胞中低丰度的RNA。

Although there are a few FRs available, however, many of these FRs are not robust enough to trace and quantify low abundant RNAs in live cells.

 

Pepper是一系列单体、多色的荧光RNA,相比其它荧光RNA,其在荧光亮度和荧光激活率上大幅提高(一个甚至多个数量级)。Pepper可以对活细胞中多种RNA进行简单而有效的成像追踪,并且几乎不会干扰目标RNA的转录、定位和翻译。

Peppers are a series of monomeric, multicolor FRs with much improved (one order of magnitude or even more) cellular fluorescence brightness and fluorescence turn-on ratio. Peppers allow simple and robust imaging of diverse RNA species in live cells with minimal perturbation of the target RNA’s transcription, localization, and translation.


由于Pepper较高的信号背景比,通过流式细胞仪或酶标仪,还可对单细胞或细胞群体中Pepper标记的RNA进行定量研究。这系列荧光RNA是活细胞内RNA实时成像的理想工具。

Due to its high signal-background ratio, it is also feasible to perform quantification of Pepper tagged RNA in single cells or assembled cells by flow cytometry and microplate readers. These FRs provide ideal tools for live imaging of cellular RNAs.



技术

Technology

RNA是生物科学和医学中发展迅速的新兴研究领域。除了分子生物学中心法则中mRNA,rRNA,tRNA的功能外,最近的研究还揭示了非编码RNA(ncRNA)的特征和功能,ncRNA数量巨大,并在各种生物过程中起着重要作用,这对RNA功能的传统概念进行了重新定义。

RNA is an emerging, rapidly growing research field in biological science and medicine. In addition to the well known functions of mRNAs, rRNAs, tRNAs in the central dogma of molecule biology, recent studies reveal the identity and functions of vast noncoding RNAs (ncRNAs) that play an important role in diverse biological processes, which are reshaping the prior conceptions about RNA functions.

 

在活细胞中,RNA存在复杂的动态过程,包括表达、降解、转录、剪接和其它化学修饰。

In living cells, RNAs exhibit complex dynamics including expression,  degradation, translocation, splicing and various chemical modifications.

 

诸如荧光原位杂交(FISH)法、酶促共价标记法的RNA可视化方法,需要将细胞固定,而不适用于活细胞成像。经过修饰的RNA可与融合了荧光蛋白的特定RNA结合蛋白(例如MCP,PCP,λN或Cas)相结合,从而实现在活细胞中对目的RNA进行标记成像,这一方法甚至可实现单分子水平的RNA检测。

Methodology to visualize RNA such as fluorescent in situ hybridization (FISH), enzymatic covalent labeling require cell fixation and are not suitable for live cell imaging. RNAs with engineered motifs can be tethered with fusions of fluorescent protein and specific RNA binding proteins (RBPs) e.g. MCP, PCP, λN or Cas may be used to image RNA in live cells at the single molecule level.

 

但是,未结合的荧光蛋白分子可在整个细胞中扩散,并产生较高背景的荧光。此外,将过大的蛋白捆缚在RNA上,是否会影响RNA的定位,稳定性和行为仍有待商榷。

However, the unbound MCP-FP molecules diffuse throughout the cells and generates high background fluorescence. In addition, whether such a heavy load of tethered protein affects the localization, stability and behavior of RNAs remains to be determined.


原位RNA荧光标记检测技术

Technology for in situ RNA fluorescent labeling and detection.


在生命科学研究上,不同颜色的荧光蛋白掀起一场巨大的变革,它可以对目的蛋白质做遗传编码标记,在活细胞中对蛋白质进行无背景追踪。而针对感兴趣的RNA,也可以采取类似的方法,利用可与荧光团结合的RNA适配体,直接进行遗传编码标记。

In the history, fluorescent proteins (FPs) of different colors had revolutionized research of life sciences, which are genetically encoded labels of proteins enabling background free tracing of proteins in live cells. RNAs of interest may also be genetically labeled similarly and straightforwardly with fluorophore-binding RNA aptamers.


这类适配体,称为荧光RNA,也应能对活细胞中的各种RNA进行简单,稳定且无背景的标记成像。

These aptamers, termed fluorescent RNAs (FRs), shall also enable easy, robust, background free imaging of diverse RNAs in living cells.


尽管荧光RNA技术看起来简单有效且前景广阔,但是目前可用的荧光RNA在实用方面却存在极大限制。

While simple and as promising as they appear, however, the utility of FRs current available is limited.


现有的荧光RNA中,部分染料配体在活细胞中呈现出显著的背景荧光,且/或难以穿过细胞质膜实现RNA标记。部分荧光RNA在活细胞中的光稳定性和荧光强度则十分有限,或者是多聚体的。

Some of the dye ligands of current FRs show significant background fluorescence in live cells and/or do not readily diffuse across plasma membranes, or the FRs. Some FRs has limited stability and brightness in live cells, or function as multimer.


与荧光蛋白相似,理想的荧光RNA应该是单体、稳定且明亮的,并具有多种可供选择的光谱,但是这一直难以实现。

Analogously to FPs, ideal FRs should be monomeric, stable, bright, and multicolored, which had been challenging to achieve.



Pepper荧光RNA技术

Technology for in situ RNA fluorescent labeling and detection.

我们以不一样的方法设计了具有膜通透性的配体染料,并经过数轮优化,获得了系列单体、高亮度和高稳定性的荧光RNA——Pepper,其发射光谱非常广泛,从青色到红色。

Peppers, a series of monomeric, highly bright, and stable FRs with a broad range of emission maxima spanning from cyan to red, were obtained by unique design of dendritic cell permeable dye ligand and multiple rounds of optimization.

 

Pepper的系列染料配体展现良好的膜通透性和低毒性,并在溶液和细胞中具有较低背景的荧光。

These dyes showed good membrane permeability, low cytotoxicity, and little fluorescence in solution or live cells.



对比目前可用的荧光RNA,Pepper在荧光强度和激活倍数上提高了一个数量级,在亲和力方面提高了一到两个数量级,温度稳定性上提高约20℃,并且拥有更好的pH耐受性和更广的光谱范围,这将更利于其在活细胞中使用。

Compared to currently available FRs, Peppers showed an order of magnitude enhanced cellular fluorescence intensity and fluorescence turn-on ratio, one or two orders of magnitude enhanced affinity, ~20 oC increased Tm, expanded pH tolerance, and a broad spectral range available for live cell studies.

 

首先,针对活细胞中的mRNA及其它多种类型的RNA,Pepper可以进行简单有效的成像,而几乎不会干扰目的RNA的转录、定位和翻译。

For the first time, Peppers allow simple and robust imaging of mRNA and other RNA species in live cells with minimal perturbation of the target RNA’s transcription, localization, and translation.

 

此外,Pepper可结合CRISPR系统示踪基因轨迹,实时追踪蛋白质-RNA的捆缚过程,并利用结构光照明显微镜对RNA进行超分辨成像。

Peppers may also be used in imaging of genomic loci through CRISPR display, real-time tracking of protein-RNA tethering, and super-resolution imaging of RNA by structured illumination microscopy.

 

由于Pepper具有较高的信噪比,还可用Pepper标记单细胞或细胞群体中的RNA,运用流式细胞仪或酶标仪,进行活细胞水平定量研究。Pepper是活细胞内RNA实时成像的理想研究工具。

Due to its high signal to background ratio, Peppers can be used in quantitative studies of RNAs in live cells, using flow cytometry or microplate reading. These Pepper FRs provide ideal tools for live imaging of cellular RNAs.