Biophysical and Structural Characterization of the Parallel-Stranded d(CGA) Triplet Repeat Motif

Deoxyribonucleic acid (DNA) has been repurposed as a building block in the construction of nanoscale assemblies. The biocompatibility, stability, programmability, structural predictability, and ability to self-assemble inherent to DNA has been leveraged to design various 2D and 3D DNA-based architectures. However, such architectures are commonly restricted by the structural rigidity and stability of the Watson-Crick duplex. Non-canonical DNA interactions can be incorporated to overcome this limitation by retaining the favorable characteristics of DNA while offering structural versatility beyond the constraints of Watson-Crick interactions. Another desirable functional advantage of non-canonical interactions are their sensitivities to the local environment, including cations, salt concentration, or pH, which allow them to undergo predictable structural changes in response to environmental perturbations. The d(CGA) triplet repeat motif is an example of a structurally dynamic non-canonical DNA motif that can transition from parallel-stranded homo-base paired duplex to anti parallel unimolecular hairpin in a pH-dependent manner. This dissertation describes the biophysical and structural characterization of the non-canonical d(CGA) repeat motif and related sequence variants. Thermodynamic parameters obtained from UV absorbance melting curves show that the structural transition resulting from decreasing the pH is accompanied by a significant energetic stabilization as hairpin structures are converted to parallel-stranded duplexes. Additionally, nuclease resistance against double strand-specific nucleases in the parallel-stranded form suggests that this motif may offer unique advantages for cellular applications. CD spectroscopy based kinetic analysis reveals that the time scale of the transition between structural forms is highly dependent upon the direction of the structural change (hairpin to parallel duplex or parallel duplex to hairpin). Biophysical characterization is complimented by the structure determination of four unique d(CGA)-based parallel-stranded duplexes across two crystal structures. Structural analysis confirms the robust structural predictability and defines the specific structural features of d(CGA) triplets in the parallel stranded form. Finally, we explored 3D DNA crystals containing parallel-stranded d(GGA) triplet repeats as a new platform for drug delivery. Together, the data presented within this dissertation will provide the foundation for the rational incorporation of non canonical based parallel-stranded interactions, specifically the d(CGA) motif, into the design of DNA based nanoarchitectures