Type of presentation: Oral

ID-3-O-1497 Single Molecule Diffusion and Conformational Dynamics by Spatial Integration of Temporal Fluctuations

Serag M. F.¹, Habuchi S.¹

¹Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, King Abdullah University of Science and Technology, Thuwal 23955-6900, SAUDI ARABIA.

mfserag@gmail.com

Providing unprecedented insights into diffusional processes of systems in life and material sciences, single molecule localization (SML) has been the battle-horse of single molecule fluorescence microscopy methods. Although, the long-established technology provides accurate spatiotemporal locations of single molecules, for estimating their translational speed, it fails to provide essential information about molecular conformation. Furthermore, localization of the center of mass remains a tedious and challenging task for big biomolecules. Direct and accurate access to conformational dynamics and integrating it with translational speed measurements provide valuable learning of molecular diffusion as a crucial life process and reptation as an important physical phenomenon in polymer physics and analytical science. Here, we introduce a new method that is designed to tackle the limitations of SML and to harness molecular diffusion measurements by integration of valuable information on conformational dynamics.

Our method is essentially a spatial quantization of temporal fluctuations of the cumulative area occupied by molecules. Typically, SML analyses express molecular motion in terms of accurate spatiotemporal positions of the molecule. Our method, however, expresses molecular motion in terms of the time-wise increase of the cumulative area occupied by the molecule in space (Fig. 1). Through careful adjustment of the number of pixels detected at each time frame, information on translational diffusion, molecular size and frequency of conformational changes can be obtained. We validated our approach by analyzing the statistical distribution of diffusion coefficients of dsDNA of different lengths and topological forms, a measurement that is critically sensitive to molecular size and conformational changes. Our method showed narrower and much better symmetrical distribution around the mean diffusion coefficient compared to SML (Fig. 2A). Furthermore, the chain relaxation time can be, easily, obtained via autocorrelation of the fluctuations of molecular area over time (Fig. 2B).

In conclusion, our new method holds great promise for far-reaching advancement of single molecule fluorescence microscopy of polymers that is relevant to widely differing scientific fields. From fundamental physics over material science to chemistry and biology, translational diffusion and conformational dynamics are, indeed, central to polymer multidisciplinary studies.

Fig. 1: A) Gradual increase in the cumulative area occupied by diffusing nanospheres (top) and 6 kbp DNA (bottom). B) The red-bordered region defines the area occupied by the molecule at each time frame. The dashed pixels represent the area increment at each time frame as a result of random movement of the molecules.

Fig. 2: Diffusional and conformational dynamics of 42 kbp DNA. A) Frequency histogram of diffusion coefficients. The values were determined via the area-based (green) and the SML-MSD (yellow) methods. B) Autocorrelation of molecular area fluctuations. The autocorrelation function fits to single exponential decay with mean correlation time of 350 ms.