DNA Methylation Detection Using Resonance and Nanobowtie-Antenna-Enhanced Raman Spectroscopy

Abstract

We show that DNA carrying 5-methylcytosine modifications or methylated DNA (m-DNA) can be distinguished from DNA with unmodified cytosine by Raman spectroscopy enhanced by both a bowtie nanoantenna and excitation resonance. In particular, m-DNA can be identified by a peak near 1000 cm and changes in the Raman peaks in the 1200-1700 cm band that are enhanced by the ring-absorption resonance. The identification is robust to the use of resonance Raman and nanoantenna excitation used to obtain significant signal improvement. The primary differences are three additional Raman peaks with methylation at 1014, 1239, and 1639 cm and spectral intensity inversion at 1324 (C=C) and 1473 cm (C=N) in m-DNA compared to that of DNA with unmodified cytosine. We attribute this to the proximity of the methyl group to the antenna, which brings the (C=C) mode closer to experiencing a stronger near-field enhancement. We also show distinct Raman spectral features attributed to the transition of DNA from a hydrated state, when dissolved, to a dried/denatured state. We observe a general broadening of the larger lines and a transfer of spectral weight from the ∼1470 cm vibration to the two higher-energy lines of the dried m-DNA solution. We attribute the new spectral characteristics to DNA softening under high salt conditions and find that the m-DNA is still distinguishable via the ∼1000 cm peak and distribution of the signal in the 1200-1700 cm band. The nanoantenna gain exceeds 20,000, whereas the real signal ratio is much less because of a low average enhanced region occupancy even with these relatively high DNA concentrations. It is improved when fixed DNA in a salt crystal lies near the nanoantenna. The Raman resonance gain profile is consistent with A-term expectations, and the resonance is found at ∼259 nm excitation wavelength.

Deep‐Ultraviolet Biomolecular Imaging and Analysis

“…Figure A,B shows resonant Raman scattering spectra of nucleosides at DUV (λ = 244 nm) excitation. Individual bands in the spectra are assigned to vibration modes of individual nucleotide bases (e.g., stretching of C5N7 and N7C8 bonds of the purine ring mode for the 1336 cm −1 band of deoxyadenosine, and stretching of N3C4 and N1C2 bonds of the pyrimidine ring mode for the 1527 cm −1 band of deoxycytidine) . The spectra of purine nucleosides (Figure A) are distinct each other despite the chemical structures of them (Figures A and C) are similar.…”

“…Methylated bases in DNA have recently been recognized as important players in the regulation of functions, homeostasis, and pathogenesis of living matters, and sensitive detection of them is crucial in biosciences and medicine. Li et al used Al bowtie nanostructures for measuring resonant SERS of DNA with and without 5‐methylcytosine . The resonant Raman and SERS effects allowed 2 orders of magnitude lower limit of detection than that of nonresonant SERS with Au nanoshells at NIR excitation …”

Investigation of genomic DNA methylation by ultraviolet resonant Raman spectroscopy

“…Recently, many vibrational spectroscopic techniques have been used to infer DNA methylation 9–11 . In this regard, techniques based on surface‐enhanced Raman scattering (SERS) have been among the most used to investigate DNA in biological systems, 12, 13 and to infer DNA damage 14 and cytosine methylations mechanisms 13, 15, 16 .…”

“…3,7 Recently, many vibrational spectroscopic techniques have been used to infer DNA methylation. [9][10][11] In this regard, techniques based on surface-enhanced Raman scattering (SERS) have been among the most used to investigate DNA in biological systems, 12,13 and to infer DNA damage 14 and cytosine methylations mechanisms. 13,15,16 The goal is achieved thanks to the enhanced sensitivity of SERS, which allows detecting Raman signal even from a minute amount of sample.…”

Detecting DNA Methylation Using Surface-Enhanced Raman Spectroscopy

“…Recently, SERS has been utilized to analyze single-base misalliances and base methylations in duplex DNA . So far, SERS has been utilized to detect methylation of adenine and more extensively of cytosine. ,− However, reported use of SERS to detect methylation of guanine is scarce. Here, we have utilized SERS to establish distinction between the methylated and nonmethylated form of guanine and to identify the position (N7 or O6) at where methylation happened.…”