Recognition and self-assembly of DNA aggregates

多年来，双链(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中，并被高电荷的糖-磷酸骨架所屏蔽。在这种相互作用的讨论中，双链DNA被显式或隐式地模拟为一个均匀带电的圆柱体。然而，这个概念是基于直觉，而不是实验或严谨的理论。事实上，实验证据表明，这个概念可能是错误的，例如，将双链DNA从一个10.5碱基对的非理想螺旋转变为一个聚集体为10.0个碱基对/转的近理想螺旋。从1980年Rhodes和Klug发表的经典论文开始，人们清楚地看到双链DNA之间的相互作用不仅依赖于双螺旋结构，而且还影响双螺旋结构。 为了解释糖磷酸骨架结构对DNA-DNA相互作用的可能影响，在过去的十年里，我们一直在发展具有表面电荷螺旋图案的大分子之间的静电相互作用理论。即使是最简单的模型，也已经为许多观测提供了可能的解释，这些模型没有考虑结构中的动态变化，例如由于热运动。后者包括上述双螺旋在聚集时的扭转变形，DNA凝聚的反离子专一性，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，这一理论预测，骨架结构对核苷酸序列的依赖性可能足够强，足以影响DNA-DNA相互作用。由此产生的效应，例如，直接识别100个碱基对(BP)或更长的双链DNA片段之间的序列同源性，显然可能具有重大的生物学意义。特别是，100-300bp的序列同源性识别对于避免导致癌症、遗传疾病等重组错误至关重要。最近报道的实验表明，完整DNA中同源序列的局部瞬时配对可能先于双链断裂，进一步被蛋白质覆盖的单链识别，以及链交叉。核小体无性区的双链DNA之间的直接相互作用被认为是可能的，但这种相互作用中序列同源性识别的机制和可能性仍不清楚。 在过去的一年里，我们完成了第一系列纯物理实验，在体外探测依赖序列的DNA-DNA相互作用。我们对两个荧光标记的双螺旋DNA分子的混合物进行了成像，它们的核苷酸组成和长度(294bp)相同，但序列不同。在轻微渗透压力的电解液中，这些DNA形成离散的液晶聚集体(球晶)。我们观察到这两种DNA在每个球晶内的自发分离，揭示了在没有蛋白质的情况下用水分离的双螺旋之间的核苷酸序列识别，这与我们的理论预测一致。虽然这些实验明确地证明了在不解压双螺旋的情况下进行序列同源识别的可能性，但要测试这种识别的机制是否真的像理论预测的那样，以及这种识别是否在体内DNA配对中发挥作用，仍有很多工作要做。进一步的实验目前正在进行中。

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.