Temperature-Controlled Gene Expression Using Thermoresponsive Polymer-DNA Conjugates

Nucleic acid technologies, including plasmids, ASO, siRNA and mRNA, are a suite of well-studied and emerging tools to study living systems and target genetic disorders <i>in vivo</i>. Controlling nucleic acid technologies by external stimulus has become a vital requirement in biotechnology and gene therapy. Among the external stimulus that can be used, temperature is an ideal candidate due to its reversibility and high tissue penetration, when combined with ultrasound¹. Temperature control can be achieved by using thermoresponsive polymers. Thermoresponsive polymers are “Smart Materials” that can switch between globule and coil states when changing the temperature. They are divided into 2 categories; polymers with a lower critical solution temperature (LCST) aggregate when temperature is above a critical temperature, and polymers exhibiting an upper critical solution temperature (UCST) become soluble above their critical temperature. Poly(N-isopropylacrylamide) (PNIPAM), which exhibits a LCST of 32○C, has been previously coupled to DNA² and preliminary studies have shown it can be used to regulate gene expression when polymerized with antisense oligonucleotides (ASOs)³. ASOs are short ssDNA that can bind to complementary mRNA then allow their cleavage in the presence of RNAse H enzyme, and have been used to control protein production in cell-free and living systems. In this project, we are exploring the conjugation the LCST and UCST (co)polymers onto DNA template and ASOs to produce a platform for temperature control.<br/>Here, different (co)polymers (PNIPAM and P(NIPAM-co-AAm)) with LCSTs in the range of 32-38○C were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization and conjugated to functional DNA and ASO by click chemistry to produce defined polymer-nucleic acid species. These conjugates aggregated upon heating, as expected, and were used to control cell free protein synthesis (CFPS). In the first instance, we conjugated PNIPAM to a DNA template, encoding a fluorescent protein. The expression from DNA-PNIPAM was significantly decreased upon heating above the LCST, compared to the non-modified DNA template. We have also generated defined conjugates of ASOs with PNIPAM, unlike the previously example that employed free radical polymerisation³. In preliminary results, these ASO-PNIPAM conjugates were able to degrade mRNA below the LCST, but exciting, showed no degradation above the LCST.<br/>These results demonstrate that it is possible to use thermoresponsive polymers to control both large (DNA template) and small (ASOs) nucleic acid technologies, using bioconjugation. Creating these bioconjugates, rather than the polydisperse nanoparticle aggregates, allows for the formation of a distinct, single molecular species, which simplifies synthesis, analysis, and pharmacokinetics. Our future aim is to attach LCST and UCST (co)polymers, which can be activated by mild hyperthermia, to many nucleic acid technologies and demonstrate their ultrasound-targeted application in biology and medicine.<br/>ACKNOWLEDGEMENTS<br/>This work was supported by funding from The Royal Society and EPSRC.<br/>REFERENCES<br/>[1] Gomes, I. P. et al., Pharm., 12(4), 2019.<br/>[2] Li, S. et <i>al.</i>, ACS. Macro. Lett., 7, 281-286, 2018.<br/>[3] Murata, Masaharu et <i>al.</i>, Chem. Lett., 32(11): 986-987, 2003.