Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscopy

Abstract

The ability to detect DNA conformation in eukaryotic cells is of paramount importance in understanding how some cells retain functionality in response to environmental stress. It is anticipated that the B to A transition might play a role in resistance to DNA damage such as heat, desiccation and toxic damage. To this end, conformational detail about the molecular structure of DNA has been derived primarily from in vitro experiments on extracted or synthetic DNA. Here, we report that a B- to A-like DNA conformational change can occur in the nuclei of intact cells in response to dehydration. This transition is reversible upon rehydration in air-dried cells. By systematically monitoring the dehydration and rehydration of single and double-stranded DNA, RNA, extracted nuclei and three types of eukaryotic cells including chicken erythrocytes, mammalian lymphocytes and cancerous rodent fibroblasts using Fourier transform infrared (FTIR) spectroscopy, we unequivocally assign the important DNA conformation marker bands within these cells. We also demonstrate that by applying FTIR spectroscopy to hydrated samples, the DNA bands become sharper and more intense. This is anticipated to provide a methodology enabling differentiation of cancerous from non-cancerous cells based on the increased DNA content inherent to dysplastic and neoplastic tissue.

Figure 1.

Fully hydrated (blue) and dehydrated (red) spectra of (A) intact chicken erythrocytes and (B) double-stranded DNA and the corresponding second derivatives for (C) intact chicken erythrocytes and (D) double-stranded DNA. Important DNA conformation bands are indicated and approximate band position given. Asterisk denotes bands which lose significant intensity upon B–A transition.

Figure 2.

Second derivative spectra of fully hydrated (blue) and dehydrated (red) (A) extracted chicken erythrocyte nuclei undergoing dehydration (ATR-FTIR), (B) extracted chicken erythrocyte nuclei undergoing rehydration (ATR-FTIR), (C) mammalian cancerous fibroblasts (L-929) undergoing rehydration (ATR-FTIR) and (D) mammalian lymphocytes undergoing rehydration (T-FTIR).

Figure 3.

Trend plot showing the shift of peak position of the antisymmetric phosphate stretching vibration from ∼1224 to 1236 cm⁻¹ with decreasing/increasing hydration. Series plotted for dehydration and rehydration are indicative of trends seen across all experiment types.

Figure 4.

Trend plot showing the shift of peak position of the C–C stretching vibration from ∼969 to ∼965 cm⁻¹ with decreasing/increasing hydration. Series plotted for dehydration and rehydration are indicative of trends seen across all experiment types.

Figure 5.

Principal component 1 loadings plots for (A) dehydrating double-stranded DNA, (B) rehydrating chicken erythrocyte nuclei, (C) dehydrating chicken erythrocytes and (D) rehydrating mammalian fibroblasts. In all cases, peaks are attributed as causing variance in hydrated sample spectra while troughs are attributed as causing variance in dehydrated sample spectra.