Via Research Recognition Day Program VCOM-Carolinas 2025

Biomedical Research

The Discovery of DNA Sequences Binding to a Cancer-Associated Protein by a Novel Technique Krishna Patel 1 , Daniel Ross 1 , Chirag Lodha 1 , Christopher Smith 1 , Levi Diggins 1 , Venkata Kolluru 1 , Olivia Lewis 1 , Christopher Syed 1 , Rachel Daley 1 , Rebecca Corallo 1 , Jacob Thaddeus 1 , Sundeep Bhanot 1 , Lauren Hiers 1 , Shane Donahue 1 , Rachael Baker 1 , Olivia McLean 1 , David Eagerton 1 , Ramu Anandakrishnan 2 , and Bidyut K Mohanty 1 1 Edward Via College of Osteopathic Medicine - Carolinas, Spartanburg, SC; 2 Edward Via College of Osteopathic Medicine - Virginia, Blacksburg, VA

Discussion

Introduction

Results

The ExoChew method demonstrates high efficiency in generating ssDNA libraries for DNA-protein interactions and DNA structural studies. Genome-wide sequencing data show that ExoChew achieves coverage (98.26%) comparable to sonicated double-stranded DNA (98.33%) with minimal regional biases and uniform read distribution. Using ExoChew, sequencing analysis successfully identified the consensus motif for hnRNP K binding in mouse DNA. This highlights its applicability in detecting protein-DNA interactions with high sensitivity . Further clustering analysis of polypyrimidine sequences revealed regions enriched in hnRNP K binding motifs. These findings validate ExoChew’s ability to map regulatory sequence patterns and binding sites. Localization of hnRNP K motifs relative to annotated genes demonstrates their association with functional regulatory regions, such as promoters and enhancers. This strengthens the clinical and biological significance of ExoChew in elucidating mechanisms of transcriptional regulation and its potential in cancer research. Conclusions • Efficient Library Preparation: ExoChew generates ssDNA libraries with genome coverage comparable to conventional dsDNA, providing uniform sequencing read distribution. • Motif Identification: ExoChew enables the detection of biologically relevant sequence motifs, such as those for hnRNP K binding, facilitating high-resolution mapping of protein-DNA interactions. • Functional Genomics Utility: Clustering analysis of polypyrimidine sequences uncovers regulatory sequence patterns linked to hnRNP K binding, with implications for gene regulation. • Clinical Relevance: ExoChew provides a scalable platform to study protein-ssDNA interactions in cancer biology, uncovering potential therapeutic targets for oncogenic pathways of hnRNP K.

• Genomic instability, a driver of cancer and various other genetic disorders, arises from errors in or during DNA replication, repair, and transcription. Single-stranded DNA (ssDNA) and secondary structures like G-quadruplexes and i-motifs (generated from polyguanine- and polycytosine-rich ssDNAs) play vital roles in gene regulation but can compromise genome integrity when mismanaged. • Proteins such as hnRNP K bind polycytosine-rich ssDNAs and I-motifs to regulate transcription and genomic stability. Overexpression of hnRNP K in cancers, including breast-, colorectal-, and pancreatic cancers, promotes tumor progression, metastasis, and chemoresistance. Despite its significance, studying protein-ssDNA interactions remains limited by inefficient ssDNA generation methods prone to reannealing and poor scalability. • HYPOTHESIS : We hypothesized that our novel ExoChew would produce high- quality ssDNA libraries suitable for investigating genome wide protein-ssDNA interactions and DNA secondary structures, advancing understanding of genomic instability in cancer and genetic disorders. • RESEARCH GOAL: Develop ExoChew , a novel enzymatic method for generating genome-wide ssDNA libraries, addressing inefficiencies of traditional approaches. • CLINICAL IMPORTANCE: ExoChew enables large-scale protein-ssDNA interactions , providing insights into cancer progression, genomic instability, and potential therapeutic targets.

Figure 3. PAGE gel showing ExoChew products of mouse genomic DNA and SSB binding. Genomic DNA was sonicated followed by ExoChew using T7 exonuclease or E. coli exonuclease III that generates single-stranded DNA. The products of sonication and ExoChew were incubated with SSB and analyzed by PAGE.

Library Type ‘‹ ƒ–‡† †• ͻͺǤ͵͵ š‘ Š‡™ •• ȋ ‘”™ƒ”† ƒ† ‡˜‡”•‡Ȍ ͻͺǤʹ͸ Figure 2. Comparison of DNA sequencing of sonication products and T7 exonuclease products of E. coli genome. Coverage (%)

Total number of reads 220,913 Number of pyrimidines in sequence

Mouse gDNA ExoChew products ↓ 6-HIS-hnRNP K ↓ Protein-ssDNA complex pulled down ↓ Extraction of DNA ↓ Conversion of ssDNA to dsDNA ↓ Sequencing

Polypyrimidine Sequence (PPS) (CT)(CT)CT)+ (CT)(CT)CT)+ (CT)(CT)CT)+ (CT)(CT)CT)+

Gap Length

1 1 2 2 1 1 2 2 5

12 15 12 15 12 15 12 15 12

131,265 88,951 163,534 121,680

CCC+ CCC+ CCC+ CCC+ CCC+

4,381 2,282 6,290 3,220

Methods

14,350

• Polypyrimidine sequence (PPS) = Sequence of 3 or more pyrimidines with no intervening purines, i.e. (C/T)(C/T)(C/T)+ • Gaps = intervening nucleotides between adjacent PPS • Gap Size = number of nucleotides in the Gaps • Cluster of PPS = series of PPS with Gap Size <= N (1 or 2) and Cluster Length >= L (12 or 15) • Cluster Length = number of Pyrimidines in the PPSs in the cluster. Does not include nucleotides in the Gaps. Table 1. Number of DNA fragments containing various pyrimidine sequences.

1 References

1.Patel, K., Lodha, C., Smith, C., Diggins, L., Kolluru, V., Ross, D., Syed, C., Lewis, O., Daley, R. and Mohanty, BK. (2023) ExoChew: An exonuclease technique to generate single-stranded DNA libraries. Biorxiv. doi: https://doi.org/10.1101/2023.10.02.560524. October 02, 2023. 2.Lu J, Gao FH. Role and molecular mechanism of heterogeneous nuclear ribonucleoprotein K in tumor development and progression. Biomed Rep. 2016 Jun;4(6):657-663. doi: 10.3892/br.2016.642. Epub 2016 Mar 29. PMID: 27284403; PMCID: PMC4887935.

Figure 4. Consensus motif of mouse ssDNA sequences pulled down using hnRNP K.

Figure 5. Location of consensus motif in sequencing reads relative to annotated genes.

Acknowledgments

Figure 1. The ExoChew technique and its uses.

The work was partially funded by VCOM’s REAP grant # 1032453 to BKM.

2025 Research Recognition Day

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