Understanding the similarity in thermophoresis between single- and double-stranded DNA or RNA.

Thermophoresis is the movement of molecules in a temperature gradient. For aqueous solutions its microscopic basis is debated. Understanding thermophoresis for this case is, however, important since it proved very useful to detect the binding affinity of biomolecules and since thermophoresis could have played an important role in early molecular evolution. Here we discuss why the thermophoresis of single- and double-stranded oligonucleotides - DNA and RNA - is surprisingly similar. This finding is understood by comparing the spherical capacitor model for single-stranded species with the case of a rod-shaped model for double-stranded oligonucleotides. The approach describes thermophoresis of DNA and RNA with fitted effective charges consistent with electrophoresis measurements and explains the similarity between single- and double-stranded species. We could not confirm the sign change for the thermophoresis of single- versus double-stranded DNA in crowded solutions containing polyethylene glycol [Y. T. Maeda, T. Tlusty, and A. Libchaber, Proc. Natl. Acad. Sci. USA 109, 17972 (2012)], but find a salt-independent offset while the Debye length dependence still satisfies the capacitor model. Overall, the analysis documents the continuous progress in the microscopic understanding of thermophoresis.

Thermophoretic manipulation of molecules inside living cells.

The complexity of biology requires that measurements of biomolecular interactions be performed inside living cells. While electrophoresis inside cells is prohibited by the cell membrane, the movement of molecules along a temperature gradient appears feasible. This thermophoresis could be used to quantify binding affinities in vitro at picomolar levels and perform pharmaceutical fragment screens. Here we changed the measurement paradigm to enable measurements inside living cells. The temperature gradient is now applied along the optical axis and measures thermophoretic properties for each pixel of the camera image. We verify the approach for polystyrene beads and DNA of various lengths using finite element modeling. Thermophoresis inside living cells is able to record thermophoretic mobilities and intracellular diffusion coefficients across the whole cytoplasm. Interestingly, we find a 30-fold reduced diffusion coefficient inside the cell, indicating that molecular movement across the cell cytoplasm is slowed down due to molecular crowding.

Why charged molecules move across a temperature gradient: the role of electric fields.

Methods to move solvated molecules are rare. Apart from electric fields, only thermal gradients are effective enough to move molecules inside a fluid. This effect is termed thermophoresis, and the underlying mechanisms are still poorly understood. Nevertheless, it is successfully used to quantify biomolecule binding in complex liquids. Here we show experiments that reveal that thermophoresis in water is dominated by two electric fields, both established by the salt ions of the solution. A local field around the molecule drives molecules along an energy gradient, whereas a global field moves the molecules by a combined thermoelectrophoresis mechanism known as the Seebeck effect. Both mechanisms combined predict the thermophoresis of DNA and RNA polymers for a wide range of experimental parameters. For example, we correctly predict a complex, nonlinear size transition, a salt-species-dependent offset, a maximum of thermophoresis over temperature, and the dependence of thermophoresis on the molecule concentration.