Experimental Measurement of Surface Charge Effects on the Stability of a Surface-Bound Biopolymer.

Quantitative experimental studies of the thermodynamics with which biopolymers interact with specific surfaces remain quite limited. In response, here we describe experimental and theoretical studies of the change in folding free energy that occurs when a simple biopolymer, a DNA stem-loop, is site-specifically attached to a range of chemically distinct surfaces generated via self-assembled monolayer formation on a gold electrode. Not surprisingly, the extent to which surface attachment alters the biopolymer's folding free energy depends strongly on the charge of the surface, with increasingly negatively charged surfaces leading to increased destabilization.

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions.

Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control.

Using the population-shift mechanism to rationally introduce "Hill-type" cooperativity into a normally non-cooperative receptor.

Allosteric cooperativity, which nature uses to improve the sensitivity with which biomolecular receptors respond to small changes in ligand concentration, could likewise be of use in improving the responsiveness of artificial biosystems. Thus motivated, we demonstrate here the rational design of cooperative molecular beacons, a widely employed DNA sensor, using a generalizable population-shift approach in which we engineer receptors that equilibrate between a low-affinity state and a high-affinity state exposing two binding sites. Doing so we achieve cooperativity within error of ideal behavior, greatly steepening the beacon's binding curve relative to that of the parent receptor.

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 suggest 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 (noninteracting) regime, an effect that presumably occurs due to steric and electrostatic repulsion arising from compact neighbors.

Determinants of the detection limit and specificity of surface-based biosensors.

Here, we employ a model electrochemical DNA sensor to demonstrate that the detection limit and specificity of surface-based sensors often are not dependent on the true affinity of the probe for its target but are simply dependent on the effective probe concentration. Under these circumstances, the observed affinity (and thus the sensor's detection limit and specificity) will depend on the density with which the probes are packed on the surface of the sensor, the surface area, and even the volume of sample employed.

Entropic and electrostatic effects on the folding free energy of a surface-attached biomolecule: an experimental and theoretical study.

Surface-tethered biomolecules play key roles in many biological processes and biotechnologies. However, while the physical consequences of such surface attachment have seen significant theoretical study, to date this issue has seen relatively little experimental investigation. In response we present here a quantitative experimental and theoretical study of the extent to which attachment to a charged-but otherwise apparently inert-surface alters the folding free energy of a simple biomolecule.