Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, often linked
to a G4C2 hexanucleotide repeat expansion in the C9orf72 gene. These repeats can form
G-quadruplex (GQ) structures, potentially contributing to disease pathology. In my
master thesis project, we investigated whether the RNA-binding protein hnRNPH1
interacts with these repeats and whether it shows preference for linear or GQ structures.
We cloned and expressed hnRNPH1 and control protein PABPC1 in E. coli, using His-
and MBP-tags for purification. Proteins were purified via affinity and ion-exchange
chromatography, with confirmation by mass spectrometry and Western blot. To assess
binding, we used Microscale Thermophoresis (MST) and Bio-Layer Interferometry
(BLI) with DNA probes mimicking RNA repeats. MST revealed that hnRNPH1 binds
both linear and GQ forms, with stronger affinity for the linear structure. PABPC1
however binds with greater afinity to the linearized GQ form. BLI method requiers
further optimisation. Our findings suggest that hnRNPH1 may preferentially bind and
potentially unfold GQ structures, providing insight into its possible role in C9orf72-
related ALS pathology. Future work will focus on testing RNA probes and additional
protein constructs to further define this interaction.
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