DNA confined by surfaces

描述(由申请人提供)：更好地了解转移病毒基因组以感染细胞的生物学过程的生物物理基础对许多相关疾病很重要 菲尔兹。预测热力学压力和力，包括将DNA--一种高度负电的弹性聚合物--限制在衣壳内(超过250倍压实)所需的渗透压，是一个不仅与传染病机制有关的问题，而且涉及到噬菌体疗法或噬菌体抗生素(1)和治疗性递送(2)。噬菌体DNA限制的实验测量包括渗透压喷射抑制实验(3)和为验证理论模型提供力或压力数据的单分子负载力测量(4，5)。(4，5)对DNA包装的结构洞察得益于与我们的合作者Chiu在NCMI上所做的冷冻电子显微镜不对称重建。(6，7)目前大多数噬菌体包装模型假定DNA表现为线性弹性聚合物，在压力下均匀弯曲，就像“反卷轴”模型。(8)这种缠绕构象的假设主要基于对通过平均数千个结构(9)获得的冷冻-EM密度图的解释，这些密度图显示了密度环、环和密度。尤其是在衣壳表面附近。最近的证据表明，在易位堆积过程中，DNA螺旋向左旋转，因此在扭曲的情况下。(10，11)众所周知，当链在序列特定的位置发生分离时，扭曲会使持续长度减少2个数量级。(12)我们的假设是，DNA扭结引起的无序可以对堆积和压力产生强烈的影响。DNA如何克服不利的热力学障碍进入衣壳并进入衣壳内取决于许多不同的分子间相互作用。因为噬菌体基因组大约有10个碱基长，我们将使用多尺度技术来模拟结构和随后的热力学。我们将从我们以前的工作中提炼出一个DNA的粗粒模型。(13)在衣壳状限制下对未连接的DNA粗粒聚合物珠进行的初步模拟已经显示出与Cryo-EM数据一致的环状密度分布。连接的聚合物路径将与数据一致地构建。我们将在禁闭中产生一组由熵驱动的、低自由能的DNA构象。最终，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.