A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA.
A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA.
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DOI：
10.1038/nmeth.2701
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发表时间：
2013-12
期刊：
影响因子：
48
通讯作者：
Jaffrey, Samie R.
中科院分区：
文献类型：
--
作者：
Strack, Rita L.;Disney, Matthew D.;Jaffrey, Samie R.
活细胞中RNA的荧光成像是细胞生物学中的技术挑战性问题。遗传编码荧光RNA的一种策略是将它们表达为与“GFP的RNA模拟物”的融合物。这些是短的适体标签，其在结合类似于GFP中发现的非荧光荧光团时显示荧光。我们发现，这些适体中最亮的Spinach在与感兴趣的RNA融合后通常表现出荧光减弱。我们发现，热不稳定性和错误折叠的倾向相结合的各种菠菜-RNA融合的低荧光帐户。使用系统诱变，我们确定了导致菠菜折叠不良的核苷酸，并产生了菠菜2，其在细胞中表现出显着改善的热稳定性和折叠。此外，我们发现Spinach 2在与各种RNA融合时很大程度上保留了其荧光。使用Spinach 2，我们详细描述了CGG三核苷酸重复的细胞动力学与脆性X震颤/共济失调综合征相关的“毒性RNA”，并表明这些RNA形成具有意想不到的形态可塑性的核灶，由细胞周期和小分子调节。总之，这些数据表明Spinach 2在活细胞中荧光标记RNA方面表现出改进的多功能性。
Fluorescent imaging of RNA in living cells is a technically challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with ‘RNA mimics of GFP’. These are short aptamer tags that exhibit fluorescence upon binding otherwise nonfluorescent fluorophores that resemble those found in GFP. We find that the brightest of these aptamers, Spinach, often exhibits reduced fluorescence after it is fused to RNAs of interest. We show that a combination of thermal instability and a propensity for misfolding account for the low fluorescence of various Spinach-RNA fusions. Using systematic mutagenesis, we identified nucleotides that account for the poor folding of Spinach, and generated Spinach2, which exhibits markedly improved thermal stability and folding in cells. Furthermore, we show that Spinach2 largely retains its fluorescence when fused to various RNAs. Using Spinach2, we detail the cellular dynamics of the CGG trinucleotide-repeat containing “toxic RNA” associated with Fragile-X tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells.
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DOI：
10.1126/science.1207339
发表时间：
2011-07-29
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Paige JS;Wu KY;Jaffrey SR
通讯作者：
Jaffrey SR
影响因子：
16.2
作者：
Todd PK;Oh SY;Krans A;He F;Sellier C;Frazer M;Renoux AJ;Chen KC;Scaglione KM;Basrur V;Elenitoba-Johnson K;Vonsattel JP;Louis ED;Sutton MA;Taylor JP;Mills RE;Charlet-Berguerand N;Paulson HL
通讯作者：
Paulson HL
影响因子：
64.5
作者：
FU, YH;KUHL, DPA;CASKEY, CT
通讯作者：
CASKEY, CT
影响因子：
4
作者：
Disney, Matthew D.;Liu, Biao;Childs-Disney, Jessica L.
通讯作者：
Childs-Disney, Jessica L.
DOI：
10.1126/science.1218298
发表时间：
2012-03-09
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Paige JS;Nguyen-Duc T;Song W;Jaffrey SR
通讯作者：
Jaffrey SR