Fluorescent RNA cytosine analogue - an internal probe for detailed structure and dynamics investigations.
Fluorescent RNA cytosine analogue - an internal probe for detailed structure and dynamics investigations.
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DOI：
10.1038/s41598-017-02453-1
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发表时间：
2017-05-24
影响因子：
4.6
通讯作者：
Wilhelmsson LM
中科院分区：
文献类型：
--
作者：
Füchtbauer AF;Preus S;Börjesson K;McPhee SA;Lilley DMJ;Wilhelmsson LM
明亮的荧光胞嘧啶类似物tCO在荧光碱基中脱颖而出，因为它在双链DNA中几乎不猝灭的荧光发射。然而，像大多数报道的碱基类似物一样，它还没有在RNA中得到彻底的表征。我们在此报道了tCO的首次合成和RNA掺入，并表征了其在RNA中的碱基模拟和荧光特性。与DNA一样，我们发现RNA双链中的高量子产率（<ΦF> = 0.22）几乎不受邻近碱基（ΦF = 0.20-0.25）的影响，导致平均亮度为1900 M−1 cm−1。RNA双链体的平均荧光寿命为4.3 ns，通常需要两个寿命才能适应指数衰减。与dsRNA相比，ssRNA的荧光特性表现为平均量子产率的小幅增加（<ΦF > = 0.24），分布范围更广（ΦF = 0.17-0.34），平均寿命略短。利用圆二色性，我们发现tco修饰的RNA双链形成规则的a型螺旋，在紫外熔融实验中，双链的稳定性仅略高于相应的天然RNA （<ΔT m> = + 2.3°C）。这些特性使tCO成为一种非常有趣的荧光RNA碱基类似物，用于详细的基于fret的结构测量，作为显微镜中明亮的内部标记，以及RNA动力学的荧光各向异性测量。
The bright fluorescent cytosine analogue tCO stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-incorporation of tCO, and characterize its base-mimicking and fluorescence properties in RNA. As in DNA, we find a high quantum yield inside RNA duplexes (<ΦF> = 0.22) that is virtually unaffected by the neighbouring bases (ΦF = 0.20–0.25), resulting in an average brightness of 1900 M−1 cm−1. The average fluorescence lifetime in RNA duplexes is 4.3 ns and generally two lifetimes are required to fit the exponential decays. Fluorescence properties in ssRNA are defined by a small increase in average quantum yield (<ΦF > = 0.24) compared to dsRNA, with a broader distribution (ΦF = 0.17–0.34) and slightly shorter average lifetimes. Using circular dichroism, we find that the tCO-modified RNA duplexes form regular A-form helices and in UV-melting experiments the stability of the duplexes is only slightly higher than that of the corresponding natural RNA (<ΔT m> = + 2.3 °C). These properties make tCO a highly interesting fluorescent RNA base analogue for detailed FRET-based structural measurements, as a bright internal label in microscopy, and for fluorescence anisotropy measurements of RNA dynamics.