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 转录为 RNA，然后翻译为蛋白质，这一过程称为表达。然后，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。可以在没有细胞存在的情况下进行表达；这称为无细胞表达 (CFE)。 CFE 系统已用于构建基因电路、DNA 计算机、芯片实验室设备和合成细胞，这些系统可用于广泛的应用，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要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组织渗透性低。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，因为它具有较高的组织穿透性，并且可以使用超声波进行时空控制。此前已经证明，可以通过加热至略高于体温的温度（也称为轻度高热）来控制细胞系统和治疗，而不会出现毒性问题。在此提出的研究中，我们的目标是使用温和的高温来控制 CFE。用温度控制治疗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。它们通过从一个温度下的可溶性线圈转变为另一温度下的不溶性球体来发挥作用。基于温度敏感的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。还存在具有上限临界溶解温度（UCST）的温度敏感聚合物；这些在温度升高时变得可溶。 LCST 和 UCST 聚合物先前已合成，其临界温度在轻度热疗范围内。在这里，CFE 的控制将通过将 UCST 聚合物附着到 DNA 来实现。许多研究已将 LCST 聚合物与 DNA 连接起来以控制其结构和功能，但只有少数研究尝试控制 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 的温和高热温度时，UCST 聚合物将从不溶性小球转变为可溶性线圈，从而激活 CFE。该过程是可逆的，并且可以通过再次将温度降低到 UCST 以下来控制。我们的研究需要使用 UCST 聚合物，而不是 LCST 聚合物，因为我们需要在温度升高时激活 CFE。我们将合成先前发表的新型 UCST 聚合物，可在轻度高温范围内发挥作用。它们的特性将在它们附着到 DNA 之前和之后进行研究。然后，附着到不同 DNA 上的最佳 UCST 聚合物将用于使用温和的高温对 CFE 进行可逆控制。之前没有关于附着在 DNA 上的 UCST 聚合物的研究，并且由于附着在 DNA 上的 LCST 聚合物已经产生了多种应用，因此研究附着在 DNA 上的 UCST 聚合物可能会导致新应用的鉴定。未来，我们使用温度敏感聚合物和温和热疗来控制 DNA 的方法可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.

基于 DNA 的人工细胞的反应扩散模式。

• DOI: http://dx.10.17863/cam.88922
• 发表时间: 2022
• 作者: Leathers A

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: http://dx.10.26434/chemrxiv-2023-ssv30
• 发表时间: 2023
• 作者: Mazzotti G

Controlling Synthetic Cell-Cell Communication.

控制合成细胞间通讯。

• DOI: http://dx.10.3389/fmolb.2021.809945
• 发表时间: 2021
• 期刊: Frontiers in molecular biosciences
• 作者: Smith JM

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、正交远程控制

• DOI: 10.1021/jacs.3c01238
• 发表时间: 2023-04-19
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Giacomo Mazzotti;D. Hartmann;M. Booth
• 通讯作者: M. Booth

Light-controlled cell-free protein synthesis using phosphorothioate-caged antisense oligonucleotides

使用硫代磷酸酯笼反义寡核苷酸进行光控无细胞蛋白质合成

• DOI: http://dx.10.26434/chemrxiv-2022-1lqc8
• 发表时间: 2022
• 作者: Hartmann D