Posttranscriptional site-directed spin labeling of large RNAs with an unnatural base pair system under non-denaturing conditions.
Posttranscriptional site-directed spin labeling of large RNAs with an unnatural base pair system under non-denaturing conditions.
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非变性条件下使用非天然碱基对系统对大 RNA 进行转录后定点自旋标记
DOI：
10.1039/d0sc01717e
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发表时间：
2020-09-21
期刊：
影响因子：
8.4
通讯作者：
Fang X
中科院分区：
文献类型：
--
作者：
Wang Y;Kathiresan V;Chen Y;Hu Y;Jiang W;Bai G;Liu G;Qin PZ;Fang X
用于电子顺磁共振（EPR）光谱的大RNA的定点自旋标记（SDSL）至今仍具有挑战性。迄今为止，用于电子顺磁共振（EPR）光谱的大RNA定点自旋标记（SDSL）仍然具有挑战性。在这里，我们证明了一个有效的和普遍适用的转录后SDSL方法的大RNA使用扩展的遗传字母表含有NaM-TPT 3非天然碱基对（UBP）。合成了一种炔修饰的TPT 3核糖核苷酸三磷酸（rTPT 3COTP），并通过体外转录将其位点特异性地掺入大RNA中，这允许通过点击化学连接含叠氮化物的氮氧化物。我们验证了这一战略SDSL的419个核苷酸的核糖核酸酶P（RNase P）的RNA嗜热脂肪芽孢杆菌在非变性条件下。通过圆二色谱法、小角X射线散射、沉降速度分析超离心和酶法测定，评价了定点UBP掺入和随后的自旋标记对RNase P的全局结构和功能的影响。连续波EPR分析表明，标记反应是有效的和具体的，和脉冲电子-电子双共振测量产生的自旋间距离分布，同意与晶体结构。所提出的标记策略克服了RNA标记的尺寸限制，为研究大RNA的结构和动力学开辟了自旋标记和EPR光谱的新途径。
Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy has remained challenging to date. Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy has remained challenging to date. We here demonstrate an efficient and generally applicable posttranscriptional SDSL method for large RNAs using an expanded genetic alphabet containing the NaM-TPT3 unnatural base pair (UBP). An alkyne-modified TPT3 ribonucleotide triphosphate (rTPT3COTP) is synthesized and site-specifically incorporated into large RNAs by in vitro transcription, which allows attachment of the azide-containing nitroxide through click chemistry. We validate this strategy by SDSL of a 419-nucleotide ribonuclease P (RNase P) RNA from Bacillus stearothermophilus under non-denaturing conditions. The effects of site-directed UBP incorporation and subsequent spin labeling on the global structure and function of RNase P are marginal as evaluated by Circular Dichroism spectroscopy, Small Angle X-ray Scattering, Sedimentation Velocity Analytical Ultracentrifugation and enzymatic assay. Continuous-Wave EPR analyses reveal that the labeling reaction is efficient and specific, and Pulsed Electron–Electron Double Resonance measurements yield an inter-spin distance distribution that agrees with the crystal structure. The labeling strategy as presented overcomes the size constraint of RNA labeling, opening new avenues of spin labeling and EPR spectroscopy for investigating the structure and dynamics of large RNAs.
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影响因子：
4.3
作者：
Baronti L;Karlsson H;Marušič M;Petzold K
通讯作者：
Petzold K
影响因子：
--
作者：
Kim, Youngchang;Bigelow, Lance;Borovilos, Maria;Dementieva, Irina;Duggan, Erika;Eschenfeldt, William;Hatzos, Catherine;Joachimiak, Grazyna;Li, Hui;Maltseva, Natalia;Mulligan, Rory;Quartey, Pearl;Sather, Alicia;Stols, Lucy;Volkart, Lour;Wu, Ruiying;Zhou, Min;Joachimiak, Andrzej
通讯作者：
Joachimiak, Andrzej
影响因子：
14.9
作者：
Cai Q;Kusnetzow AK;Hubbell WL;Haworth IS;Gacho GP;Van Eps N;Hideg K;Chambers EJ;Qin PZ
通讯作者：
Qin PZ
影响因子：
6.1
作者：
Konarev, PV;Volkov, VV;Svergun, DI
通讯作者：
Svergun, DI
影响因子：
14.9
作者：
Falb M;Amata I;Gabel F;Simon B;Carlomagno T
通讯作者：
Carlomagno T