Amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder resulting in motor neuron loss in
the brain and spinal cord, and frontotemporal dementia (FTD), a degenerative condition of the brain frontal and
anterior temporal lobes, are multisystem disorders with overlapping functional and genetic causes. In 2011 it
was discovered that a hexanucleotide GGGGCC expansion in the C9ORF72 gene is a common genetic cause
for ALS and FTD, however, the mechanisms by which this repeat expansion leads to ALS/FTD are not clearly
understood. The proposed research will contribute to our understanding of these molecular mechanisms by
providing direct information on the structures adopted by the sense/antisense RNA of variable lengths
produced through the transcription of the C9ORF72 GGGGCC expansion repeats, as these transcripts have
been proposed to play central roles in the pathology of these diseases. This study advances the hypothesis
that peptide nucleic acids could be developed as molecular tools to characterize the structures formed by the
C9ORF72 sense (G4C2)/antisense (G2C4) transcript RNAs and potentially to disrupt their interactions with RNA
binding proteins, and has the following specific aims:
Specific Aim I. Characterization of the C9ORF72 hexanucleotide expansion sense (G4C2) and antisense
(G2C4) transcript structure(s). Our preliminary results indicate that the sense (G4C2) expansion repeat RNA
forms G quadruplex structures that co-exist in equilibrium with hairpin structures. We will use an array of
biophysical methods to characterize the structures formed by the C9ORF72 sense (G4C2) expansion repeat
RNA, specifically analyzing the influence the repeat length and other factors (salt dependence, folding
conditions and time) have upon the equilibrium between them. Additionally, to determine how RNA binding
proteins affect this equilibrium we will analyze by both biophysical and computational chemistry methods the
interactions of the sense (G4C2) expansion repeat with the fragile X mental retardation protein and with FUS,
proteins that others and we showed bind G quadruplex RNA structures. Subsequently, similar biophysical
methods will be used to characterize the structure(s) formed by the antisense (G2C4) RNA transcript that has
not previously been structurally characterized.
Specific Aim II. Development of peptide-nucleic acids that bind specifically to either G quadruplex or
hairpin structures formed by the C9ORF72 expansion sense (G4C2) and antisense (G2C4) transcripts.
Organic synthetic methods will be used to synthesize ¿-modified peptide-nucleic acid (¿PNA) molecules
designed to recognize specifically the structures formed by the C9ORF72 expansion sense/antisense
expansion repeat transcripts. The interactions of the ¿PNAs with their designated targets will be characterized
by biophysical and computational chemistry methods, and subsequently their ability to prevent the interactions
of the C9ORF72 expansion sense/antisense transcripts with RNA binding proteins will be investigated.
