Stampfl Sabine, Lempradl Adelheid, Koehler Gottfried, Schroeder Renée
Department of Biochemistry, Max F. Perutz Laboratories, Campus Vienna Biocenter 5/1, 1030 Vienna, Austria.
Chembiochem. 2007 Jul 9;8(10):1137-45. doi: 10.1002/cbic.200700030.
Understanding the interactions of small molecules like antibiotics with RNA is a prerequisite for the development of novel drugs. In this study we address structural and thermodynamic features of such interactions by using a simple model system: the binding of the highly charged antibiotic neomycin B to a short hairpin RNA molecule. Nucleotide A16, which acts as a flap over the neomycin B binding pocket, was substituted by the fluorescent adenine analogue 2-aminopurine (2-AP). Steady-state and time-resolved fluorescence measurements were complemented by UV-melting and circular dichroism studies. The binding of neomycin B at three sites was found to have a strong inverse correlation with Na(+) concentration. For the highest-affinity site, both fluorescence and UV absorption experiments were consistent with a model assuming at least three neomycin NH(3) (+) groups participating in addition to hydrogen bonds in electrostatic interactions with the RNA. The variation of fluorescence intensity and lifetime upon neomycin B binding indicated unstacking of 2-AP16 from neighbouring bases as it flipped over the binding pocket. RNA conformational changes upon binding of the antibiotic were confirmed by circular dichroism. The two weaker binding sites were characterized as unspecific binding to the aptamer, while the high-affinity binding event was shown to be highly specific even at high ionic concentration. In addition, 2-AP was confirmed to be a noninvasive fluorescent probe; it serves as a sensitive spectroscopic tool to investigate details of the interactions between small molecules and RNA.
了解抗生素等小分子与RNA的相互作用是开发新型药物的前提条件。在本研究中，我们通过使用一个简单的模型系统来研究此类相互作用的结构和热力学特征：带高电荷的抗生素新霉素B与一个短发夹RNA分子的结合。作为新霉素B结合口袋上方襟翼的核苷酸A16被荧光腺嘌呤类似物2-氨基嘌呤（2-AP）取代。稳态和时间分辨荧光测量通过紫外熔解和圆二色性研究得到补充。发现新霉素B在三个位点的结合与Na(+)浓度有很强的负相关。对于最高亲和力位点，荧光和紫外吸收实验均与一个模型一致，该模型假设除了与RNA发生静电相互作用的氢键外，至少还有三个新霉素NH(3) (+)基团参与其中。新霉素B结合时荧光强度和寿命的变化表明，2-AP16在翻转越过结合口袋时从相邻碱基上解堆叠。抗生素结合后RNA的构象变化通过圆二色性得到证实。两个较弱的结合位点被表征为与适体的非特异性结合，而高亲和力结合事件即使在高离子浓度下也显示出高度特异性。此外，2-AP被确认为一种非侵入性荧光探针；它作为一种灵敏的光谱工具，用于研究小分子与RNA之间相互作用的细节。
