Recognition and self-assembly of DNA aggregates

多年来，双链DNA之间的相互作用被认为与DNA结构和碱基对序列无关，因为核苷酸被埋在双螺旋内，并被高度带电的糖-磷酸骨架屏蔽。在讨论这种相互作用时，双链DNA被明确或隐含地建模为均匀带电的圆柱体。然而，这个概念是基于直觉的感知，而不是实验或严格的理论。实际上，实验证据，例如，双链体DNA从溶液中10.5个碱基对/转角的非理想螺旋转化为聚集体中10.0 bp/转角的接近理想螺旋，表明这种概念可能是错误的。从罗兹和Klug在1980年发表的经典论文开始，双链DNA之间的相互作用不仅取决于双螺旋结构，而且影响双螺旋结构。 为了解释糖磷酸骨架的结构对DNA-DNA相互作用的可能影响，在过去的十年里，我们一直在发展一种具有螺旋表面电荷模式的大分子之间的静电相互作用理论。即使是最简单的模型，它没有考虑结构中的动态变化，例如，由于热运动，已经为许多观测提出了可能的解释。这些观察结果包括上述聚合时双螺旋的扭转变形、DNA凝聚的反离子特异性、DNA聚集体中的多个液晶相以及测量的分子间力。因此，我们继续发展这一理论，并将其应用于各种现象。 最重要的是，该理论预测骨架结构对核苷酸序列的依赖性可能足够强，从而影响DNA-DNA相互作用。结构特异性DNA-DNA相互作用是由带负电荷的糖磷酸骨架与结合在相对分子上的凹槽中的抗衡离子的优先并置引起的。我们的X射线衍射实验分析证实了这种平行的DNA分子在纤维状，水合聚集体的并列。此外，统计分析和比较已知结构的DNA寡核苷酸在晶体（由X-射线衍射测定）和溶液（由NMR测定）揭示了DNA结构的变化与该理论的预测一致的晶体内，使我们能够评估的理论的基本参数。然而，目前还不清楚序列依赖性相互作用如何受到热波动的影响，特别是DNA弯曲。 在过去的一年里，我们完成了一个全面的统计理论，预测了热弯曲的戏剧性和违反直觉的影响。与我们的预期相反，DNA的热波动强烈地放大而不是削弱序列依赖的相互作用。这种起伏增强了DNA的结构适应性，导致相邻分子更好地对齐，并使DNA骨架的几何形状更接近理想螺旋的几何形状。定量比较显示，良好的协议的理论预测与测得的渗透压在DNA聚集体和所观察到的X-射线衍射图案从水合DNA纤维。 序列对双链DNA之间相互作用的影响，例如，预测的100个碱基对（bp）或更长序列之间的序列同源性的直接识别显然具有重要的生物学意义。在2008年，我们发表了第一个实验证据同源配对的300 bp，完整的DNA双螺旋在液晶聚集体。这些实验和发表的报告表明，同源的，无核小体的双链体DNA区域可能优先在体内相互作用，表明在完整的DNA中同源序列的局部，瞬时配对可能先于双链断裂，进一步识别蛋白质覆盖的单链，和链交换。2009年底，M.来自哈佛大学的Prentiss发表了优雅的单分子研究，揭示了1-5 kb双链DNA片段与长得多的分子上的同源区域的选择性结合，进一步支持了我们的理论。然而，令人惊讶的是，他们在单价盐中观察到这种结合，在这种条件下，以前从未观察到双链体DNA的配对或聚集，并且认为理论上是不可能的。在我们的理论中，两个同源DNA双链体的稳定平行并置预期在一些二价和大多数多价抗衡离子的存在下，而不是在单价盐中。 为了解决我们的预测与M.普伦蒂斯小组，去年我们重新审视了这个理论。具体来说，我们排除了DNA双链体在形成稳定对时保持直的和彼此平行的简化假设。我们发现，在这些实验条件下，DNA分子将倾向于超螺旋，形成辫子。我们开发了一个理论，这种辫子的静电能，并证明，形成稳定的编织对同源双螺旋可能是积极有利的，即使在单价盐，根据具体的counterweight。我们的预测，这种配对的反离子，盐浓度和温度的依赖性密切匹配的实验观察。目前，我们正在设计实验，以更详细地测试这些想法。

For years, interactions between double stranded (duplex) DNA were presumed to be independent of the DNA structure and base pair sequence because the nucleotides are buried inside the double helix and shielded by the highly charged sugar-phosphate backbone. In discussion of such interactions, duplex DNA was explicitly or implicitly modeled as a uniformly charged cylinder. However, this concept was based on intuitive perception rather than experiments or rigorous theory. In reality, the experimental evidence, e.g., transformation of duplex DNA from a non-ideal helix with 10.5 base pairs/turn in solution into a nearly ideal helix with 10.0 bp/turn in aggregates, suggested that this concept may be wrong. Starting from the classical paper of Rhodes and Klug published in 1980, it became clear that interactions between duplex DNA not only depend on but also affect the double helix structure. To account for possible effects of the structure of the sugar phosphate backbone on DNA-DNA interactions, over the last decade we have been developing a theory of electrostatic interactions between macromolecules with helical patterns of surface charges. Even the simplest models, which did not account for dynamic variations in the structure, e.g., due to the thermal motion, already suggested possible explanations for many observations. Such observations included the torsional deformation of the double helix upon aggregation mentioned above, counterion-specificity of DNA condensation, multiple liquid crystalline phases in DNA aggregates, and measured intermolecular forces. We, therefore, continued development of this theory and its applications to various phenomena. Most importantly, this theory predicted that the dependence of the backbone structure on the nucleotide sequence might be sufficiently strong to affect DNA-DNA interactions. The structure-specific DNA-DNA interactions result from preferential juxtaposition of the negatively charged sugar phosphate