Nanopore unstacking of single-stranded DNA helices
Nanopore unstacking of single-stranded DNA helices
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DOI：
10.1002/smll.200700049
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发表时间：
2007-07-01
期刊：
影响因子：
13.3
通讯作者：
Li, Chang Ming
中科院分区：
文献类型：
--
作者：
Chen, Peng;Li, Chang Ming
核酸的二级结构和力学性质在其功能以及DNA复制等相关生物过程中起着重要作用。单分子力谱已成为研究分子特性的一种前所未有的强大工具，而这些特性在总体平均宏观测量中往往是模糊不清的最近的一系列实验和理论研究表明，利用电压偏压纳米孔径作为快速单分子力谱仪，可以依次解压缩双链dna、折叠rna或结构肽，并检查它们的性质和结构。[2-5]大多数纳米孔研究都依赖于α-溶血素，α-溶血素自组装形成高保真度的蛋白质通道，穿过分离两个离子溶液室的脂质双分子层。施加在纳米孔上的电压偏置建立了离子电流，并驱动带电生物聚合物（如dna）通过孔的易位。测量由这种分子易位引起的离子电流的瞬态减少，提供了关于易位分子的特性的丰富信息，以及当它被迫通过窄孔时破坏其结构的动力学，这几乎比核苷酸大。[6-8]在这里，我们通过电泳证明了在α-溶血素蛋白纳米孔易位过程中单个单链（ss）均聚DNA螺旋的力诱导解堆。通过对易位动力学的分析，推导出解叠能和协同度。对ssDNA分子通过α-溶血素孔的易位的研究已经观察到缓慢的易位动力学，而不是预期的快速、自由的电泳易位。[6,9]这种反常行为一直是人们不断猜测的话题。由于随机盘绕的DNA分子的构象熵，可以用自由能势垒来解释ssdna的意外缓慢易位然而，在涉及长dsDNA分子通过相对较大的、人造的、固态纳米孔的快速电泳运输的实验中，没有观察到这种熵效应。[9,11]因此，有人提出易位动力学是由DNA -溶血素相互作用控制的，这种相互作用延缓了DNA的运动，尽管这种相互作用的性质仍然需要进一步研究单链均聚核酸已被证明具有刚性的二级螺旋结构，该结构通过沿糖-磷酸主链连续碱基之间的芳基堆叠来稳定。x射线散射研究表明，RNA均聚物polyA和polyC的螺旋直径分别约为2.1 nm和1.9 nm[13,14]，与dsDNA的螺旋直径（2.4 nm）相当。ssDNA的螺旋结构也已经在实验和理论上得到了鉴定[15-17]Luzzati等人发现了一种中间形式的DNA，它可能是螺旋形式的ssDNA，其轴向半径是dsDNA的70%。为了支持这一点，使用
The secondary structures and mechanical properties of nucleic acids play important roles in their functions as well as in relevant biological processes, such as DNA replication. Single-molecule force spectroscopy has emerged as a tool of unprecedented power for the study of molecular properties that are often obscured in ensemble-averaged macroscopic measurements.[1] A series of recent experimental and theoretical studies has demonstrated the utilization of voltagebiased nanometer-sized pores as a rapid single-molecule force spectroscope to sequentially unzip double-stranded (ds) DNAs, folded RNAs, or structured peptides and examine their properties and structures.[2–5] Most nanopore studies have relied on α-hemolysin, which self-assembles to form a protein channel with high fidelity across a lipid bilayer separating two ionic solution chambers. Imposition of a voltage bias across the nanopore establishes an ionic current and drives the translocation of charged biopolymers, such as DNAs, through the pore. Measurement of the transient reduction in the ionic current induced by this molecular translocation provides rich information regarding the properties of the translocated molecule as well as the dynamics of disrupting its structure as it is forced through the narrow aperture, which is barely larger than a nucleotide.[6–8] Here, we demonstrate the force-induced unstacking of individual single-stranded (ss) homopolymeric DNA helices during translocation through an α-hemolysin protein nanopore by electrophoresis. The unstacking energy and cooperativity are inferred from an analysis of the translocation kinetics.Studies of the translocation of ssDNA molecules through α-hemolysin pores have observed slow translocation kinetics rather than the expected rapid, free electrophoretic translocation.[6, 9] This anomalous behavior has been a topic of continuing speculation. The unexpectedly slow translocation of ssDNAs has been interpreted in terms of the free-energy barrier due to the conformational entropy of randomly coiled DNA molecules.[10] However, no such entropic effect has been observed in experiments involving the rapid electrophoretic transport of long dsDNA molecules through relatively large, fabricated, solid-state nanopores.[9, 11] Therefore, it has been proposed that the translocation kinetics is controlled by DNA–hemolysin interactions that retard the DNA motion, although the nature of such interactions still needs scrutiny.[12] Single-stranded homopolymeric nucleic acids have been shown to assume a rigid secondary helical structure that is stabilized by aromatic base stacking between consecutive bases along the sugar–phosphate backbone. X-ray scattering studies show that the helical diameters of RNA homopolymers polyA and polyC are about 2.1 and 1.9 nm, respectively,[13, 14] which are comparable to the diameter of the dsDNA helix (2.4 nm). The helical structures in ssDNA have also been identified experimentally [15–17] and theoretically.[18] Luzzati et al.[19] discovered an intermediate form of DNA, which may be a ssDNA in helical form and assumes an axial radius 70% of that of dsDNA. In support of this, by using