熒(ying)光(guang)蛋(dan)白廣泛用于報(bao)告基因表(biao)達，蛋(dan)白質(zhi)動態(tai)變化(hua)和(he)代謝活動。與(yu)蛋(dan)白質(zhi)類似，RNA在(zai)細胞中位置分布(bu)，行為和(he)功能(neng)極其復雜。鑒(jian)于此，作為熒(ying)光(guang)蛋(dan)白的(de)模擬物，熒(ying)光(guang)RNA（FR）被(bei)用來(lai)進行RNA的(de)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(qian)已開發出幾個可用(yong)的熒光RNA，但是，這其中多數都不(bu)夠穩定，無法用(yong)于(yu)追蹤和定量(liang)活(huo)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(dan)體(ti)、多(duo)(duo)色的熒(ying)(ying)(ying)光(guang)(guang)RNA，相比(bi)其它熒(ying)(ying)(ying)光(guang)(guang)RNA，其在熒(ying)(ying)(ying)光(guang)(guang)亮度和熒(ying)(ying)(ying)光(guang)(guang)激活(huo)率上大幅提高（一個甚至多(duo)(duo)個數量級）。Pepper可以對活(huo)細(xi)胞中多(duo)(duo)種(zhong)RNA進行簡單(dan)而有(you)效的成像追蹤，并且幾乎不(bu)會干擾目(mu)標RNA的轉錄(lu)、定位(wei)和翻(fan)譯(yi)。

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(de)信號背景比(bi)，通過流(liu)式細(xi)胞(bao)儀或(huo)酶標儀，還可對單細(xi)胞(bao)或(huo)細(xi)胞(bao)群(qun)體中Pepper標記的(de)RNA進行(xing)定量(liang)研究(jiu)。這(zhe)系列熒光RNA是活細(xi)胞(bao)內RNA實時(shi)成像的(de)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(wu)科學(xue)和醫學(xue)中(zhong)發展迅速的新(xin)興研究(jiu)(jiu)領(ling)域。除了(le)分子生物(wu)學(xue)中(zhong)心法則中(zhong)mRNA，rRNA，tRNA的功(gong)能外，最近的研究(jiu)(jiu)還揭示了(le)非(fei)編碼RNA（ncRNA）的特征和功(gong)能，ncRNA數量巨大，并在各種生物(wu)過程中(zhong)起著重要(yao)作(zuo)用，這對RNA功(gong)能的傳(chuan)統概念進行(xing)了(le)重新(xin)定義(yi)。

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(fu)雜的動態過程(cheng)，包(bao)括表達(da)、降解、轉錄、剪接和其它化學修飾。

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

諸如熒(ying)光(guang)原位雜交（FISH）法、酶促(cu)共價(jia)標(biao)記(ji)法的(de)RNA可(ke)視化方法，需要將(jiang)細(xi)(xi)胞固(gu)定，而(er)(er)不(bu)適用于活(huo)細(xi)(xi)胞成像。經過修(xiu)飾的(de)RNA可(ke)與融(rong)合(he)了熒(ying)光(guang)蛋白(bai)的(de)特定RNA結合(he)蛋白(bai)（例如MCP，PCP，λN或(huo)Cas）相結合(he)，從而(er)(er)實現(xian)在活(huo)細(xi)(xi)胞中對目的(de)RNA進行標(biao)記(ji)成像，這一(yi)方法甚至可(ke)實現(xian)單分子水平(ping)的(de)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(shi)，未結合的(de)熒(ying)光蛋白(bai)分子可(ke)在整個細胞中擴散(san)，并產生(sheng)較(jiao)高背景的(de)熒(ying)光。此外，將過大的(de)蛋白(bai)捆縛在RNA上，是(shi)否會影響RNA的(de)定位，穩(wen)定性和行為仍(reng)有待商榷(que)。