backbone with counterions bound in grooves on the opposing molecule. Our analysis of x-ray diffraction experiments confirmed such juxtaposition of parallel DNA molecules in fibrous, hydrated aggregates. Furthermore, statistical analysis and comparison of known structures of DNA oligonucleotides in crystals (determined by x-ray diffraction) and in solution (determined by NMR) revealed changes in DNA structure within the crystals consistent with predictions of this theory and allowed us to evaluate essential parameters of the theory. However, it remained unclear how sequence-dependent interactions might be affected by thermal fluctuations, particularly by DNA bending. During the last year, we completed a comprehensive statistical theory, which predicted dramatic and counterintuitive effects of thermal bending. Contrary to our expectations, thermal undulations of DNA strongly amplify rather than weaken the sequence-dependent interactions. The undulations enhance the structural adaptation of DNA, leading to better alignment of neighboring molecules and pushing the geometry of the DNA backbone closer to that of an ideal helix. Quantitative comparison revealed good agreement of the theoretical predictions with measured osmotic pressures in DNA aggregates and with observed x-ray diffraction patterns from hydrated DNA fibers. The effects of the sequence on interactions between duplex DNAs, e.g., the predicted direct recognition of sequence homology between 100 base pair (bp) or longer sequences, may obviously have significant biological implications. In 2008, we published the first experimental evidence for homologous pairing of 300 bp, intact DNA double helices in liquid crystalline aggregates. These experiments and published reports, which indicated that homologous, nucleosomes-free regions of duplex DNA might preferentially interact in vivo, suggested that local, transient pairing of homologous sequences in intact DNAs may precede double strand breaks, further recognition by protein-covered single strands, and strand crossover. In late 2009, the group of M. Prentiss from Harvard University published elegant single-molecule studies, which revealed selective binding of 1-5 kb duplex DNA fragments to homologous regions on much longer molecules, further supporting our theory. Surprisingly, however, they observed this binding in monovalent salt, at conditions at which pairing or aggregation of duplex DNAs was never observed before and was considered to be theoretically impossible. In our theory, a stable parallel juxtaposition of two homologous DNA duplexes was expected to in the presence of some divalent and most polyvalent counterions, but not in monovalent salt. To resolve the potential discrepancy between our predictions and the experimental observations of M. Prentiss group, during the last year we revisited the theory. Specifically, we eliminated the simplifying assumption that DNA duplexes remain straight and parallel to each other when they form a stable pair. We found that under the conditions of these experiments DNA molecules will tend to supercoil, forming a braid. We developed a theory for the electrostatic energy of such braids and demonstrated that formation of stable braided pairs of homologous double helices may be energetically favorable even in monovalent salt, depending on the specific counterion. Our predictions for the dependence of such pairing on counterions, salt concentration and temperature closely matched the experimental observations. Currently, we are designing experiments for more detailed testing of these ideas.