Institute of Organic Chemistry, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 52a, 6020 Innsbruck, Austria.
J Am Chem Soc. 2011 Oct 12;133(40):16161-7. doi: 10.1021/ja2063583. Epub 2011 Sep 14.
A precise tertiary structure must be adopted to allow the function of many RNAs in cells. Accordingly, increasing resources have been devoted to the elucidation of RNA structures and the folding of RNAs. 2-Aminopurine (2AP), a fluorescent nucleobase analogue, can be substituted in strategic positions of DNA or RNA molecules to act as site-specific probe to monitor folding and folding dynamics of nucleic acids. Recent studies further demonstrated the potential of 2AP modifications in the assessment of folding kinetics during ligand-induced secondary and tertiary RNA structure rearrangements. However, an efficient way to unambiguously identify reliable positions for 2AP sensors is as yet unavailable and would represent a major asset, especially in the absence of crystallographic or NMR structural data for a target molecule. We report evidence of a novel and direct correlation between the 2'-OH flexibility of nucleotides, observed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probing and the fluorescence response following nucleotide substitutions by 2AP. This correlation leads to a straightforward method, using SHAPE probing with benzoyl cyanide, to select appropriate nucleotide sites for 2AP substitution. This clear correlation is presented for three model RNAs of biological significance: the SAM-II, adenine (addA), and preQ(1) class II (preQ(1)cII) riboswitches.
为了使许多 RNA 在细胞中发挥功能，必须采用精确的三级结构。因此，人们投入了更多的资源来阐明 RNA 的结构和折叠。2-氨基嘌呤（2AP）是一种荧光核苷类似物，可以取代 DNA 或 RNA 分子中的战略位置，作为特定位置的探针，来监测核酸的折叠和折叠动力学。最近的研究进一步证明了 2AP 修饰在评估配体诱导的二级和三级 RNA 结构重排过程中的折叠动力学方面的潜力。然而，目前还没有一种有效的方法可以明确识别 2AP 传感器的可靠位置，这将是一项重大资产，尤其是在缺乏目标分子的晶体学或 NMR 结构数据的情况下。我们报告了一种新的、直接的相关性的证据，即核苷酸 2'-OH 的灵活性，通过选择性 2'-羟基酰化分析引物延伸（SHAPE）探测来观察，以及核苷酸取代为 2AP 后荧光反应之间的相关性。这种相关性为三种具有生物学意义的模型 RNA 带来了一种简单的方法，即使用苯甲酰氰化物进行 SHAPE 探测，以选择合适的核苷酸位点进行 2AP 取代。这种明显的相关性在三个模型 RNA 中得到了验证：SAM-II、腺嘌呤（addA）和 preQ(1)类 II（preQ(1)cII）核糖开关。