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(zai)生命科(ke)學(xue)研究(jiu)上，不同顏色的熒(ying)光蛋(dan)白(bai)(bai)掀起一(yi)場巨大的變(bian)革，它(ta)可(ke)以對(dui)目的蛋(dan)白(bai)(bai)質(zhi)做遺傳編碼標(biao)(biao)記，在(zai)活細胞(bao)中對(dui)蛋(dan)白(bai)(bai)質(zhi)進行(xing)無背景追蹤。而針對(dui)感興趣的RNA，也可(ke)以采取類(lei)似(si)的方法(fa)，利用可(ke)與熒(ying)光團(tuan)結合的RNA適配體，直接進行(xing)遺傳編碼標(biao)(biao)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(zhe)類適配體，稱為熒光RNA，也應能對活細胞中的各種RNA進行簡單，穩(wen)定且無背景的標(biao)記成(cheng)像(xiang)。

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

盡管熒光(guang)(guang)RNA技術(shu)看起來簡單(dan)有(you)效且(qie)前景廣(guang)闊(kuo)，但是(shi)目前可用的(de)熒光(guang)(guang)RNA在實用方面卻存(cun)在極(ji)大限制。

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

現(xian)有(you)的(de)熒(ying)光(guang)(guang)RNA中(zhong)，部(bu)(bu)分染料配體(ti)在活細(xi)胞(bao)中(zhong)呈現(xian)出顯著的(de)背景熒(ying)光(guang)(guang)，且(qie)/或難(nan)以穿過細(xi)胞(bao)質膜實現(xian)RNA標記。部(bu)(bu)分熒(ying)光(guang)(guang)RNA在活細(xi)胞(bao)中(zhong)的(de)光(guang)(guang)穩定(ding)性和熒(ying)光(guang)(guang)強度(du)則(ze)十分有(you)限，或者是多聚體(ti)的(de)。

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(yu)熒(ying)(ying)光蛋白(bai)相(xiang)似，理想(xiang)的(de)熒(ying)(ying)光RNA應該是(shi)單體(ti)、穩定且明(ming)亮(liang)的(de)，并具有多(duo)種(zhong)可供選(xuan)擇的(de)光譜，但是(shi)這(zhe)一直難以實現。

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

我(wo)們以不一樣(yang)的方法(fa)設計了(le)具有膜(mo)通透性的配體(ti)染料，并經過數輪(lun)優化，獲得(de)了(le)系列(lie)單體(ti)、高(gao)亮度(du)和(he)高(gao)穩(wen)定性的熒光RNA——Pepper，其發射光譜(pu)非常廣泛(fan)，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(he)低毒性，并在溶(rong)液和(he)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(dui)比目前可用(yong)的熒(ying)光RNA，Pepper在(zai)熒(ying)光強度(du)和激活倍(bei)數(shu)上提(ti)高(gao)了(le)一個(ge)數(shu)量級，在(zai)親和力方面(mian)提(ti)高(gao)了(le)一到(dao)兩個(ge)數(shu)量級，溫度(du)穩(wen)定性上提(ti)高(gao)約(yue)20℃，并且擁(yong)有更(geng)好的pH耐(nai)受性和更(geng)廣的光譜范圍(wei)，這將更(geng)利于其在(zai)活細胞(bao)中使用(yong)。

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(shou)先，針對(dui)活細(xi)胞(bao)中的(de)(de)mRNA及(ji)其它(ta)多種類(lei)型(xing)的(de)(de)RNA，Pepper可(ke)以進行(xing)簡單有效(xiao)的(de)(de)成像，而幾乎(hu)不會(hui)干擾目的(de)(de)RNA的(de)(de)轉錄(lu)、定位(wei)和(he)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(xi)統(tong)示蹤基因(yin)軌跡，實時追蹤蛋(dan)白質-RNA的(de)捆縛過程，并利用結構(gou)光照明顯微鏡對RNA進(jin)行超(chao)分辨成像(xiang)。

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(xin)噪比，還可(ke)用(yong)Pepper標記單細(xi)(xi)(xi)胞(bao)(bao)或(huo)細(xi)(xi)(xi)胞(bao)(bao)群體中(zhong)的RNA，運用(yong)流(liu)式細(xi)(xi)(xi)胞(bao)(bao)儀(yi)或(huo)酶標儀(yi)，進(jin)行活細(xi)(xi)(xi)胞(bao)(bao)水平定量研(yan)究。Pepper是活細(xi)(xi)(xi)胞(bao)(bao)內RNA實時成像(xiang)的理想研(yan)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.