RNA modification via novel fluorescent base analogues: Effect on the structure of RNA-containing lipid nanoparticles and their sub-populations

Nucleic acid-based therapies are a rapidly expanding field. To study delivery and performance of nucleic acids in cells, they must often be fluorescently labelled, usually by attaching large fluorescent dyes. However, this can significantly perturb the nucleic acid’s biological functions and structural integrity. To address this, the Wilhelmsson group has developed chemically modified nucleic acid building blocks which are inherently fluorescent, so-called “fluorescent base analogues”. These can be efficiently incorporated into RNA which closely mimics the behaviour of the native RNA, and can be translated to proteins within cells. LNPs are enormously successful mRNA delivery vectors, but relationships between their structure and function are not yet well understood. To leverage the benefits of fluorescent base analogue labeled RNA to explore critical questions in the field (e.g. how cargo is released from LNPs), the effect of incorporating these novel labelled RNAs on LNP structure needs to be validated. In this experiment, we characterise the structures of LNPs loaded with fluorescent base analogue labeled RNA, and compare them to LNPs loaded with unlabeled and conventionally labeled RNA. We will also characterise sub-populations within these LNP formulations, separated using AF4. The results will deepen our understanding of the impact of fluorescent labelling strategies on LNP architecture, and mark a pivotal step forward in correlating LNP structure to performance.