Understanding and controlling the cellular fate of fluorine-modified biologics

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PROJECT SUMMARY Organofluorine compounds possess attractive chemical, pharmacological and biological properties that have allowed them to make paradigm shifts in the design of biopharmaceuticals and biologic materials. The introduction of fluorine atoms into amino acids and nucleic acids opens a vast new chemical landscape with which to alter the folding, stability, oligomerization propensity and bioactivity of peptides, proteins and DNA. However, although shown to impart favorable properties, the impact of adding fluorine into biologic scaffolds is rarely predictable. Further, increasing evidence suggests perfluorinated compounds promiscuously adsorb to many of the fundamental building blocks of cells - including lipids, proteins and DNA - to elicit a plurality of bioeffects. One of these effects, recently discovered by the PI’s lab, is the ability of organofluorine molecules to direct protein and DNA assembly into fluorous microdomains that phase separate into fluorinated liquids without denaturing the biologic. The PI has recently exploited these emergent properties to enable ultrasound-guidance of fluorinated proteins in three-dimensional tissues. Building upon these preliminary findings, the proposed research program will mechanistically explore how organofluorine compounds influence the structure and function of adsorbed proteins and DNA and use these insights to guide the design of new supramolecular assembled biomaterials. Our overarching hypothesis is that organofluorine compounds non-covalently adsorb to proteins and DNA to direct their separation into fluorine-rich phases, which in turn alters their oligomeric assembly, cellular fate and bioactivity. To test this assertion, we will expand our perfluorinated compound (PFC) library to include a diversity of molecules with basic/acidic functionalities and heterocyclic moieties. We will use this library to establish structure-activity relationships governing the ability of PFCs to adsorb proteins, and investigate how PFC complexation alters protein cellular uptake, intracellular trafficking and bioactivity. In parallel, we will use this library to study the molecular mechanisms mediating PFC-DNA interactions and examine how PFC complexation alters DNA stability and metabolic homeostasis in exposed cells. Together, these studies will establish a comprehensive mechanistic understanding of how PFCs interact with proteins and DNA and will allow us to rationally design fluorous biotechnologies that exploit the unusual assembly phenomenon and phase- separation properties that emerge. As an example, we will create ultrasound-sensitive fluorine nanoemulsions loaded with PFC-modified ribonucleoproteins (RNPs) to enable imaging-guided gene editing in kidney tissue. Success of this research will advance the use of PFCs as a new molecular motif to control protein and DNA assembly, and the methods developed applied to discover new reagents for intracellular transduction of fluorinated biomacromolecules. Ultimately, advancing knowledge on how organofluorine compounds interact with proteins and DNA, and its effects on cells, will guide the rational design of new PFC enabled technologies with desirable functional properties for drug discovery and nanomedicine applications.