Peptide Nucleic Acid (PNA) Oligomers for Targeted Disruption of Biomolecular Condensates in ALS/FTD

PROJECT SUMMARY The formation of biomolecular condensates (BMCs) is an important phenomenon in biology, creating transient compartments that can accelerate biochemical reactions or sequester molecules during times of stress. Aberrations in BMC formation are implicated in a growing number of diseases. Thus, molecular tools that enable

researchers to monitor and manipulate BMCs are in great demand. Ideally, these tools could be deployed without engineering the biomolecules that drive BMC formation in order to avoid artifacts introduced by altering the sequence of the RNA or protein components. Our proposal is focused on developing RNA-targeting probes that

can be used to (a) fluorescently label RNAs suspected of participating in BMCs and (b) disrupt specific RNAs from entering BMCs. The molecular probes we will use for these experiments are based on peptide nucleic acid (PNA), a synthetic version of DNA in which the natural sugar-phosphodiester backbone is replaced by an

extended peptide. Hybridization of the PNA to its RNA target will be used to introduce a fluorescent dye for visualization in microscopy. Moreover, PNA hybridization should prevent that region of the RNA from either binding to a protein or interacting with other RNAs, blocking its incorporation into a BMC. The advantage of this

approach is that the RNA target need not be modified in any way, i.e. we will target endogenous RNAs. Additionally, the PNA can be introduced in a reversible manner, meaning it can be removed from the RNA at any time, releasing the RNA to participate in BMC formation when the researcher deems it appropriate.

Our model system will be the C9orf72 gene bearing expanded repeats having the sequence G4C2/C4G2. The resulting RNAs, both of which are present due to bidirectional transcription, have been implicated in numerous toxic gain-of-function phenomena that are central to the pathology of amyotrophic lateral sclerosis (ALS) and

frontotemporal dementia (FTD). PNAs that can block the entry of these RNAs into BMCs can help assign roles to specific molecular components of these complex, multicomponent condensates. Moreover, since numerous neurodegenerative diseases feature expanded repeats of other sequences, the tools we develop in this project

can be readily adapted to other targets and diseases, greatly enhancing the potential impact of our proposed research. Finally, while antisense approaches targeting the C9orf72 RNA have not yet led to viable therapies, it is possible that the high affinity of PNA and a focus on targeting both of the expanded repeat transcripts will offer

better outcomes.