Stabilization of RNA oligomers through reverse micelle encapsulation.
Stabilization of RNA oligomers through reverse micelle encapsulation.
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DOI：
10.1021/ja8084753
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发表时间：
2009-03
影响因子：
15
通讯作者：
H. Workman;P. Flynn
中科院分区：
文献类型：
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作者：
H. Workman;P. Flynn
细胞环境富含简单和复杂分子的多样性，而且也相当拥挤。相比之下，用于生物物理学和结构生物学体外研究的典型样品浓度利用简单缓冲系统中的纯化大分子，浓度范围从微摩尔到毫摩尔。尽管这种配方已被证明适合广泛的生物学和结构研究，但它与细胞内通常存在的相对拥挤的条件有很大不同。一段时间以来，人们已经认识到这些拥挤效应对蛋白质的重要性，但在核酸中的等效分析尚未得到充分探索。基于表面活性剂的反胶束封装已成为一种有效的生物物理工具，可以在多种条件下研究离子强度、pH、水合和拥挤对生物活性大分子的影响。我们封装了来自 HIV 的 TAR RNA 和 U4 snRNA 的 5' 茎环寡核苷酸的寡核苷酸模型。观察亚氨基 (1)H 共振是评估核酸寡核苷酸稳定性的既定方法，这意味着存在堆叠的氢键碱基对。对 RNA 分子的 (1)H NMR 谱的检查表明，几个亚氨基共振的强度在封装后增加。在溶液中游离寡核苷酸的光谱中未观察到的其他共振支持了分子已获得稳定性的建议。这些结果表明，RNA 寡核苷酸可能在细胞水平拥挤的情况下获得显着的稳定性。
The cellular milieu is rich in diversity of both simple and complex molecules and is also quite crowded. By contrast, typical sample concentrations employed for in vitro investigation of biophysics and structural biology make use of purified macromolecules in simple buffer systems at concentrations that range from micromolar to millimolar. Although this formulation has proven to be compatible with a wide range of biological and structural studies, it is quite different from the relatively crowded conditions typically found within cells. The importance of these crowding effects for proteins has been recognized for some time, but the equivalent analysis is underexplored in nucleic acids. Encapsulation with surfactant-based reverse micelles has emerged as an effective biophysical tool, allowing study of the influence of ionic strength, pH, hydration, and crowding on biologically active macromolecules over a wide range of conditions. We have encapsulated an oligonucleotide model of TAR RNA from HIV and the 5' stem loop oligonucleotide of the U4 snRNA. Observation of imino (1)H resonances is an established method for evaluating the stability of nucleic acid oligonucleotides, implying the presence of stacked, hydrogen bonded base pairs. Inspection of (1)H NMR spectra of the RNA molecules reveals that the intensity of several of the imino resonances increases upon encapsulation. Additional resonances not observed in spectra of the oligonucleotides free in solution support the suggestion that the molecules have gained stability. These results indicate that RNA oligonucleotides may acquire significant stability in the presence of cellular levels of crowding.