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

基于我们最近的发现（Nature Nanotechnology 13，730（2018））的ssDNA的序列特异性相分离-ssDNA的聚合物性质-该提案的中心科学目标是理解和调节富含嘌呤的ssDNA的相分离行为并激活ssDNA的这种聚合物性质以用于自组装系统的设计，包括胶态凝聚层和嵌段共聚物（BCP）结构。这些调查是针对合并BCP科学领域已知的合成聚合物和DNA纳米科学与其高度发达的链置换反应，适体技术，酶操作的可能性与使命交叉施肥的方法和实施最好的两个世界到新的系统。一个决定性的方面也是走出平衡，并研究化学燃料和瞬时聚合诱导的自组装，其中结构可以自主建立和破坏使用拮抗酶。首先，我们的目标是定量了解ssDNA相分离的序列依赖性，然后确定响应于不同化学信号的等温液/液相分离的途径，特别是使用具有受控动力学的链置换反应，以及使用适体的ATP。我们的目标是使用受控的成核和生长过程获得均匀的凝聚液滴。第三，我们将设计和合成ssDNA-b-（合成聚合物）BCP与非常长的ssDNA（共）聚合物，以及编码序列的分子识别能力的适体和链置换反应。在此基础上，我们将研究热和等温触发形成的BCP型自组装，然而，与以前的工作形成鲜明对比，ssDNA块将是疏溶剂块。第四，通过融合前三部分的理解，我们将通过相分离ssDNA块的活性酶促聚合（在相分离条件下）来设计聚合诱导自组装过程（比萨），此外，我们将通过添加能够以延迟时间降解所形成的ssDNA的外切核酸酶1来发展这种方法以制造瞬时比萨结构。该项目将把DNA纳米科学方法的独特优势引入聚合物相分离和自组装，还将把新的聚合物化学和聚合物科学工具引入DNA自组装。在一个更抽象的方式，我们希望能够启发生物学家思考ssDNA相分离的重要性，在细胞核和亚细胞冷凝物的背景下，并可能提供新的工具，研究基本的生物过程中使用软物质的方法。

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.