熒光蛋白的簡(jiǎn)述

熒光蛋白廣泛用于報告基因表達，蛋白質(zhì)動(dòng)態(tài)變化和代謝活動(dòng)。與蛋白質(zhì)類(lèi)似，RNA在細胞中位置分布，行為和功能極其復雜。鑒于此，作為熒光蛋白的模擬物，熒光RNA（FR）被用來(lái)進(jìn)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.

盡管目前已開(kāi)發(fā)出幾個(gè)可用的熒光RNA，但是，這其中多數都不夠穩定，無(wú)法用于追蹤和定量活細胞中低豐度的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是一系列單體、多色的熒光RNA，相比其它熒光RNA，其在熒光亮度和熒光激活率上大幅提高（一個(gè)甚至多個(gè)數量級）。Pepper可以對活細胞中多種RNA進(jìn)行簡(jiǎn)單而有效的成像追蹤，并且幾乎不會(huì )干擾目標RNA的轉錄、定位和翻譯。

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較高的信號背景比，通過(guò)流式細胞儀或酶標儀，還可對單細胞或細胞群體中Pepper標記的RNA進(jìn)行定量研究。這系列熒光RNA是活細胞內RNA實(shí)時(shí)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.

RNA是生物科學(xué)和醫學(xué)中發(fā)展迅速的新興研究領(lǐng)域。除了分子生物學(xué)中心法則中mRNA，rRNA，tRNA的功能外，最近的研究還揭示了非編碼RNA（ncRNA）的特征和功能，ncRNA數量巨大，并在各種生物過(guò)程中起著(zhù)重要作用，這對RNA功能的傳統概念進(jìn)行了重新定義。

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.

在活細胞中，RNA存在復雜的動(dòng)態(tài)過(guò)程，包括表達、降解、轉錄、剪接和其它化學(xué)修飾。

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

諸如熒光原位雜交（FISH）法、酶促共價(jià)標記法的RNA可視化方法，需要將細胞固定，而不適用于活細胞成像。經(jīng)過(guò)修飾的RNA可與融合了熒光蛋白的特定RNA結合蛋白（例如MCP，PCP，λN或Cas）相結合，從而實(shí)現在活細胞中對目的RNA進(jìn)行標記成像，這一方法甚至可實(shí)現單分子水平的RNA檢測。

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，未結合的熒光蛋白分子可在整個(gè)細胞中擴散，并產(chǎn)生較高背景的熒光。此外，將過(guò)大的蛋白捆縛在RNA上，是否會(huì )影響RNA的定位，穩定性和行為仍有待商榷。

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.

在生命科學(xué)研究上，不同顏色的熒光蛋白掀起一場(chǎng)巨大的變革，它可以對目的蛋白質(zhì)做遺傳編碼標記，在活細胞中對蛋白質(zhì)進(jìn)行無(wú)背景追蹤。而針對感興趣的RNA，也可以采取類(lèi)似的方法，利用可與熒光團結合的RNA適配體，直接進(jìn)行遺傳編碼標記。

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.

這類(lèi)適配體，稱(chēng)為熒光RNA，也應能對活細胞中的各種RNA進(jìn)行簡(jiǎn)單，穩定且無(wú)背景的標記成像。

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

盡管熒光RNA技術(shù)看起來(lái)簡(jiǎn)單有效且前景廣闊，但是目前可用的熒光RNA在實(shí)用方面卻存在極大限制。

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

現有的熒光RNA中，部分染料配體在活細胞中呈現出顯著(zhù)的背景熒光，且/或難以穿過(guò)細胞質(zhì)膜實(shí)現RNA標記。部分熒光RNA在活細胞中的光穩定性和熒光強度則十分有限，或者是多聚體的。

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.

與熒光蛋白相似，理想的熒光RNA應該是單體、穩定且明亮的，并具有多種可供選擇的光譜，但是這一直難以實(shí)現。

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

我們以不一樣的方法設計了具有膜通透性的配體染料，并經(jīng)過(guò)數輪優(yōu)化，獲得了系列單體、高亮度和高穩定性的熒光RNA——Pepper，其發(fā)射光譜非常廣泛，從青色到紅色。

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的系列染料配體展現良好的膜通透性和低毒性，并在溶液和細胞中具有較低背景的熒光。

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

對比目前可用的熒光RNA，Pepper在熒光強度和激活倍數上提高了一個(gè)數量級，在親和力方面提高了一到兩個(gè)數量級，溫度穩定性上提高約20℃，并且擁有更好的pH耐受性和更廣的光譜范圍，這將更利于其在活細胞中使用。

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，針對活細胞中的mRNA及其它多種類(lèi)型的RNA，Pepper可以進(jìn)行簡(jiǎn)單有效的成像，而幾乎不會(huì )干擾目的RNA的轉錄、定位和翻譯。

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可結合CRISPR系統示蹤基因軌跡，實(shí)時(shí)追蹤蛋白質(zhì)-RNA的捆縛過(guò)程，并利用結構光照明顯微鏡對RNA進(jìn)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具有較高的信噪比，還可用Pepper標記單細胞或細胞群體中的RNA，運用流式細胞儀或酶標儀，進(jìn)行活細胞水平定量研究。Pepper是活細胞內RNA實(shí)時(shí)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.