Investigation of genomic DNA methylation by ultraviolet resonant Raman spectroscopy

Abstract

Cytosine plays a preeminent role in DNA methylation, an epigenetic mechanism that regulates gene expression, the misregulation of which can lead to severe diseases. Several methods are nowadays employed for assessing the global DNA methylation levels, but none of them combines simplicity, high sensitivity, and low operating costs to be translated into clinical applications. Ultraviolet (UV) resonant Raman measurements at excitation wavelengths of 272 nm, 260 nm, 250 nm, and 228 nm have been carried out on isolated deoxynucleoside triphosphates (dNTPs), on a dNTP mixture as well as on genomic DNA (gDNA) samples, commercial from salmon sperm and non-commercial from B16 murine melanoma cell line. The 228 nm excitation wavelength was identified as the most suitable energy for enhancing cytosine signals over the other DNA bases. The UV Raman measurements performed at this excitation wavelength on hyper-methylated and hypo-methylated DNA from Jurkat leukemic T-cell line have revealed significant spectral differences with respect to gDNA isolated from salmon sperm and mouse melanoma B16 cells. This demonstrates how the proper choice of the excitation wavelength, combined with optimized extraction protocols, makes UV Raman spectroscopy a suitable technique for highlighting the chemical modifications undergone by cytosine nucleotides in gDNA upon hyper-and hypo-methylation events.

Strategies and Perspectives for UV Resonance Raman Applicability in Clinical Analyses of Human Sperm RNA

“…In order to enhance adenine and guanine contributions and also to avoid the spectral fluorescence background affecting the UVRR spectrum of the nitrogenous bases as previously observed in Reference [ 23 ] and furtherly shown in Figure 2 A, we chose to work with a 250 nm-radiation source. In fact, as shown in the inset of Figure 2 A, the UVRR spectrum of 3T3J2 cells collected using a 266 nm-radiation source is completely dominated by a strong fluorescence background.…”

“…In medicine, the damages and methylations of nitrogen bases are now of increasing interest when specifically affecting RNA, and the translational potential of Raman spectroscopy is in this field is still poorly investigated. We already reported that UV Resonance Raman spectroscopy represents a valuable tool for studying DNA damages [ 1 ] and its epigenetic modifications such as cytosine methylation, which can lead to the development of several diseases [ 23 ]. Based on this, in this work, we specifically evaluated the applicability of UVRR to study the RNA extracted from spermatozoa for a future possible application in reproductive medicine.…”

“…To this purpose, D’Amico and colleagues recently exploited the UV Resonance Raman (UVRR) setup and the tunability of the synchrotron radiation source available at Elettra Synchrotron Facility (IUVS beamline) to successfully investigate cytosine methylation pattern of the DNA in model cell lines, which is the most abundant epigenetic change in eukaryotic cells related to several diseases. More specifically, UVRR has been used as a label-free and less time-consuming manner to detect cytosine methylation in cells, tuning the radiation source wavelength to 228 nm in order to selectively and resonantly excite the vibrational modes of cytosine, observing significant vibrational modifications depending on the presence of the methylated form [ 23 ]. Working at the same time in resonance condition and in the UV range provide the combined advantages of detecting vibrational modifications in low concentrated samples and to mainly enhance the molecular fingerprints addressed to nucleic acids, respectively.…”

“…We tuned the synchrotron radiation source to a 250 nm-radiation wavelength in order to investigate the quality of RNA extracted from spermatozoa (derived from 5 patients), compared to RNA isolated from commercial immortalized cell lines, willing to reach the lowest detection limit for RNA and possibly enhancing the spectral regions that could be affected by nucleotide modifications. Additionally, the use of the 250 nm-radiation source is actually a good compromise between the reduction of the fluorescence contribution dominating the Raman spectra at lower energies, where adenine vibrations are resonantly enhanced (i.e., 266 nm) and the relatively good enhancement of adenine peaks occur at 250 nm [ 23 ].…”

Scanning Probe Microscopies: Imaging and Biomechanics in Reproductive Medicine Research

“…Other label-free techniques based on vibrational signals, such as Raman microspectroscopy and Fourier transform infrared spectroscopy, represent valuable complementary techniques to obtain information about the biochemical compositions of molecular elements (DNA damage, lipid component, etc.) of gametes and embryos [ 2 , 5 , 6 , 8 ], with relevant contributions also in the studies of reproductive organs [ 9 ].…”