Abnormal intracellular phase transitions in mutant hnRNP A1 may underlie the development of several neurodegenerative diseases. The risk of these diseases increases upon C9Orf72 repeat expansion and the accumulation of the corresponding G-quadruplex (G4)-forming RNA, but the link between this RNA and the disruption of hnRNP A1 homeostasis has not been fully explored so far. Our aim was to clarify the mutual effects of hnRNP A1 and C9Orf72 G4 in vitro. Using various optical methods and atomic force microscopy, we investigated the influence of the G4 on the formation of cross-beta fibrils by the mutant prion-like domain (PLD) of hnRNP A1 and on the co-separation of the non-mutant protein with a typical SR-rich fragment of a splicing factor (SRSF), which normally drives the assembly of nuclear speckles. The G4 was shown to act in a holdase-like manner, i.e., to restrict the fibrillation of the hnRNP A1 PLD, presumably through interactions with the PLD-flanking RGG motif. These interactions resulted in partial unwinding of the G4, suggesting a helicase-like activity of hnRNP A1 RGG. At the same time, the G4 was shown to disrupt hnRNP A1 co-separation with SRSF, suggesting its possible contribution to pathology through interference with splicing regulation.

Pharmacological chaperones of heterogeneous nuclear ribonucleoproteins (hnRNPs) show promise as potential neuroprotective drug candidates. They are expected to prevent the accumulation of neurotoxic hnRNP biocondensates and aggregates, which are hallmarks of severe degenerative diseases. Here, we present the first rational design of oligonucleotide chaperones of hnRNP A1. This design was inspired by previous studies on the specificity of the RNA recognition motif (RRM) and the RGG motif of hnRNP A1 for endogenous nucleic acids. To obtain robust and specific chaperones, we combined an RRM-binding sequence with an RGG-binding G-quadruplex oligonucleotide that inhibits hnRNP A1 aggregation and introduced various modifications into the sugar-phosphate backbone of the oligonucleotide. Modifications that locked the RRM-binding sequence in a conformational state characteristic of RNA improved chaperone affinity and activity. The former was assessed using microscale thermophoresis assays, while the latter was evaluated using fluorimetry and microscopy. The leading chaperone bound to hnRNP A1 at micromolar concentrations and inhibited the assembly of its condensates and amyloid-like aggregates (fibrils) by over 90%.