Infrared vibrational fingerprinting for the detection of nucleic acid methylation with structural validation by...

Nucleic acid methylation is a fundamental epigenetic regulation that influences gene expression and is increasingly explored as a biomarker in cancer biology. However, conventional methods employed for methylation analysis are often labor-intensive, require comprehensive sample preparation, and lack real-time structural sensitivity. Herein, we report an experimentally validated spectroscopic framework for quantitative, label-free detection of methyl (-CH₃) signatures and structural features of nucleic acids using attenuated total reflection Fourier transform infrared (ATR-FTIR) and circular dichroism (CD) spectroscopic techniques. Accordingly, concentration-dependent detection thresholds and spectral resolution limits for DNA and RNA methylation were determined. ATR-FTIR data revealed distinct IR peaks attributed to the asymmetric and symmetric stretching and bending vibrations of the CH₃ groups, and displayed improved spectral separation appearing at intermediate concentrations (~500 ng/μL) of nucleic acids. Comparison of methylated RNA oligonucleotides and cell-derived RNA samples confirmed the robustness of methyl-sensitive spectral markers. CD spectroscopy demonstrated a strong linear correlation between DNA concentration and the ellipticity at 277 nm, consistent with excitonic coupling theory. RNA gave rise to detectable CD signals at lower concentrations (~25 ng/μL), whereas DNA required around 100 ng/μL for reliable structural interpretation, reflecting intrinsic differences between A- and B-form helices. Our study provides (i) experimentally determined lower detection limits, (ii) validated IR vibrational fingerprints of methylation-associated groups, and (iii) a combined spectroscopic analysis suitable for rapid and cost-effective examination of nucleic acid methylation. Thus, the current study lays the groundwork for future applications targeting site-specific modifications such as m⁶A and 5-methylcytosine in biomolecules.