VCOM Carolinas Research Day 2023

Biomedical Studies

ExoChew: A novel technique using exonuclease activity to generate a single-stranded DNA library *Chirag Lodha, OMS-II, *Krishna Patel, OMS-I, *Bidyut Mohanty, PhD VCOM Carolinas, Spartanburg SC

Abstract # BIOM-9

Abstract

Results

Conclusions

Polyguanine and polycytosine-rich sequences can form secondary structures known as G quadriplex (G4) and I-motifs that can cause genomic instability and DNA damage, which can eventually lead to the development of cancer and other genetic disorders. These structures are formed by single stranded DNA (ssDNA). This study generated an ssDNA library from the genomic DNA of bacteria, yeast, and mammalian cells. Generally, genomic ssDNA is generated using heat or alkaline treatment of double-stranded DNA (dsDNA), which are both quick, effective, and affordable methods. The difficulty in analyzing these protein-DNA interactions is that ssDNA fragments tend to reanneal with complementary ssDNA to form dsDNA. By using specific exonucleases, we can overcome this problem and create a library of higher quality ssDNA. Sample dsDNA extracted from mouse DNA was first mechanically sheared. The generation of dsDNA fragments was done by using both a manual as well as an automatic sonicator, and the method was standardized using both devices. The polyguanine and polycytosine-rich sequences that form G4 and I-motifs or bind to ssDNA-binding proteins are less than 100 bases long and easy for genome-wide sequencing. The dsDNA fragments were created between 200-300bp in length so that ssDNAs generated from them will be 100-150 bases long. To create ssDNAs, two exonucleases were used: E. coli Exonuclease III (EIII), which degrades each strand of a dsDNA from the 3’ to 5’ direction, and T7 exonuclease, which works in the 5’ to 3’ direction. The DNA amount was reduced, indicating that both exonucleases successfully created ssDNA. The ssDNA library generated using this novel technique will be used for genome wide binding by ssDNA binding proteins, structural protein studies, and additional relevant studies that require an ssDNA library.

Based on our gel runs, we conclude that mouse genomic DNA was fragmented into ~200 bp size DNA fragments by both sonication methods; however, while it took several cycles of sonication by the manual sonicator, the automatic fragmentation was completed in a few minutes. The exonuclease treatment generated ssDNA as was seen by reduction in fluorescence intensity of DNA. This indicates that the EIII and T7 exonucleases worked as expected, and an ssDNA genomic library was created. Because of its ease of operability as well as simple standardization, the automatic ultrasonicator should be strongly considered if available when creating a genomic library of ssDNA. Additionally, because there is a shared Covaris SonoLab TM program for the automatic sonicator, it allows anyone with the same device to easily create their own ssDNA library. Manual sonication is a viable option, but is prone to overheating of the sample, as well as changes in positioning during sonication. This can lead to increased variability in the base pair length of DNA fragments. With automatic sonication, ssDNA libraries can be created in less than one hour, which is significantly longer than the few minutes required for heat denaturation. However, because of the inherent nature of exonuclease activity, there is no risk of DNA fragments reannealing, allowing libraries to be created and stored indefinitely. In terms of future applications of this method, ssDNA binding studies are already underway, such as those of G4 and-motifs as well as ssDNA-protein interactions. An accompanying poster by Krishna Patel is being presented in this conference in which I am a co-author. Using the ssDNA library generated by this technique, we will be able to see why and how these specific structures form and how they bind to specific proteins. This will help us to explore the biochemical mechanisms of genomic instability and DNA damage in cancer and other diseases, opening the door to eventual therapeutic interventions that can be done at the DNA level.

Covaris M220 Focused ultrasonicator

QSONICA Q125 Sonicator

25% 50% 75% 100% L C

C MS L

Introduction or Methods

Automatic sonication protocol: 1. 50 µg dsDNA (500 µL) was taken a Covaris vial and placed in the sample holder in a water bath. 2. Covaris SonoLab TM 7 program was used to break the dsDNA to a length of 200bp, (default settings: power of 75.0, duty factor 20. and cycles/burst 200). 3. Exonucleases were added and incubated for 30 minutes (37 ℃ for EIII and 25 ℃ for T7). 4. DNA was analyzed by agarose gel electrophoresis.

Manual sonication protocol: 1. 50 µg dsDNA (500 µL) was taken in a 1.5 mL microcentrifuge tube. 2. The tube was placed into an ice bath and sonicated at 70% power in 30 second intervals, with 1 minute break between cycles until broken down to ~200bp of dsDNA. 3. Exonucleases were added and incubated for 30 minutes at their respective temperatures. 4. DNA was analyzed by agarose gel electrophoresis.

References

Automatically sonicated dsDNA C: control dsDNA

Manually sonicated dsDNA C: control dsDNA MS: manually sonicated dsDNA L: 100bp DNA ladder

25%: 25% sonicated 50%: 50% sonicated 75%: 75% sonicated 100%: 100% sonicated L: 100bp DNA ladder

1. Mohanty, B. K. et al (2021) Heterogeneous nuclear ribonucleoprotein E1 binds polycytosine DNA and monitors genome integrity. Life Sci Alliance. 4(9):e202000995. doi: 10.26508/lsa.202000995. 2. Peña et al., (2022) Human genomic DNA is widely interspersed with i-motif structures. doi: https://doi.org/10.1101/2022.04.14.488274.

Exonuclease Activity

C 100% EIII T7 L

Automatically sonicated dsDNA C: control dsDNA 100%: 100% sonicated dsDNA EIII: EIII exonuclease treated DNA T7: T7 exonuclease treated DNA L: 100bp DNA ladders

Chromosomal DNA

Sonication

Sonication

5’ 3’

5’ 3’

5’ 3’

5’ 3’

Double-stranded DNA

T7 exonuclease (5’ → 3’)

E. coli Exonuclease III (3’ → 5’)

Acknowledgements

dA dC

Single-stranded DNA and nucleotides

5’ 3’

5’ 3’

5’ 3’

5’ 3’

dC dA

Special thanks to the VCOM Carolinas campus for the access to both sonicator devices.

5’ 3’

5’ 3’

5’ 3’

5’ 3’

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