Lipid nanoparticles (LNPs) are small self-assembled particles of approximately 100 nanometres in diameter, which are able to encapsulate and protect biologically active molecules such as nucleic acids (RNA or DNA) so to deliver the payload intact to the inside of cells. LNPs are used to deliver COVID-19 mRNA vaccines, with spectacular success. However, how it works is still something of a 'black-art', due to a lack of detailed understanding at a molecular level of the factors which control each stage of this very complex process. The protein output of messenger RNA (mRNA) has long been understood to be dependent on the rate of translation initiation and secondary structures formed by mRNA were thought to hinder elongation. Unexpectedly, recent studies found that greater mRNA translation is correlated to highly structured coding sequences, resulting in increased functional mRNA half-life as well as protein output. Currently, we have not only screened hundreds of internal AstraZeneca ionisable lipids that form part of the LNPs using in vitro cellular assays, but also optimised RNA primary sequence to improve its translatability, stability and immunogenicity in order to increase the biological performance. To our knowledge, the mRNA higher order structure inside LNPs and its effect on the rate of mRNA endosomal escape inside cells have not been studied. This information is beneficial in the rational design of the optimal mRNA sequence to enable more efficient and safer intracellular delivery of mRNAs and other nucleic acids for vaccines and other advanced treatments such as gene editing. In this project, we will initiate the first attempt to understand the effects of mRNA primary sequence and its resultant secondary/ tertiary structure on endosomal escape, which impacts mRNA expression. We aim to take advantage of the state-of-the-art facilities at the Rutherford Appleton Laboratory to generate high quality structural data from LNPs formulated with a set of mRNA sequences developed by AstraZeneca. The techniques we will use are Small Angle X-ray Scattering (SAXS) , Small Angle Neutron Scattering (SANS), and cryogenic Transmission Electron Microscopy (cryo-TEM). Synchrotron SAXS at Diamond Light Source is a very powerful way of probing the mRNA structure in solution as well as identifying and characterising liquid-crystalline or other types of ordering of the LNPs. SANS at the ISIS Neutron and Muon Source has the unique ability to determine the distribution of the different lipids and mRNA within the LNPs. This approach takes advantage of a unique feature of neutron scattering, whereby molecules can be 'highlighted' by substituting deuterium for hydrogen in the chemical structure of the synthesized molecule and/or that of the buffer. Cryo-TEM is able to give structural details of mRNA in solution and in an LNP. The experimental work will be complemented by computer simulations of the interaction of mRNA with the lipids.