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

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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.