The Role of Structural Enthalpy in Spherical Nucleic Acid Hybridization

Abstract

DNA hybridization onto DNA-functionalized nanoparticle surfaces (e.g., in the form of a spherical nucleic acid (SNA)) is known to be enhanced relative to hybridization free in solution. Surprisingly, via isothermal titration calorimetry, we reveal that this enhancement is enthalpically, as opposed to entropically, dominated by ∼20 kcal/mol. Coarse-grained molecular dynamics simulations suggest that the observed enthalpic enhancement results from structurally confining the DNA on the nanoparticle surface and preventing it from adopting enthalpically unfavorable conformations like those observed in the solution case. The idea that structural confinement leads to the formation of energetically more stable duplexes is evaluated by decreasing the degree of confinement a duplex experiences on the nanoparticle surface. Both experiment and simulation confirm that when the surface-bound duplex is less confined, i.e., at lower DNA surface density or at greater distance from the nanoparticle surface, its enthalpy of formation approaches the less favorable enthalpy of duplex formation for the linear strand in solution. This work provides insight into one of the most important and enabling properties of SNAs and will inform the design of materials that rely on the thermodynamics of hybridization onto DNA-functionalized surfaces, including diagnostic probes and therapeutic agents.

Figure 1

(A) van ’t Hoff plots from which thermodynamic constants are extracted for binding between complementary linear strands and either linear 12-mer DNA (blue) or SNAs (red). (B) Comparison of enthalpic gain and entropic cost derived from the van’t Hoff plots. (C) Isothermal titration calorimetry of 12-mer DNA duplex hybridization free in solution and (D) 12-mer DNA duplex hybridization on SNAs functionalized with ~46 strands per particle. Upper panel: differential heating power ΔP vs time. Lower panel: integrated heats of reaction Q vs molar ratio.

Figure 2

(A) Coarse-grained MD simulations of 12-mer DNA before (E_U) and after (E_H) duplex formation free in solution and on an SNA functionalized with 46 strands. (B) Comparison of simulation-derived enthalpies of hybridization for a duplex formed free in solution and one formed on an SNA. (C) Breakdown of contributions to the enthalpy of hybridization.

Figure 3

Enthalpy of hybridization onto SNAs for the first four complementary strands as a function of (A) DNA surface density and (B) linker length. Linear DNA hybridization enthalpy (blue) is provided for comparison.

Scheme 1

Comparison of Complementary DNA Hybridization to Either Linear DNA (left) or Spherical Nucleic Acids (SNAs, right) to Elucidate the Thermodynamic Origin of Binding Enhancement Observed on SNAs