Liquid-liquid phase separation (LLPS) is a thermodynamic process by which a mixture of solutions de-mixes into separate, coexisting phases. In cells, macromolecules such as proteins and nucleic acids can undergo LLPS to form membraneless structures known as biomolecular condensates. These condensates are usually liquid-like droplets highly concentrated in the species they are composed of. Condensates are crucial to many cellular processes and functions, such as protein assembly, gene regulation, subcellular organization, storage, and stress response. On a larger scale, condensates are also implicated in various neurodegenerative diseases, such as Huntington's and Alzheimer's disease.
\r\n\r\nDNA condensates are easy to engineer due to the programmable nature of the molecule. DNA nanostars are multi-armed junctions with double-stranded arms and typically single-stranded, palindromic overhangs or 'sticky ends' that allow for transient interactions between the molecules. They can phase-separate to form liquid-like droplets at the microscopic scale. Centrifugation of a high concentration solution of nanostars coalesces condensates into a macroscopic liquid that is visible to the naked eye.
\r\n\r\nIn biology, one of the key functions of condensates is to act as compartments and localize various molecules, such as enzymes and nucleic acids. Our goal is to engineer macroscopic DNA liquids to similarly act as compartments that can separate multiple biomolecular targets. We propose that these macroscopic DNA liquids are a potentially novel method for multiplexed separation that is fast, simple to use, and biocompatible with various high-value targets, such as protein therapeutics.
\r\n\r\nWe designed multiple immiscible DNA liquids of different densities to act as macroscopic compartments. We present a set of design principles for engineering nanostars to create liquid layers in a microcentrifuge tube. We show via UV absorbance measurements how various nanostar features, such as arm length, sticky end strength, and valency, affect liquid phase density, which determines the order of layering between liquids. We further show that the interface quality between pairwise liquids is determined by a combination of density differences between liquids and the orthogonality of nanostar sticky ends, and we devise a metric to calculate this orthogonality. Using these design principles, we are currently able to create up to five orthogonal liquid layers in a tube.
\r\n\r\nWith these DNA liquid layers, we envision separation to be a two step process. The first step is to localize specific target biomolecules into these layers. We demonstrate localization of oligos in a multilayer DNA liquid system by modifying nanostars with tag regions complementary to the target strand. We also demonstrate localization of fluorescent streptavidin to a single DNA liquid layer by modifying nanostars with a streptavidin-binding aptamer. The second step is to release targets from the liquid layers so that they can be collected for downstream use. After localization of the aforementioned targets, we added strands complementary to either the tag region or the aptamer. This causes targets to be displaced from their binding moiety and released into the supernatant.
", "doi": "10.7907/3639-5v91", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17203", "collection": "thesis", "collection_id": "17203", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05052025-211734908", "type": "thesis", "title": "Enriching Architectures for Biosensing and Motor-Filament Systems Through the Programmability of DNA", "author": [ { "family_name": "Guareschi", "given_name": "Matteo Michele", "orcid": "0000-0002-5197-3158", "clpid": "Guareschi-Matteo-Michele" } ], "thesis_advisor": [ { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Qian", "given_name": "Lulu", "orcid": "0000-0003-4115-2409", "clpid": "Qian-Lulu" }, { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "orcid": "0000-0002-1653-3202", "clpid": "Rothemund-P-W-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Since its inception, the field of DNA nanotechnology has focused on studying the fundamental behaviors and capabilities of engineered nucleic acids. A deep understanding of this toolkit has enabled advancements in several fields, for research tools and in translational applications. Together with its programmability and nanometric resolution, the great promise of DNA nanotechnology lies in the incorporation of structure and function in a single molecule. In this work, we show how these advantages can be leveraged to expand the capabilities of two different systems: a sensor for biomarkers and a motor-filament architecture. During our exploration, we also discover and work to overcome some of the less obvious limitations of the technology, shining light on more foundational questions.
\r\n\r\nWe demonstrate an electrochemical biosensor based on a DNA origami that can detect and quantify nucleic acids and proteins in a package easily adaptable to different analytes by simply replacing the binder molecules. Upon target binding, the structure undergoes a large conformational change, bringing a multitude of redox reporters to the electrode surface where an electric current can be measured. The high number of reporter molecules on a single detector results in improved signal gain per binding event, allowing for the detection of low analyte concentrations, while the conformational change yields an unprecedented gain between the off and on state. We demonstrate how the system can be readily adapted to different analyte molecules and reused over several cycles to analyze multiple samples. We then run simulations of the detector molecule to understand structural deformations intrinsic to this design, in order to optimize the number and placement of the redox reporters. We discover and investigate a phenomenon that causes significant curling of the DNA origami, possibly limiting the contribution of many of the reporter molecules. We explore experimental directions to mitigate the issue by changing the configuration of the redox molecules and by designing stiffer sensors.
