Via Research Recognition Day Program VCOM-Carolinas 2025

Biomedical Research

Unlocking the Promoter Puzzle: G-Quadruplex and I-Motif Secondary DNA Structures in VEGF Gene Regulation and Therapeutic Potential Levi Diggins OMS-IV, Krishna Patel OMS-III, Jake Rodriguez OMS-II, Sundeep Bhanot OMS-II, Daniel Ross, Olivia

Lewis OMS-I, Rachel Daley OMS-III, Rebecca Corallo OMS-III, and Bidyut K Mohanty. Edward via College of Osteopathic Medicine, 350 Howard St. Spartanburg SC

Introduction

Results

Conclusions

References Acknowledgements 6) Del Villar- Guerra, Rafael, et al. “G -Quadruplex Secondary Structure Obtained from Circular Dichroism Spectroscopy.” Angewandte Chemie (International Ed. in English) , U.S. National Library of Medicine, 11 June 2018, www.ncbi.nlm.nih.gov/pmc/articles/PMC5920796/. 7) ER;, Swenson. “Hypoxia and Its Acid - Base Consequences: From Mountains to Malignancy.” Advances in Experimental Medicine and Biology , U.S. National Library of Medicine, 2016, pubmed.ncbi.nlm.nih.gov/27343105/. 8) Lawson, Teegan, et al. “A Structural Perspective on the Regulation of Human Single -Stranded DNA Binding Protein 1 (Hssb1, OBFC2B) Function in DNA Repair.” Computational and Structural Biotechnology Journal , U.S. National Library of Medicine, 28 Mar. 2019, www.ncbi.nlm.nih.gov/pmc/articles/PMC6451162/. Acknoledgements BKM was partially funded by VCOM REAP grants 1032453 and 1302559. 1) Arrigo, A., Aragona, E., & Bandello, F. (2022). VEGF-targeting drugs for the treatment of retinal neovascularization in diabetic retinopathy. Annals of medicine , 54 (1), 1089 – 1111. https://doi.org/10.1080/07853890.2022.2064541 2) Song, D., Liu, P., Shang, K., & Ma, Y. (2022). Application and mechanism of anti-VEGF drugs in age related macular degeneration. Frontiers in bioengineering and biotechnology , 10 , 943915. https://doi.org/10.3389/fbioe.2022.943915 3) Patel, Sonia A, et al. “Molecular Mechanisms and Future Implications of VEGF/VEGFR in Cancer Therapy.” Clinical Cancer Research: An Official Journal of the American Association for Cancer Research , U.S. National Library of Medicine, 4 Jan. 2023, www.ncbi.nlm.nih.gov/pmc/articles/PMC10274152/. 4) Kendrick, S. & Hurley, L. (2010). The role of G-quadruplex/i-motif secondary structures as cis-acting regulatory elements. Pure and Applied Chemistry, 82(8), 1609-1621. https://doi.org/10.1351/PAC-CON 09-09-29 5) Wright, Elisé P, et al. “Identification of Multiple Genomic DNA Sequences Which Form I -Motif Structures at Neutral Ph.” Nucleic Acids Research , U.S. National Library of Medicine, 7 Apr. 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5605235/. The promoter-proximal region of VEGF contains polycytosine-rich DNA sequence capable of producing I-motif and a complimentary guanine rich sequence capable of producing G-quadruplex. Our CD analysis created the expected absorption peaks of iM at 280-287 nm and G4 at 260-265 nm, which is consistent with the literature 5,6 . The formation of iM and G4 were favored at different pH. Whereas i-motif displayed an acidic pH requirement, G4 formation was favored at near-neutral pH (Figure 2). This aligns with the hypothesis that hypoxia induced cellular acidity, as seen in PDR and AMD, leads to increased formation of i-motif, supporting a regulatory role of iM and G4 in the pathological and physiological expression of VEGF. Equally, a similar mechanism could be possible in the acidic microenvironments present in tumor cells 7 . Following linearization by boiling, both samples displayed small but significant changes to their pH dependent formation. The formation of both iM and G4 at higher pH (>6.0) displayed a higher-level of formation following linearization (Figure 3), indicating a propensity for structure formation during periods of denaturation such as translation. The emergence of iM and G4 was significantly influenced by the increasing concentration of complementary strand (Figure 4) while keeping the iM-forming sequence at a constant concentration. When the G4-forming sequence was added at 1:1 (orange) and 2:1 ratios (green), iM formation was sustained (Figure 4A). However, at 3:1 and 4:1 ratios, the iM peak decreased, and G4 formation was favored. Conversely, when the G4 forming sequence was held constant with increasing iM sequence, different outcomes were observed, with G-quadruplex formation being favored in all ratios except for 1:1 (orange) (Figure 4B). This indicates the capability of these sequences to coexist and support structure formation in the complementary strand. The formation of iM also displayed a dose-dependent linearization in the presence of SSB protein (Figure 5). SSB represents one of the intracellular proteins 8 . This represents a potential role of protein binding in iM formation and, consequently, in VEGF expression intracellularly. Similarly, this hints at a new target for VEGF targeting therapies. In summary, G4 and iM structures are regulators of gene expression in VEGF and a variety of other oncogenes, tumor suppressor genes and other non-cancer related diseases. The discovery of these structures represent a promising area for future drug development and disease prevention. In the case of VEGF, this could impact treatments for wet age-related macular degeneration, proliferative diabetic retinopathy and cancer.

