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被转录为RNA，然后在称为表达的过程中转化为蛋白质。然后，从表达产生的RNA和蛋白质与各种细胞过程有关，从膜信号到控制表达本身。可以在不存在细胞的情况下进行表达。这被称为无细胞表达（CFE）。 CFE系统已被用于构建基因回路，DNA计算机，芯片上的实验室和合成细胞，这些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剂。使用外部刺激对CFE的控制对于将来的应用至关重要，因为它将允许按需精确激活和抑制表达。当前的控制方法依赖于小分子激活剂和光，这些激活剂和光分别缺乏时空控制和低组织穿透力。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，因为它具有高组织穿透性，并且可以使用超声进行空间控制。以前已经证明，可以通过加热到高于体温（否则称为轻度的高温，而没有毒性问题）来控制细胞系统和治疗剂。在此处提出的研究中，我们旨在使用温和的高温温度来控制CFE。用温度控制治疗剂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。这些通过从一个温度下的可溶线圈变为另一个温度下的不溶性球的功能。基于温度敏感的聚合物的药物输送技术已成功地用于临床试验中，证明了它们的安全性和功效。最广泛的温度敏感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中变得不溶。温度敏感的聚合物具有上部临界溶液温度（UCST）；这些可溶于温度升高。 LCST和UCST聚合物以前均已合成，在轻度高温范围内具有临界温度。在此，CFE的控制将通过将UCST聚合物连接到DNA来实现。许多研究已将LCST聚合物与DNA相关联，以控制其结构和功能，尽管只有少数人试图控制CFE。我们的目标是创建一个系统，在体温下，连接到DNA的UCST聚合物将形成抑制CFE的球体。在加热至温和的高温温度（在UCST上方）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，我们需要使用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。我们将合成在轻度热疗范围内发挥作用的新颖和先前发表的UCST聚合物。在将它们附着在DNA上之前和之后，它们的性质将进行研究。然后，使用轻度的高温温度将使用连接到不同DNA的最佳UCST聚合物可逆控制CFE。以前没有关于附着在DNA附加的UCST聚合物的研究，并且由于来自与DNA相连的LCST聚合物已经产生了多种应用，因此研究与DNA相关的UCST聚合物可能会导致对新应用的识别。将来，我们使用对温度敏感的聚合物和轻度高温控制DNA的方法可用于开发可控的无细胞技术或控制替代性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

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

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

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

• DOI: 10.26434/chemrxiv-2022-1lqc8
• 发表时间: 2022-12-05
• 期刊: chemRxiv
• 作者: Hartmann，Denis;Booth，Michael J.
• 通讯作者: Booth，Michael J.