Label-free surface-enhanced Raman spectroscopy detection of DNA methylation with single-base sensitivity

DNA methylation is one of the earliest known modification pathways, and it regulates gene expression, which in turn influences many biological processes. Direct, label-free detection of DNA methylation with high sensitivity remains a great challenge. Surface-enhanced Raman scattering (SERS), a non-invasive and label-free vibration spectroscopy technique, offers sensitive intrinsic chemical information that makes it an attractive option for DNA analysis. In this study, we employed iodide-modified silver nanoparticles to generate highly consistent SERS signals of DNA at micromolar concentrations in aqueous solutions. This enabled the acquisition of single-base sensitive DNA fingerprint details pertaining to base methylations (such as 5-methylcytosine). As a proof of concept, the SER spectra of a DNA and its methylated counterpart were compared and analyzed, resulting in an obvious identification of DNA methylation. In particular, we first designed two DNA sequences, the sole change being that one of the nucleotides, cytosine, is replaced with a 5-methylcytosine. Further SERS experimental study revealed that methylating a single cytosine in the DNA strand caused a subtle but evident alteration in the SERS spectrum. A new Raman peak emerged at 760 cm^-1 the Raman peak at 792 cm^-1 moved to 790 cm^-1, and there was a noticeable drop in the peak intensity ratio between 1572 cm^-1 and 1634 cm^-1. This approach may provide a novel and easy-to-use tool for the label-free identification of single-molecule DNA mutations or modifications, furthering the advancement of ultra-sensitive genomic research in the future.