Via Research Recognition Day Program VCOM-Carolinas 2025

Biomedical Research

Circular Dichroism (CD) Spectra Analysis of Secondary Structures formed by Hexanucleotide Repeats in C9orf72 gene Sundeep Bhanot, OMS-2; Levi Diggins, OMS-4; Daniel Ross, BS, NREMT; Rebecca Corallo, OMS-3; Rachel Daley, OMS-3; Krishna Patel, OMS-3; Olivia Lewis, OMS-1; Shane Donahue, OMS-2; Jacob Thaddeus, OMS-2; Lauren Hiers, OMS-2; Christopher Syed, OMS-4; Rachael Baker, BA; David Eagerton, PhD; Bidyut Mohanty, PhD . Edward Via College of Osteopathic Medicine, 350 Howard St, Spartanburg, SC 29316 Introduction Results Discussion

Formation of i-motif occurs at 288 nm with positive peaks & 262 nm with negative peaks in low pH. G4 formation is favored in neutral to low alkaline pH levels, with max and min peaks seen at 264 nm and 245 nm, respectively. • All oligos demonstrated a dependence on pH levels. As pH levels were increased, the ability to form secondary structures decreased. • Variability in size of tandem repeats also showed differences in CD spectra findings. • Overall, boiling and cooling of oligos demonstrated a reduction in i-motif population. • Boiling and cooling of G4 oligos showed changed in ellipticity along with change in secondary structure formation, with MES buffer showing the greatest change among all buffers. • G4 structures demonstrated the most stability among all buffers when compared to i-motif structures. Continued and more detailed analysis of G4 and i-motif formation among GGGGCC/CCCCGG hexanucleotide repeats has the potential to be very promising. FTD and ALS have a documented clinical course, but little is known about the biomolecular background of the disease states. This area of research can be resourceful in determining potential therapeutic options. Further exploration of the effects of various factors on a greater number of tandem repeats is currently underway. Conclusions

Mutations in the C9orf72 gene are known to be associated with frontotemporal dementia (FTD) & amyotrophic lateral sclerosis (ALS.) This mutation is commonly the expansion of hexanucleotide repeats of GGGGCC/CCCCGG. Normally, 20 copies of this repeat are found, however, are expanded to hundreds to thousands of copies in patients with ALS or FTD. These repeats can form secondary structures known as G-quadruplex (G4) and intercalating motif (i-motif or iM). These secondary structures are influenced by several regulatory factors, including pH, temperature, ions, and various DNA binding proteins. CD spectroscopy was used to find the conditions considered most optimal for formation of secondary structures with the presence of these repeats, particularly sequences containing 2, 4, 6, and 14 tandem repeats of GGGGCC and GGCCCC found in intron 1 of C9orf72.

C9ORF72 G Repeats

Sequence

Nucleotides

Predicted G4s

12

0

C9ORF72 G-2

5'-CCG GGG CCG GGG-3'

24

1

C9ORF72 G-4

5'-CCG GGG CCG GGG CCG GGG CCG GGG-3'

36

1

C9ORF72 G-6

5'-CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG-3'

5'-CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG-3'

84

4

C9ORF72 G-14

Figure 3. Prediction of G4 formation and gel analysis of the oligos

Figure 4. Effects of various pH on G4 formation

C9ORF72 C Repeats

Sequence

Nucleotides Predicted i-motifs

iM Density

12

0

0%

C9ORF72 C-2

5'-CCC CGG CCC CGG-3'

24

1

4.17%

C9ORF72 C-4

5'-CCC CGG CCC CGG CCC CGG CCC CGG-3'

5'-CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG-3'

36

1

2.78%

C9ORF72 C-6

5'-CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CG GCCC CGG-3'

84

3

3.57%

C9ORF72 C-14

Figure 5. Prediction of iM formation and gel analysis of the oligos

Methods

For this study, G4 and i-motif structures were formed with the use of buffers of varying pH. CD spectroscopy was then used to study the presence of these structures.

Figure 6. Effects of various pH on i-motif formation

Figure 1. Hexanucleotide repeats in intron 1.

References

(GGGGCC)x4

(CCCCGG)x4

Figure 7. Effects of boiling on G4 and i-motif formation

Diggins, L., Ross, D., Bhanot, S., Corallo, R., Daley, R., Patel, K., Lewis, O., Donahue, S., Thaddeus, J., Hiers, L., Syed, C., Eagerton, D. and Mohanty, BK (corresponding author) (2024) CD spectra reveal the state of G-quadruplexes and i-motifs in repeated and other DNA sequences. Biophys Rep (NY) 5(1):100187. doi: https://doi.org/10.1016/j.bpr.2024.100187.

Acknowledgements

Figure 8. Effect of single-strand DNA binding protein (SSB) on iM formation

Figure 1. Secondary structure models.

Figure 2. Utilized Buffers.

BKM was funded by VCOM REAP grants 1032453 and 1302559.

2025 Research Recognition Day

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