Nanoscale Investigation of DNA Demethylation in Leukemia Cells by Means of Ultrasensitive Vibrational Spectroscopy

Abstract

DNA methylation is a crucial epigenetic hallmark of cancer development but the experimental methods able to prove nanoscale modifications are very scarce. Over time, Raman and its counterpart, surface-enhanced Raman scattering (SERS), became one of the most promising techniques capable to investigate nanoscale modifications of DNA bases. In our study, we employed Raman/SERS to highlight the differences between normal and leukemia DNA samples and to evaluate the effects of a 5-azacytidine treatment on leukemia cells. To obtain spectral information related to DNA base modifications, a DNA incubation step of 4 min at 94 °C, similar to the one performed in the case of RT-PCR experiments, was conducted prior to any measurements. In this way, reproducible Raman/SERS spectra were collected for all genomic DNA samples. Our Raman results allowed discrimination between normal and cancer DNAs based on their different aggregation behavior induced by the distinct methylation landscape present in the DNA samples. On the other hand, the SERS spectra collected on the same DNA samples show a very intense vibrational band located at 1008 cm−1 assigned to a rocking vibration of 5-methyl-cytosine. The intensity of this band strongly decreases in cancer DNA due to the modification of the methylation landscape occurring in cancers. We believe that under controlled experimental conditions, this vibrational band could be used as a powerful marker for demonstrating epigenetic reprogramming in cancer by means of SERS.

Keywords

• leukemia
• 5-azacytidine
• Raman spectroscopy
• SERS
• DNA demethylation