Monitoring the Conformation and Concentration of DNA in Live Cells using Fourier Transform Infrared Spectroscopy

Abstract

The ability to accurately detect DNA both quantitatively and qualitatively inside cells using Fourier transform infrared (FTIR) spectroscopy has been disputed. Recently, we have demonstrated that the variability of DNA absorptions is due to the dehydrated nature of biological samples prepared for FTIR spectroscopic measurement [1]. We have further demonstrated that in the dehydrated and fixed state DNA in cells assumes an A-DNA conformation instead of the native B-DNA form. Importantly, as well as being detected in eukaryotes which were invariably destroyed during dehydration, this B-A DNA transition has also been observed in desiccation-resistant, dormant bacteria and the native B- conformation has been detected upon rehydration of these cells. This previously undetected reversible transition raises several interesting questions about the role of A-DNA as a defense mechanism and its role in the evolution of nucleic acids.The B-A conformational transition has also been identified as causing changes to the molar extinction coefficients of several DNA bands explaining previous observations of unexpectedly low DNA absorption intensities. The Beer-Lambert nature of these absorptions was demonstrated by infrared spectroscopy of avian erythrocytes and extracted nuclei in conjunction with Partial Least Squares regression analysis to quantify cellular DNA [2]. Furthermore, spectra of hydrated single cells throughout interphase were also used to investigate the quantitative and qualitative biochemical changes involved in the G1, S and G2 phases of the cell cycle [3]. Using Principal Component Analysis cells only two hours apart were successfully clustered based on changes to the concentration and conformation of lipid, protein and DNA.1. D. R. Whelan, et. al., Nucleic Acids Research, 39, 5439-5448 (2011).2. D. R. Whelan, et. al., Journal of Biophotonics, 10, 775-784 (2013).3. D. R. Whelan, et al., Analyst, 138, 3891-3899 (2013).