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.
展开▼
机译:明亮的胞嘧啶类似物tC O sup>在荧光碱基中脱颖而出,因为它在双链DNA中几乎没有猝灭荧光。但是,像大多数报道的碱基类似物一样,它尚未在RNA中被彻底表征。我们在这里报告了tC O sup>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。就像在DNA中一样,我们发现RNA双链体内的量子产率很高(<ΦF> = 0.22),而实际上不受相邻碱基的影响(ΦF= 0.20-0.25),导致平均亮度为1900 M -1 < / sup> cm -1 sup>。 RNA双链体的平均荧光寿命为4.3µns,通常需要两个寿命才能适应指数衰减。与dsRNA相比,ssRNA的荧光性质是由平均量子产率的小幅提高(<ΦF> = 0.24)定义的,分布范围更广(ΦF= 0.17-0.34),平均寿命略短。使用圆二色性,我们发现tC O sup>修饰的RNA双链体形成规则的A型螺旋,并且在UV熔解实验中,双链体的稳定性仅略高于相应的天然RNA( <ΔTm> == + 2.3℃。这些特性使tC O sup>成为非常有趣的荧光RNA基础类似物,用于基于FRET的详细结构测量,显微镜下的明亮内部标记以及RNA动力学的荧光各向异性测量。
展开▼