Entropic and Electrostatic Effects on the Folding Free Energy of a Surface-Attached Biomolecule: An Experimental and Theoretical Study

Abstract

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. Specifically, we have measured the folding free energy of a DNA stem loop both in solution and when site-specifically attached to a negatively charged, hydroxyl-alkane-coated gold surface. We find that, whereas surface attachment is destabilizing at low ionic strength it becomes stabilizing at ionic strengths above ~130 mM. This behavior presumably reflects two competing mechanisms: excluded volume effects, which stabilize the folded conformation by reducing the entropy of the unfolded state, and electrostatics, which, at lower ionic strengths, destabilizes the more compact folded state via repulsion from the negatively charged surface. To test this hypothesis we have employed existing theories of the electrostatics of surface-bound polyelectrolytes and the entropy of surface-bound polymers to model both effects. Despite lacking any fitted parameters, these theoretical models quantitatively fit our experimental results, suggesting that, for this system, current knowledge of both surface electrostatics and excluded volume effects is reasonably complete and accurate.

DNA Hairpin Stabilization on a Hydrophobic Surface

“…Although DNA hybridization has been extensively studied in solution, much less is known about interfacial hybridization 17. Recent efforts have addressed interfacial contributions such as electrostatics, conformational restriction due to confinement, and surface coverage 18–20. Relative to solution, surfaces were found to drive equilibrium either towards or away from the helical state depending on both the DNA sequence and surface properties, highlighting that surface effects represent combined contributions from many DNA‐surface interactions 19–21.…”

“…Recent efforts have addressed interfacial contributions such as electrostatics, conformational restriction due to confinement, and surface coverage 18–20. Relative to solution, surfaces were found to drive equilibrium either towards or away from the helical state depending on both the DNA sequence and surface properties, highlighting that surface effects represent combined contributions from many DNA‐surface interactions 19–21. The present work uses single‐molecule fluorescence tracking to observe both the conformational state and dynamic trajectory of a hairpin‐forming DNA sequence on two different surface chemistries: a model hydrophobic surface (trimethylsilane, TMS) and hydrophilic oligo(ethylene glycol) (OEG).…”

3D single-molecule tracking enables direct hybridization kinetics measurement in solution

“…The entropic effects on surface-tethered ssDNA (i.e. reduction in conformational freedom) can also influence duplex stability as single-stranded hairpin formation is favored on the surface 20 . Since these measurement conditions are not physiologically relevant, it is questionable whether those results faithfully reflect the true NA annealing and melting kinetics in solution.…”

Bioelectrochemical Switches for the Quantitative Detection of Antibodies Directly in Whole Blood

“…In the presence of saturating amount of anti-DNP antibody (30 nM), this current falls by ~50% within a few minutes (Figure 2b). The detection limit of this electrochemical switch displays similar subnanomolar detection limit than the optical switch suggesting that surface attachment does not significantly alter the binding-induced structural change taking place upon antibody binding 31 . Of note, this detection limit is well below clinically relevant antibody concentrations 35,36 .…”