Programmable Synthesis and Supramolecular Self-assembly of Stable DNA Nanoparticles

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DNA originates from nature where it carries genetic information for the development and functioning of an organism. As a polymer, single-stranded DNA (ssDNA) can undergo base pairing with complementary strands through the Watson-Crick A-T and G-C base pairs. Therefore, DNA has the inherent advantage of being programmable, in addition to its biodegradability. Indeed, the use of DNA as a building material has initiated broad interest in nanotechnology research not only for applications in the biomedical field including biosensing, diagnostics, and drug delivery, but also for applications in fundamental engineering research including as templates for nanofabrication, in molecular robotics, and for generating plasmonic architectures. My research is motivated by the need for all-nucleotide architectures ranging from hundreds of nanometers to micrometers with high stability and programmability that have potential applications in healthcare and biomedicine. Current DNA nanoparticle systems suffer from fast degradation in biological milieu and low drug-loading efficiency, thus limiting the transformation of these nanotechnologies into clinical usage. Furthermore, the existing bottom-up strategies to modify the surface of DNA nanoparticles, including DNA origami, with polymers are limited to only thin layers due to the low initiation efficiency of chemical synthesis. In contrast, template-free enzymatic DNA synthesis methods can produce polynucleotide chains that contain hundreds to a few thousand bases with a variety of functional groups. This enables the synthesis of polynucleotide brush-modified DNA platforms with programmable features and with high stability towards enzymatic degradation. Current methods to assemble precise DNA supramolecular structures mainly use molecular recognition base-pairing which entails complicated design, often low yields, and the resulting supramolecular mesostructures potentially lack resilience due to the relatively “brittle” linkages. The research described in this dissertation aims to develop toolsets to synthesize all-nucleotide nanoparticle and supramolecular platforms with programmable sizes and shapes, that are significantly more stable against nuclease digestion than currently available DNA self-assembled structures. These platforms will be useful for a variety of applications ranging from drug delivery to biosensing. We exploit the ability of a template-free DNA polymerase —terminal deoxynucleotidyl transferase (TdT)— to catalyze the polymerization of both natural and non-natural nucleotides. This enzymatic reaction which is termed TdT-catalyzed enzymatic polymerization (TcEP) and surface-initiated TdT-catalyzed enzymatic polymerization (SI-TcEP) can proceed in solution and on surfaces, respectively. The research described in this dissertation addresses three specific research aims. In Specific Aim 1, I investigated the formation of DNA amphiphiles containing cytostatic nucleotides, which self-assembled into micelles and showed efficacy for drug delivery applications. I used aptamers to initiate the TcEP reaction to synthesize the polymeric form of FdUTP, which is a cytostatic nucleotide that can treat various types of cancers. I found that a range of therapeutic nucleotide analogs can be added to the aptamer initiator with a degree of polymerization ranging from tens to thousands of bases. Under the same reaction conditions, the degree of polymerization depends on the identity of the nucleotide which affects its affinity with TdT. I then used TdT to add non-natural hydrophobic nucleotides to the 3’ end of the hydrophilic polynucleotide chains, which resulted in ssDNA amphiphiles that were able to self-assemble into micelles. I was able to synthesize monodisperse DNA micelles of ~100 nm, which have a half-life of more than 24 hours in 50% FBS (fetal bovine serum). Furthermore, studies with cancer cell lines that overexpress PTK7 receptors showed that the binding specificity of the aptamers was maintained even after self-assembly and that targeted, micellar delivery resulted in lower cancer cell viability compared to free drugs. In Specific Aim 2, I explored the site-specific functionalization of DNA origami nanostructures (DONs) with polynucleotide brushes using SI-TcEP. We employed the programmability of DNA origami to generate site-specific initiation sites on the DON surfaces. Due to the “living chain growth polycondensation mechanism” of TcEP reactions, the polynucleotide brush modification is highly controllable, and can reach degrees of polymerization exceeding 1 kb. The height of the brush corona and the morphology of the brush-modified DONs observed from AFM imaging were consistent with predictions from coarse-grained simulations. By incubating the samples with nucleases or without divalent cations we found that compared to unmodified counterparts, the nuclease and structural stability of the polynucleotide-modified DONs increased significantly. Because of the programmability of the initiation sites on DNA origami, I was able to exert spatiotemporal control over the digestion of partially modified DONs. To this end, we designed more complex, double-stranded DNA (dsDNA) sequences as the initiators and I investigated the spatiotemporal control of polynucleotide brush growth through the activation and removal of the initiators by restriction enzyme cutting. These strategies enabled the synthesis of bifunctional and asymmetric DONs through sequential growth of polynucleotide brushes. Building on the DON modification strategies, in Specific Aim 3, I studied the self-assembly of DNA origami into supramolecular structures. By harnessing the control over the location and number of initiation sites, I generated spherical supramolecular assemblies composed of hydrophobic patch-functionalized DONs with different sizes. While these supramolecular assemblies have a morphology that is similar to that of polymeric micelles, the corona and the core here are composed of amphiphilic DNA origami nanorods instead of polynucleotides or other, flexible polymer chains. By selectively removing staple strands in the origami structure, the flexibility and morphology of the corona can be controlled. Furthermore, we site-specifically modified the faces of origami nanocubes to enable their entropic self-assembly into two-dimensional chains with different lengths. The assembly behavior of the nanocubes was controlled by the number of faces modified with hydrophobic patches and the area of the hydrophobic patches. The morphology of the resulting supramolecular assemblies, as imaged by tapping mode AFM, agreed with the molecular conformation predictions from coarse-grained simulations. In summary, we developed widely adaptable platforms using enzymatic synthesis methods (i.e., TcEP and SI-TcEP) to generate all-nucleotide nanoparticles and supramolecular assemblies. These platforms are exciting because they are highly programmable, adaptable for different applications, and significantly more resistant to nuclease degradation than single-stranded polynucleotides and DNA origami alone. Compared with current DNA origami supramolecular assembly methods, our method of harnessing hydrophobic interactions is easier to achieve and will likely yield structurally resilient supramolecular architectures. I anticipate that the toolsets developed through my research will advance materials science and bionanotechnology.





Yang, Yunqi (2023). Programmable Synthesis and Supramolecular Self-assembly of Stable DNA Nanoparticles. Dissertation, Duke University. Retrieved from


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