Vascular endothelial growth factor (VEGF) plays a monumental role in the pathogenesis of wet age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). In both AMD and PDR, VEGF mRNA and growth factor receptors (VEGFR1, VEGFR2) are upregulated due to retinal hypoxia, ultimately resulting in vascular proliferation that can cause blindness, vitreous hemorrhage, and traction-related retinal detachment. VEGF is a glycopeptide homodimer with several isoforms primarily responsible for pathologic angiogenesis in AMD and PDR 1,2 . Equally, pathological VEGF expression and subsequent angiogenesis plays a key role in tumor growth throughout the body, serving as the key mechanism for vessel development within tumors, which are necessary for uncontrolled proliferation of cancer cells 3 . G-quadruplex (G4) and i-motif (iM) producing (polyG- and polyC-rich) DNA sequences have been discovered within the promoter-proximal region of VEGF. The presence of these structures have also been implicated as key regulators of gene expression. Hypothesis: Cellular influences on G4 and iM formation represent potential sites for the development of dysregulated angiogenesis in eye disease and tumor growth 4 . Elucidating the cellular influences on the development of G4 and iM within the promoter proximal sequence of VEGF represent avenue for future treatment development and risk stratification for disease involving dysregulated angiogenesis, including ophthalmological conditions such as PDR and AMD as well as tumor growth. The goal of this project is to analyze the intracellular factors involved in the development of G4 and iM, including: o Cellular acidity which is influenced by tissue hypoxia as described in the pathogenesis of AMD and PDR. o Protein binding dynamics.

G-quadruplex and i-motif formation at VEGF promoter by CD analysis

Figure 2A: VEGF-C Strand with i-Motif formation at absorption peak ~280-285 (nm).

Figure 2B: VEGF-G Strand with G4 formation at absorption peak ~260-263 (nm).

Figure 3A: VEGF-C Strand with i-Motif formation at absorption peak ~280-285 (nm) following linearization by boiling.

Figure 3B: VEGF-G Strand with G4 formation at absorption peak ~260-263 (nm) following linearization by boiling.

o Complimentary double-stranded DNAbinding dynamics. o Structure formation following linearization by boiling.

Methods

For this work, iM and G4 were formed in-vitro using buffers with varying pH, which preferentially promoted the corresponding structure formation. The presence of these structures were then studied using Circular Dichroism (CD), a spectroscopic method that measures the absorption of right and left circularly polarized light in optically active molecules including DNA and proteins. Once the absorption peaks of G4 and iM were standardized, the formation of these structures in various conditions could be analyzed. The formation of G4 and iM was studied in the presence of competing guanine and cytosine-rich sequences as well as E. coli derived single strand binding protein (SSB) and following linearization by boiling. VEGF C and VEGF G oligodeoxynucleotides (DNA oligos) were purchased from IDT and Sigma Aldrich. The oligos were dissolved in water to a concentration of 100 picomoles/microliter. Appropriate amounts of oligos were mixed with appropriate buffers to carry out CD measurements. Buffers included: Sodium cacodylate pH 5.5 and 7.4, Tris-acetate pH 6.0, MES pH6.5, Tris-KCL pH 7.4. E. coli SSB was purchased from MCLAB with a concentration of 5 mg/ml.

Figure 4B: VEGF-G Strand competition with VEGF-C strand at varying concentrations. 1:1 ratio (Orange).

Figure 4A: VEGF-C Strand competition with VEGF-G strand at varying concentrations. 1:1 ratio (Orange).

Figure 1A: VEGF gene promoter with rich cytosine and guanine strands

Figure 1B: G-quadruplex and i-Motif structure

Figure 5: VEGF- C strand i-motif formation competition with SSB protein binding.

2025 Research Recognition Day

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