Uncovering the Origins of the Binding Properties of Spherical Nucleic AcidsPublic Deposited
Spherical nucleic acids (SNAs) are a class of structures composed of spherical nanoparticle cores that are densely functionalized with radially oriented, linear DNA. SNAs exhibit properties that are distinct from those of their linear counterparts. These constructs can readily enter cells, evade nuclease degradation, and bind complementary DNA targets with binding constants orders of magnitude larger than those of linear DNA of the same sequence. These fundamentally interesting properties have led to the use of SNAs as medical diagnostic probes, gene regulation agents, and building blocks for programmable colloidal crystal engineering. The unifying property that enables the application of SNAs in all of these fields is their ability to bind complementary DNA sequences with a higher affinity constant than linear DNA. Despite how essential this binding property is to the practical implementation of SNAs, little is known about its origin and how it changes with the SNA architecture, e.g. nanoparticle shape and DNA surface density. ', 'This thesis answers fundamental questions about how the architecture of DNA-nanoparticle conjugates affects their ability to bind complementary DNA, and what considerations should be taken in order to move beyond the spherical architecture of SNAs in order to use optically interesting anisotropic cores. In Chapter 2, the difference between linear DNA hybridization and hybridization to a DNA-functionalized spherical nanoparticle was explored. Hybridization onto DNA-functionalized particles is shown to be enthalpically enhanced due to the structural confinement of the DNA duplex on the surface. This confinement prevents DNA from adopting conformationally unfavorable states, and is further enhanced by high DNA surface density. This understanding challenges the idea that molecular crowding is detrimental to duplex formation, and led us to investigate the effect of DNA surface density and nanoparticle radius of curvature on the types of DNA displacement reactions that govern the efficacy of SNA-based intracellular detection probes. The work in Chapter 3 shows that when a complementary DNA strand is tightly bound to a densely functionalized spherical particle, the propensity for displacement is highly tunable with nanoparticle size. The size of the spherical nanoparticle was varied to demonstrate that complementary DNA strands are more weakly bound on large nanoparticles and displacement is therefore more favorable. The enthalpy of hybridization can be varied by 10-20 kcal/mol simply by changing the SNA architecture (e.g., particle size and DNA surface density) without ever modifying the DNA sequence. This result emphasizes the idea that moving to anisotropic nanoparticle cores, which are larger and have a smaller radius of curvature than spherical nanoparticles, imparts DNA-nanoparticle conjugates with a knob of binding tunability that is independent of the DNA shell. Finally, one limitation to the implementation of anisotropic DNA-functionalized cores is the ability to synthesize certain anisotropic nanoparticles uniformly and in high yields. In Chapter 4, the mechanism of formation of anisotropic gold triangular prisms was studied to understand why reaction conditions favor a broad product distribution. Nanoparticle probes of varying chemical and structural compositions were used to test the hypothesis that prisms form by heterogeneous nucleation. Interestingly, triangular prisms form by homogenous nucleation, in stark contrast to the accepted heterogeneous nucleation pathway. This finding creates a route for the optimization of synthesis yields, because it indicates that to improve product yield and uniformity for some anisotropic nanoparticle shapes, efforts should be placed toward studying reaction conditions that promote homogenous nucleation.', 'This work demonstrates that fundamental knowledge of DNA hybridization thermodynamics on SNAs and nanoparticle synthesis specifically addresses limitations to the construction and implementation of anisotropic DNA-nanoparticle conjugates. Chapter 5 summarizes these findings and points to several future directions for this research area.