Via Research Recognition Day Program VCOM-Carolinas 2025

Biomedical Research

Genome-wide identification of single-stranded DNA sequences that bind to hnRNP K in humans Rachael Baker, B.A., Daniel Ross, B.S., Olivia Lewis, OMS-I, Krishna Patel, OMS-III, Lauren Heirs, OMS-II, Shane Donahue, OMS-II, Tarah Anasseri, OMS-I, Payal Arora, OMS-II, David Eagerton, Ph.D., and Bidyut K. Mohanty, Ph.D. Edward Via College of Osteopathic Medicine, 350 Howard St, Spartanburg, SC 29316

Introduction and Objective

Results

A. The human genome is interspersed with polycytosine rich (polyC-rich) and their complimentary polyguanine rich DNA sequences that can form noncanonical secondary structures. These sequences and secondary structures can affect genome integrity. Regulatory proteins, such as polyC-binding heterogeneous nuclear riboproteins (hnRNPs), can bind to single stranded DNA sequences in a site and structure-specific manner to these DNA sequences. These regulatory proteins are implicated in the pathogenic mechanisms including cancer and other diseases. One of the hnRNPs, namely hnRNP K, shows a binding preference for polyC-rich sequences, and regulates transcription, mRNA stability, splicing, and translation- and promotes cancer. It has been shown that hnRNP K binds to multiple sequences in the mouse genome. Our recent data shows that hnRNP K binds to thousands of polyC-rich DNA sequences in the mouse genome. B. HYPOTHESIS: We predict that hnRNP K binds to polyC-rich DNA sequences in the human genome to promote cancer and other genetic diseases. C. Our OBJECTIVE is to identify the single-stranded DNA sequences within the human genome that bind to hnRNP K and play a role in canc er.

Methods Figure 3. Consensus motif for mouse ssDNA sequences pulled down using hnRNP K (Patel et a l, unpublished).

Discussion Figure 5. ExoChew. (A) A 3% agarose gel showing comparison of human genomic DNA (gDNA), 200 bp sonicated gDNA, and sonicated gDNA that has been treated with T7 Exonuclease. (B) A 6% polyacrylamide gel showing comparison of sonicated human gDNA with and without single stranded binding protein (SSB), and its Exonuclease products with and without SSB.

Discussion and Further Directions

human

Genomic Human DNA

A. Our results indicate the successful transformation of human double-stranded DNA to single-stranded DNA. A pull-down assay was performed using the T7 Exonuclease treated DNA product and His tagged hnRNP K. B. The pull-down products were converted back to double-stranded DNA. The DNA library is currently being sequenced in-house using the Oxford Nanopore MinION MK1D. C. The identified sequences will be analyzed.

Figure 6. Oxford Nanopore MinION MK1D DNA Sequencer.

Figure 1. Map of hnRNP K.

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.​

hnRNP K 0

Figure 2. A 6% polyacrylamide gel shift of C9orf72 C6 ((C 4 G 2 ) 6 ) DNA demonstrating the ability of hnRNP K to bind to a human polycytosine-rich DNA sequence.

Acknowledgements

Figure 4. Protocol for isolating and identifying hnRNP K binding DNA sites.

Free DNA

BKM was funded by VCOM REAP grants 1032453 and 1302559.

2025 Research Recognition Day

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