The Effects of Crowding on the Stability of a Surface-Tethered Biopolymer: An Experimental Study of Folding in a Highly Crowded Regime

The high packing densities and fixed geometries with which biomolecules can be attached to macroscopic surfaces suggests that crowding effects may be particularly significant under these, often densely-packed conditions. Exploring this question experimentally we report here the effects of crowding on the stability of a simple, surface-attached DNA stem-loop. We find that crowding by densely-packed, folded biomolecules destabilizes our test-bed biomolecule by ~2 kJ/mol relative to the dilute (non-interacting) regime, an effect that presumably arises due to steric and electrostatic repulsion arising from compact neighbors. Crowding by a dense brush of unfolded biomolecules, in contrast, enhances its stability of by ~6 kJ/mol, presumably due to excluded volume and electrostatic effects that reduce the entropy of the unfolded state. Finally, crowding by like copies of the same biomolecule produces a significantly broader unfolding transition, likely because, under these circumstances, the stabilizing effects of crowding by unfolded molecules increases (and the destabilizing effects of neighboring folded molecules decreases) as more and more neighbors unfold. The crowding of surface-attached biomolecules may thus be a richer, more complex phenomenon than that seen in homogeneous solution.