DNA confined by surfaces

描述（由申请人提供）：更好地理解转移病毒基因组以感染细胞的生物过程的生物物理基础对于许多疾病相关的 领域的预测热力学压力和力，包括将DNA-高度带负电荷的弹性聚合物-限制到衣壳中（超过250倍压缩）所需的渗透压，是一个不仅与感染性疾病机制有关，而且与噬菌体治疗或噬菌体抗生素（1）和治疗递送（2）有关的问题。噬菌体DNA限制的实验测量包括渗透压喷射抑制实验（3）和单分子加载力测量，其提供用于验证理论模型的力或压力数据。（4，5）通过与我们的合作者Chiu在NCMI中完成的冷冻电子显微镜不对称重建，有助于对DNA包装的结构洞察。（6，7）大多数噬菌体包装的当前模型假设DNA表现为在应力下均匀弯曲的线性弹性聚合物，如“逆线轴”模型。(8)这种缠绕构象的假设主要基于对冷冻EM密度图的解释，通过对数千个结构进行平均获得（9），其显示密度环，特别是在衣壳表面附近。最近的证据表明，在易位包装过程中，DNA螺旋在左手方向上旋转，从而使其处于扭曲状态。(10已知当在序列特定位置发生链分离时，欠扭转使持续长度减少2个数量级。(12)我们的假设是，DNA扭结诱导的障碍，可以有很强的影响包装和压力。DNA如何克服不利的热力学障碍进入并包装在衣壳内取决于许多不同的分子间相互作用。由于噬菌体基因组大约有10千碱基对长，我们将采用多尺度技术来模拟结构和随之而来的热力学。我们将从我们以前的工作中改进一个粗粒度的DNA模型。(13)初步模拟的未连接的DNA粗粒聚合物珠在captured的限制已经显示出环状的密度分布与cryo-EM数据一致。将根据数据构建连接的聚合物路径。我们将产生一个熵驱动的，低自由能构象的DNA约束的合奏。最后，我们将测试与无序量，离子屏蔽和DNA-蛋白质限制相互作用的贡献相关的假设。

DESCRIPTION (provided by applicant): Better understanding the biophysical basis of the biological process to transfer a viral genome to infect a cell is important to many disease related fields. Predicting the thermodynamic pressures and forces including the osmotic pressure necessary to confine DNA-a highly-negatively charged, elastic polymer-into capsids (over a 250-fold compaction) is a problem with implications not only relevant to infectious disease mechanism but in phage therapy or phage antibiotics(1) and therapeutic delivery(2). Experimental measurements of phage DNA confinement include osmotic pressure ejection-inhibition experiments(3) and single-molecule loading force measurements that provide force or pressure data for validation of theoretical models.(4, 5) Structural insight into DNA packaging is aided by cryo-electron microscopy asymmetric reconstructions done in the NCMI with our collaborator Chiu.(6, 7) Most current models of phage packing assume DNA behaves as a linearly elastic polymer that bends uniformly under stress, like the 'inverse spool' model.(8) The assumption of such spooled conformations is based primarily on interpretations of cryo-EM density maps, obtained by averaging thousands of structures(9), which show density rings, especially near the capsid surface. Recent evidence shows that during translocation packing, the DNA helix is rotated in a left-handed direction thus under twisting it.(10, 11) It Is known tha under twisting reduces persistence length by 2 orders of magnitude when strand separation occurs in sequence specific places.(12) Our hypothesis is that DNA kinking induced disorder can have a strong effect on packing and pressures. How DNA overcomes the unfavorable thermodynamic barrier to enter and pack inside a capsid depends on many different intermolecular interactions. Because phage genomes are around ten kilo-basepairs long, we will employ a multi scale technique to model the structure and consequent thermodynamics. We will refine a coarse-grained model of DNA from our previous work.(13) Preliminary simulations of unconnected DNA coarse grained polymer beads in capsid-like confinement already show ringed density distributions consistent with cryo-EM data. Connected polymer paths will be constructed consistent with data. We will produce an ensemble of entropically-driven, low free energy conformations of DNA in confinement. Ultimately, we will test hypotheses related to the amount of disorder, ion screening and the contribution of DNA-protein confinement interactions.