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

使用温度敏感聚合物-DNA 缀合物控制无细胞表达

• 批准号: EP/V030434/1
• 负责人: Michael Booth
• 金额: $ 36.21万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FV030434%2F1
• 关键词: Controlling; cell; free; expression; temperature

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.

在活细胞中，基因组DNA被转录成RNA，然后在一个称为表达的过程中翻译成蛋白质。然后，通过表达产生的RNA和蛋白质参与所有形式的细胞过程，从膜信号到表达本身的控制。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，可用于从研究细胞如何工作到开发和筛选治疗药物的广泛应用。使用外部刺激控制CFE对于未来的应用是至关重要的，因为它将允许根据需要精确地激活和抑制表达。目前的控制方法依赖于小分子激活剂和光，它们分别存在缺乏时空控制和组织渗透率低的问题。解决这两个限制的一个外部刺激因素是温度。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渗透率，并且可以使用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，也就是所谓的轻度热疗来控制，而不会出现毒性问题。在这里提出的研究中，我们的目标是使用温和的热疗温度控制CFE。用温度控制治疗药物的一种常见方法是使用由温度敏感聚合物制成的智能材料。它们的功能是从一种温度下的可溶卷曲变成另一种温度下的不溶球状。温度敏感型聚合物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型聚合物具有较低的临界溶液温度(LCST)，这意味着它们在温度升高时变得不溶。还存在具有较高临界溶液温度(UCST)的温度敏感型聚合物；随着温度的升高，这些聚合物变得可溶。之前已经合成了临界温度在轻度高温范围内的LCST和UCST聚合物。在这里，通过将UCST聚合物连接到DNA上来控制CFE。许多研究已经将LCST聚合物连接到DNA上来控制其结构和功能，尽管只有少数几个人试图控制CFE。我们的目标是创造一个系统，在这个系统中，在体温下，连接到DNA的UCST聚合物将形成抑制CFE的球体。当加热到UCST以上的温和热疗温度时，UCST聚合物将从不溶的球状变为可溶的盘状，激活CFE。这一过程将是可逆的，可以通过再次降低UCST以下的温度来控制。对于我们的研究，使用UCST聚合物而不是LCST聚合物是必要的，因为我们需要在温度上升时激活CFE。我们将合成在轻度热疗范围内起作用的新型和以前发表的UCST聚合物。它们的性质将在它们连接到DNA之前和之后进行研究。然后，连接到不同DNA上的最佳UCST聚合物将用于使用温和热疗温度对CFE进行可逆控制。以前还没有关于连接到DNA上的UCST聚合物的研究，由于连接到DNA上的LCST聚合物已经出现了多种应用，所以研究连接到DNA上的UCST聚合物可能会导致识别新的应用。在未来，我们使用温度敏感聚合物和温和热疗控制DNA的方法可以用于开发可控的无细胞技术或控制替代的DNA和RNA疗法。

项目成果

Reaction-Diffusion Patterning of DNA-Based Artificial Cells.

• DOI: 10.1021/jacs.2c06140
• 发表时间: 2022-09-28
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Leathers, Adrian;Walczak, Michal;Brady, Ryan A.;Al Samad, Assala;Kotar, Jurij;Booth, Michael J.;Cicuta, Pietro;Di Michele, Lorenzo
• 通讯作者: Di Michele, Lorenzo

Precise, orthogonal remote-control of cell-free systems using photocaged nucleic acids

使用光笼核酸对无细胞系统进行精确、正交的远程控制

• DOI: 10.26434/chemrxiv-2023-ssv30
• 发表时间: 2023
• 作者: Mazzotti G

Controlling Synthetic Cell-Cell Communication.

• DOI: 10.3389/fmolb.2021.809945
• 发表时间: 2021
• 期刊: Frontiers in molecular biosciences
• 影响因子: 5
• 作者: Smith JM;Chowdhry R;Booth MJ
• 通讯作者: Booth MJ