DNA Methylation and Hydroxymethylation Quantification Using Vibrational Spectroscopy.
- 1Optoelectronics Research Centre, Building 53, University of Southampton, University Road, Southampton, Hampshire SO17 1BJ, United Kingdom.
- 2The Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, United Kingdom.
- 3The Royal Marsden NHS Foundation Trust, Fulham Road, London SW3 6JJ, United Kingdom.
Abstract
Global quantification of DNA cytosine modifications, including 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), is important for understanding cancer biology, though established methods require multistep workflows and costly instrumentation. Here, we show that attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy combined with regression modeling enables rapid, label-free, and nondestructive quantification of both modifications from DNA samples. Using Adenomatous Polyposis Coli (APC) promoter DNA standards spanning 0-100% modification, we identified modification-sensitive spectral features and observed that 5-hmC produces greater spectral changes than 5-mC. A univariate peak-ratio approach yielded strong linearity for both modifications (R ² = 0.97), while partial least-squares regression (PLSR) improved quantification accuracy to R ² = 0.99 (RMSE = 2.6%) for 5-hmC and R ² = 0.97 (RMSE = 5.7%) for 5-mC. In composite mixtures containing all three cytosine states, 5-hmC remained highly quantifiable (R ² = 0.97; RMSE = 5.1%), while 5-mC accuracy decreased (R ² = 0.90; RMSE = 9.6%), consistent with the greater spectral distinctiveness secondary to the hydroxymethyl group. Transferability was assessed using circulating tumor DNA (ctDNA), short cell-free DNA fragments shed from tumor cells into the bloodstream, comprising multiplexed reference material spanning seven genomic regions and a polydisperse fragment-length distribution (155-220 bp). After domain adaptation between synthetic and ctDNA spectra, we obtained a quantitative methylation calibration with R ² = 0.98 and RMSE = 5.2% under cross-validation. These results support ATR-FTIR spectroscopy as a viable platform for global cytosine modification quantification and establish proof-of-concept applicability to ctDNA analysis.
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BibTeX
@article{fatayer2026methylation,
title = {{DNA Methylation and Hydroxymethylation Quantification Using Vibrational Spectroscopy.}},
author = {Rashad Fatayer and Stephen-John Sammut and Ganapathy Senthil Murugan},
journal = {ACS measurement science au},
year = {2026},
month = jul,
doi = {10.1021/acsmeasuresciau.6c00167},
url = {https://europepmc.org/article/MED/42921427},
note = {CC BY},
}
RIS
TY - JOUR
TI - DNA Methylation and Hydroxymethylation Quantification Using Vibrational Spectroscopy.
AU - Rashad Fatayer
AU - Stephen-John Sammut
AU - Ganapathy Senthil Murugan
PY - 2026
DA - 2026/07/24
JO - ACS measurement science au
DO - 10.1021/acsmeasuresciau.6c00167
UR - https://europepmc.org/article/MED/42921427
AB - Global quantification of DNA cytosine modifications, including 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), is important for understanding cancer biology, though established methods require multistep workflows and costly instrumentation. Here, we show that attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy combined with regression modeling enables rapid, label-free, and nondestructive quantification of both modifications from DNA samples. Using Adenomatous Polyposis Coli (APC) promoter DNA standards spanning 0-100% modification, we identified modification-sensitive spectral features and observed that 5-hmC produces greater spectral changes than 5-mC. A univariate peak-ratio approach yielded strong linearity for both modifications (R ² = 0.97), while partial least-squares regression (PLSR) improved quantification accuracy to R ² = 0.99 (RMSE = 2.6%) for 5-hmC and R ² = 0.97 (RMSE = 5.7%) for 5-mC. In composite mixtures containing all three cytosine states, 5-hmC remained highly quantifiable (R ² = 0.97; RMSE = 5.1%), while 5-mC accuracy decreased (R ² = 0.90; RMSE = 9.6%), consistent with the greater spectral distinctiveness secondary to the hydroxymethyl group. Transferability was assessed using circulating tumor DNA (ctDNA), short cell-free DNA fragments shed from tumor cells into the bloodstream, comprising multiplexed reference material spanning seven genomic regions and a polydisperse fragment-length distribution (155-220 bp). After domain adaptation between synthetic and ctDNA spectra, we obtained a quantitative methylation calibration with R ² = 0.98 and RMSE = 5.2% under cross-validation. These results support ATR-FTIR spectroscopy as a viable platform for global cytosine modification quantification and establish proof-of-concept applicability to ctDNA analysis.
SN - 2694-250X
ER -