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，不考虑结构中的动态变化，例如，由于热运动，已经为许多观测提供了可能的解释。后者包括上面提到的聚集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及其在各种现象中的应用。 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。由此产生的影响，例如，直接识别100个碱基对（bp）或更长的双链体DNA片段之间的序列同源性显然具有重要的生物学意义。特别是，100-300 bp的序列同源性识别是必不可少的，以避免重组错误，导致癌症，遗传性疾病等实验最近在文献中报道的建议，在完整的DNA同源序列的本地，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，和链交叉。双链体DNA在无核小体区的直接相互作用被认为是可能的，但在这种相互作用中序列同源性识别的机制和可能性仍然未知。 在过去的一年里，我们完成了第一系列纯物理实验，探索体外序列依赖的DNA-DNA相互作用。 我们成像的两个荧光标记的混合物，双螺旋DNA分子具有相同的核苷酸组成和长度（294 bp），但不同的序列。在电解质溶液中，在轻微的渗透应力，这些DNA形成离散的液晶聚集体（球晶）。我们观察到两种DNA在每个球晶内的自发分离，揭示了在不存在蛋白质的情况下由水分离的双螺旋之间的核苷酸序列识别，与我们的理论预测一致。虽然这些实验明确地证明了不解链双螺旋的序列同源性识别的可能性，但仍有许多工作要做，以测试这种识别的机制是否确实如理论所预测的那样，以及这种识别是否在体内DNA配对中起任何作用。目前正在进行进一步的实验。

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 do not account for dynamic variations in the structure, e.g., due to the thermal motion, already suggest possible explanations for many observations. The latter include 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, continue development of this theory and its applications to various phenomena. Most importantly, this theory predicts that the dependence of the backbone structure on the nucleotide sequence may be sufficiently strong to affect DNA-DNA interactions. The resulting effects, e.g., direct recognition of sequence homology between 100 base pair (bp) or longer fragments of duplex DNA, may obviously have significant biological implications. In particular, 100-300 bp sequence homology recognition is essential for avoiding recombination mistakes that lead to cancer, genetic disorders, etc. Experiments recently reported in the literature 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. Direct interactions between duplex DNAs in nucleosome-free regions were proposed to be involved, but the mechanism and possibility of sequence homology recognition in such interactions remained unknown. During the last year, we completed the first series of purely physical experiments probing sequence-dependent DNA-DNA interactions in vitro. We imaged a mixture of two fluorescently tagged, double helical DNA molecules with identical nucleotide composition and length (294 bp), but different sequences. In electrolytic solution at minor osmotic stress these DNAs formed discrete liquid-crystalline aggregates (spherulites). We observed spontaneous segregation of the two kinds of DNA within each spherulite, revealing nucleotide sequence recognition between double helices separated by water in the absence of proteins, consistent with our theoretical predictions. While these experiments unequivocally demonstrate the possibility of sequence homology recognition without unzipping the double helix, much work remains to be done to test whether the mechanism of this recognition is indeed as predicted by the theory and whether such recognition plays any role in DNA pairing in vivo. Further experiments are currently in progress.