Controlling cell-free expression with temperature-sensitive polymer-DNA conjugates

在活细胞中，基因组DNA被转录成RNA，然后翻译成蛋白质，这一过程被称为表达。然后，由表达产生的RNA和蛋白质参与各种细胞过程，从膜信号传导到表达本身的控制。它可以在没有细胞存在的情况下进行表达；这被称为无细胞表达（CFE）。CFE系统已被用于构建基因电路、DNA计算机、芯片实验室设备和合成细胞，可用于广泛的应用，从研究细胞如何工作到开发和筛选治疗方法。使用外部刺激控制CFE对于未来的应用至关重要，因为它将允许根据需求精确激活和抑制表达。目前的控制方法依赖于小分子激活剂和光，它们分别缺乏时空控制和低组织穿透。解决这两个限制的外部刺激是温度。温度是体外和体内使用的最佳刺激，因为它具有很高的组织穿透性，并且可以通过超声波进行时空控制。以前已经证明，细胞系统和治疗方法可以通过加热到略高于体温来控制，也就是所谓的轻度热疗，没有毒性问题。在这里提出的研究中，我们的目标是使用轻度热疗温度来控制CFE。用温度控制治疗的一种常用方法是使用由温度敏感聚合物制成的智能材料。它们的功能是从在一个温度下的可溶线圈变为在另一个温度下的不可溶球体。基于温度敏感聚合物的给药技术已成功应用于临床试验，证明了其安全性和有效性。最广泛使用的温度敏感聚合物具有较低的临界溶解温度（LCST），这意味着它们在温度升高时变得不溶。具有较高临界溶液温度（UCST）的温度敏感聚合物也存在；温度升高，这些物质就可溶解。LCST和UCST聚合物以前都已被合成，其临界温度在轻度高温范围内。在这里，控制CFE将通过将UCST聚合物连接到DNA来实现。许多研究将LCST聚合物与DNA联系起来，以控制其结构和功能，尽管只有少数研究试图控制CFE。我们的目标是创建一个系统，在体温下，与DNA连接的UCST聚合物将形成抑制CFE的小球。当加热到高于UCST的轻度高温温度时，UCST聚合物将从不溶的小球变为可溶的线圈，从而激活CFE。这一过程是可逆的，并且可以通过再次将温度降低到UCST以下来控制。使用UCST聚合物，而不是LCST聚合物，对于我们的研究是必要的，因为我们需要在温度升高时激活CFE。我们将合成新的和以前发表的UCST聚合物，在轻度高温范围内发挥作用。它们的性质将在它们附着到DNA之前和之后进行研究。最佳的UCST聚合物附着在不同的dna上，然后使用温和的高温温度用于可逆控制CFE。目前还没有关于UCST聚合物与DNA结合的研究，由于LCST聚合物与DNA的结合已经产生了多种应用，研究UCST聚合物与DNA的结合可能会导致新的应用。在未来，我们使用温度敏感聚合物和轻度热疗控制DNA的方法可用于开发可控的无细胞技术或控制替代DNA和RNA治疗方法。

In living cells, genomic DNA is transcribed to RNA, then translated to protein, in a process called expression. The RNA and protein produced from expression is then involved in all manner of cellular processes, from membrane signalling to control of expression itself. It is possible to carry out expression without the presence of a cell; this is known as cell-free expression (CFE). CFE systems have been used to construct gene circuits, DNA computers, lab-on-a-chip devices, and synthetic cells, which can be used in a wide range of applications, from studying how cells work to developing and screening therapeutics. Control of CFE using external stimuli is vital for future applications because it will allow precise activation and repression of expression upon demand. Current methods of control rely on small-molecule activators and light, which suffer from a lack of spatiotemporal control and low tissue penetration, respectively. An external stimulus that addresses both these limitations is temperature. Temperature is an optimal stimulus for both in-vitro and in-vivo use as it has high tissue penetration and can be spatiotemporally controlled using ultrasound. It has previously been demonstrated that cellular systems and therapeutics can be controlled by heating to just above body temperature, otherwise known as mild hyperthermia, without toxicity issues. In the research proposed here, we aim to control CFE using mild hyperthermia temperatures. A common way of controlling therapeutics with temperature is to use smart materials made from temperature-sensitive polymers. These function by changing from soluble coils at one temperature to insoluble globules at another temperature. Temperature-sensitive polymer-based drug delivery technologies have been successfully used in clinical trials, demonstrating their safety and efficacy. The most widely-used temperature-sensitive polymers have a lower critical solution temperature (LCST), meaning they become insoluble upon an increase in temperature. Temperature-sensitive polymers with an upper critical solution temperature (UCST) also exist; these become soluble upon an increase in temperature. Both LCST and UCST polymers have previously been synthesised that have critical temperatures in the mild hyperthermia range.Here, control of CFE will be achieved by attaching UCST polymers to DNA. Many studies have connected LCST polymers to DNA to control its structure and function, although only a few have attempted to control CFE. Our goal is to create a system where, at body temperature, UCST polymers connected to DNA will form globules that inhibit CFE. Upon heating to mild hyperthermia temperatures, above the UCST, the UCST polymers will change from insoluble globules to soluble coils, activating CFE. This process will be reversible and can be controlled by again reducing the temperature below the UCST. The use of UCST polymers, rather than LCST polymers, is necessary for our studies as we require activation of CFE upon an increase in temperature. We will synthesise novel and previously published UCST polymers that function in the mild hyperthermia range. Their properties will be studied before and after they have been attached to DNA. Optimal UCST polymers attached to different DNAs will then be used for reversible control of CFE using mild hyperthermia temperatures. There has been no previous research on UCST polymers attached to DNA and, since multiple applications have arisen from LCST polymers attached to DNA, studying UCST-polymers attached to DNA might lead to the identification of novel applications. In the future, our method of controlling DNA using temperature-sensitive polymers and mild hyperthermia could be used to develop controllable cell-free technologies or to control alternative DNA and RNA therapeutics.