Applying spectral phasor analysis to DNA environments

Abstract

Two contradictory models exist that describe the condensation of interphase chromatin into mitotic chromosomes. Fixed cell and indirect methods describe a hierarchical folding of interphase chromatin. In contrast, developments in living cell characterisation have described a highly dynamic, disordered packing of chromatin at different concentration densities in both interphase and mitotic states. This presents a need to develop living cell applicable techniques for the elucidation of chromatin architecture and dynamics. The technique spectral phasor analysis is capable of characterising spectral shift associated with environmental changes throughout a live cell. Spectral phasor analysis can unmix fluorescent molecules without prior knowledge of their contribution to the spectra and elucidate subtle spectral shifts in single dyes as they associate with different cellular environments. Here, spectral phasor analysis was tested for its ability to characterise DNA environments using the non-intercalating, membrane permeable dye, H33342. This enabled chromatin dynamics to be elucidated as minor groove bound H33342 condensed in fixed and live cell environments. Spectral phasor analysis was performed on DNA bound H33342 in solution following different treatment conditions designed to change the binding mode and affinity of H33342. Spectral characterisation was also performed on L6 myoblasts fixed at different stages post serum starvation. Fixed L6 cells were also spectrally analysed for the elucidation of cellular environments exhibiting discrete emissions. Cellular environments were inferred morphologically. Multiple data acquisitions were performed through the z-plane in a fixed cell for the quantification of discrete H33342 emission volumes. Live L6 myoblasts were also characterised by spectral phasor analysis following cell degradation by UV irradiation. Spectral phasor analysis elucidated shifts in H33342 emission spectra as a product of H33342, DNA and MgCl2 concentration increases in solution. Shifts in H33342 emissions were also identified following heat denaturation of gDNA in solution and found to be dependent on minor groove flanking sequence. Areas of convolution in solutions of self-complementary oligonucleotides also exhibited distinct spectral characteristics from the solutions themselves, suggesting a local DNA concentration dependent spectral shift. In fixed cells, spectral phasor analysis revealed cell-wide cyclical fluctuation of both the I»max and width of H33342 emission spectra, up to 30 hours post exposure to serum starved conditions. It was observed that, over the serum starved time course, the width of H33342 emissions broadened and the I»max decreased. Nuclear microenvironments were elucidated by shifts in I»max and spectral width and a clear difference between H33342 emission spectra in the nucleus and cytoplasm was apparent. Shifts in the emission spectra of H33342 were identified at different stages of the cell cycle, indicating that spectral shift can occur as a consequence of chromatin condensation. The intensity of H33342 emissions was found to exhibit a negative and linear correlation to spectral width, whereas no such correlation was found for I»max. By comparing emissions of H33342 bound to chromatin in different density states, the intensity of H33342 emissions was found to increase and the spectral width narrowed as chromatin density increased. Analysis of discrete emissions in 3D revealed fluorescence intensity increases and spectral width narrowing towards the centre of the chromosomal mass in prophase. These emissions occupied smaller volumes as intensity increased and width narrowed, further supporting the model that spectral shift in H33342 emissions is chromatin density dependent. Analysis of live and fixed cell H33342 emissions revealed differences in emission characteristics. Upon inducing cell degradation by irradiation, nuclear and cytoplasmic emission differences began to converge. Spectral phasor analysis represents a distinctive analysis tool for the characterisation of DNA environments in solution and in fixed and live cells. With future development spectral phasor analysis can be applied to the elucidation of live cell chromatin condensation and dynamics in 3D, positioning it as a powerful technique in the biophysical toolkit.

Date of Award	2017
Original language	English