Graphene Biosensors for Probing Molecular Interactions of Peptides and RNA Forming Liquid-Liquid Phase Separation

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease affecting both upper and lower motor neurons. Frontotemporal dementia (FTD) is a type of brain disorder with degeneration of the frontal and temporal lobes of the cerebrum. These neurogenerative diseases share the expansion of GGGGCC hexanucleotide repeats in the C9orf72 gene, identified as a major cause of the diseases [1]. The gene produces dipeptide repeats proteins (DPRs) that cause cell death. These peptides contained arginine (Arg) and counter amino acid in the dipeptide sequence and were found to form liquid-liquid phase separation (LLPS), closely related to their cytotoxicity. A previous report indicated that poly-R is highly charged and interacts with anionic molecules such as RNA but exerts no cytotoxic effect. Poly(PR) and poly(GR), on the other hand, entraps proteins via multivalent interaction in LLPS, impairing protein translation. Thus, it is known that the position of Arg in DPRs profoundly affects the interaction [2]. These findings indicate that the interactions between DPRs and RNA should be unique to form LLPS and entrapment the proteins. However, the understanding of the interactions of DPRs is still limited because of the lack of measurement techniques that can distinguish the weak interactions among them.<br/>In this study, we applied graphene field effect transistors (GFETs) to evaluate the biomolecular interactions. Graphene has excellent electrical properties and high specific surface area and has recently shown promise as an ultra-sensitive biosensor. The interaction between biomolecules in the vicinity of the graphene surface can be detected as an electrical conduction change of graphene. We immobilized RNA on the graphene surface via pyrene anchors, as same in other works, and measured the interactions of RNA with DPRs known to form LLPS. The interactions are measured by incubating the DRPs of interest across the RNA-functionalized surface. Atomic force microscopy (AFM) was used to ensure these surface modifications. In electrical measurements, the peptides interacting with the RNA were quantified by the shift of the charge-neutral points in their gate responses of GFETs, and differences in interactions due to peptide species were discussed. From the results, we succeeded in detecting the difference in interaction with RNA by peptide species with high sensitivity at the nano-molar level. We believe that this result is helpful in quantitatively discussing the effect of small amino acid domains of biomolecules on LLPS droplet formation, an event that occurs on a microscopic scale.<br/> <br/>References<br/>[1] DeJesus-Hernandez, Mariely et al. Neuron, Volume 72, Issue 2, 2011, 245 – 256<br/>[2] Yuhei Hayamizu, Kohsuke Kanekura et al. J Cell Biol 1 November 2021, 220 (11)