LCST-Type Polymer Behavior of Single-Stranded DNA: From Fundamentals to Switchable Block Copolymers and Chemically Fueled, Transient Polymerization-Induced Self-Assembly

在我们最近发现(自然纳米技术13,730(2018))的序列特定的单链DNA的相分离--单链DNA的一种聚合物性质--的基础上，了解和调节富含嘌呤的单链DNA的相分离行为，并激活单链DNA的这种聚合物特性，用于设计自组装系统，包括胶体凝聚体和嵌段共聚物(BCP)结构，是这项提议的中心科学目标。这些研究旨在将合成聚合物和DNA纳米科学中已知的BCP科学领域与其高度发达的链置换反应、适体技术和酶操作的可能性融合在一起，任务是交叉受精方法并将两个世界中的最好实施到新系统中。一个决定性的方面也是走出平衡，研究化学燃料和瞬时聚合诱导的自组装，其中的结构可以使用拮抗酶自动构建和破坏。首先，我们将致力于定量了解单链DNA相分离的顺序依赖性，然后确定响应不同化学信号的等温液/液相分离的途径，特别是使用动力学受控的链置换反应，以及使用适体的ATP。第二，通过使用序列编码来控制单链DNA的浊点和相分离的动力学，我们将通过受控的成核和生长过程来实现均匀凝聚的液滴。第三，我们将设计和合成具有很长的单链DNA(Co)聚合物的单链DNA-b-(合成聚合物)BCP，以及用于适体和链置换反应的分子识别能力的编码序列。在此基础上，我们将研究BCP型自组装的热触发和等温触发的形成，然而，与以前的工作不同的是，单链DNA块将是疏水的块。第四，通过融合前三部分的理解，我们将设计聚合诱导自组装过程(PISA)，通过(在相分离条件下)相分离的单链DNA片段的活性酶聚合，并且，我们将通过加入能够延迟降解形成的单链DNA的核酸外切酶1来进化这一方法来制造瞬时的PISA结构。该项目将介绍DNA纳米科学方法在聚合物相分离和自组装方面的独特优势，并将新的聚合物化学和聚合物科学工具引入DNA自组装。以一种更抽象的方式，我们希望能够启发生物学家思考单链DNA在细胞核和亚细胞凝聚物背景下的重要性，并潜在地提供新的工具，利用软物质方法研究基本的生物过程。

Building on our recent discovery (Nature Nanotechnology 13, 730 (2018)) of the sequence-specific phase-segregation of ssDNA – a polymer property of ssDNA – it is the central scientific objective of this proposal to understand and tune the phase-segregation behavior of purine-rich ssDNA and activate this polymer property of ssDNA for the design of self-assembling systems, including colloidal coacervates and block copolymer (BCP) structures. The investigations are directed towards merging the fields of BCP science known from synthetic polymers and DNA nanoscience with its highly developed strand displacement reactions, aptamer technologies, and possibilities for enzymatic manipulations with a mission to cross fertilize approaches and implement the best of two worlds into new systems. A decisive aspect is also to step outside equilibrium and investigate chemically fueled and transient polymerization induced self-assembly, where structures can be autonomously built and destroyed using antagonistic enzymes. First, we will aim for a quantitative understanding on the sequence dependence of the phase-segregation of ssDNA and then identify pathways for isothermal liquid/liquid phase-separation in response to distinct chemical signals, in particular using strand displacement reactions with controlled kinetics, as well as ATP using aptamers.Second, by controlling the ssDNA cloud points and kinetics of phase-segregation using sequence coding, we will aim for uniform coacervate droplets using controlled nucleation and growth processes. Third, we will design and synthesize ssDNA-b-(synthetic polymer) BCPs with very long ssDNA (co)polymers, as well as with encoded sequences for molecular recognition capability for aptamers and strand displacement reactions. Based upon those, we will study thermally and isothermally triggered formation of BCP-type self-assemblies, yet, in striking contrast to previous work, the ssDNA block will be the solvophobic block. Fourth, by merging the understanding of the first three parts, we will design polymerization-induced self-assembly pro-cesses (PISA) by living enzymatic polymerization of phase-segregating ssDNA blocks (under phase-segregation condi-tions) and, moreover, we will evolve this approach to make transient PISA structures by adding exonuclease 1 that is able to degrade the formed ssDNA with a time delay. The project will introduce distinct advantages of DNA nanoscience approaches into polymer phase-segregation and self-assembly, and will also introduce new polymer chemistry and polymer science tools to DNA self-assembly. In a more abstract fashion, we hope to be able to inspire biologists to think about the importance of ssDNA phase-segregation in the nucleus and subcellular condensate context, and potentially provide new tools to study fundamental biological processes using soft matter approaches.