\r\n\r\nWe then set out to integrate DNA origami-based nanostructures with an engineered dynein protein that can bind to and kick double-stranded DNA instead of tubulin. Motor-filament architectures have been studied as the main mechanism for cellular transport and as a system that can exhibit mesoscopic active matter behaviors in biology, but the relative difficulty of engineering microtubules has hindered the exploration of their properties. The high-resolution programmability of DNA nanostructures makes them prime candidates to overcome this obstacle and this study has been enabled by the recent development of new protein motors where the tubulin binding domain is replaced by a DNA binding domain. We first look at DNA nanotubes, structures that resemble microtubules, but that retain a level of programmability that is typical of DNA nanotechnology. By exploiting the DNA strand displacement technique, we incorporate machinery that enables new behaviors, with a focus on different ways to turn gliding on and off by stopping the DNA nanotubes.
\r\n\r\nWe then turn our focus to more complex gliders designed with DNA origami. We explore the space of DNA origami polymers in order to assemble superstructures that can be detected under light microscopy, encountering again issues of deformations due to the addition of overhangs. We then assess the gliding capabilities of DNA origami, designing ways to incorporate motor binding sequences on them, but we find that DNA origami sticks nonspecifically to the engineered dynein motors. After testing several different hypotheses, we gather evidence that this interaction might be caused by the large sequence variability of the scaffold strand in DNA origami, coupled with the recognition of spurious binding sequences by the motor proteins.
", "doi": "10.7907/fmhp-r892", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:13753", "collection": "thesis", "collection_id": "13753", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312020-234931669", "type": "thesis", "title": "Using DNA Origami to Create Hybrid Nanophotonic Architectures for Single-Photon Emitters", "author": [ { "family_name": "Mitskovets", "given_name": "Anna", "orcid": "0000-0003-1967-5334", "clpid": "Mitskovets-Anna" } ], "thesis_advisor": [ { "family_name": "Atwater", "given_name": "Harry Albert", "clpid": "Atwater-H-A" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "clpid": "Rothemund-P-W-K" } ], "thesis_committee": [ { "family_name": "Gopinath", "given_name": "Ashwin", "clpid": "Gopinath-Ashwin" }, { "family_name": "Atwater", "given_name": "Harry Albert", "clpid": "Atwater-H-A" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "clpid": "Rothemund-P-W-K" }, { "family_name": "Vahala", "given_name": "Kerry J.", "clpid": "Vahala-K-J" }, { "family_name": "Faraon", "given_name": "Andrei", "clpid": "Faraon-A" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The limitations in physical dimensions of silicon transistors give us a stimulus to explore alternative systems for better computational performance. The most promising system that received a lot of attention in the past few years is a quantum computer. Ideally, a nanophotonic quantum computer would consist of hundreds of single-photon emitters, optical or plasmonic resonators, optical waveguides and interconnects. The main difficulty in large-scale production of such quantum photonic networks is the integration and deterministic coupling of single-photon sources to photonic elements.
\r\n\r\nIn the first part of this thesis, we utilize spontaneous parametric down-conversion to create correlated pairs of indistinguishable photons. These photons are generated by bismuth borate nonlinear crystal and then are coupled to a photonic chip where they interfere at directional couplers to produce a path-entangled state. Our photonic chip consists of waveguides, directional couplers, and a single Mach-Zender interferometer with a thermo-optic phase shifter. When a part of the waveguide connecting directional couplers is replace with a plasmonic waveguide, quantum state of photons is converted to plasmonic state. Here we report a measurement of path entanglement between surface plasmons with 95% contrast, confirming that a path-entangled state can indeed survive without measurable decoherence. Our measurement suggests that elastic scattering mechanisms of the type that might cause pure dephasing in plasmonic systems must be weak enough not to significantly perturb the state of the metal under the experimental conditions we investigated.
\r\n\r\nThe second part of this work is dedicated to the study of a novel DNA origami self-assembly technique for creating hybrid nanophotonic architectures to create single-photon emitters. DNA origami is a modular platform for the combination of molecular and colloidal components to create optical, electronic, and biological devices. We present a DNA origami molecule that can be deterministically positioned on a silicon chip within 3.2\u00b0 alignment. Orientation is absolute (all degrees of freedom are speci\ufb01ed) and arbitrary (every molecule\u2019s orientation is independently speci\ufb01ed). The use of orientation to optimize device performance is shown by aligning \ufb02uorescent emission dipoles within microfabricated optical cavities. Large-scale integration is demonstrated via an array of 3,456 DNA origami with 12 distinct orientations, which indicates the polarization of the excitation light. Following this experiment, we explore how many molecular emitters can be coupled to this DNA origami shape and discover interesting interactions between ssDNA extensions that can cause origami to fold along its seam. Finally, we examine DNA origami self-assembly methods that can be used to deterministically couple single-photon emitters to resonators in order to decrease pure-dephasing rates and increase indistinguishability of emitted photons.
", "doi": "10.7907/kqaj-ex65", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:7772", "collection": "thesis", "collection_id": "7772", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302013-123041371", "type": "thesis", "title": "Beyond Watson and Crick: Programming the Self-Assembly and Reconfiguration of DNA Nanostructures Based on Stacking Interactions", "author": [ { "family_name": "Woo", "given_name": "Sungwook", "clpid": "Woo-Sungwook" } ], "thesis_advisor": [ { "family_name": "Rothemund", "given_name": "Paul W. K.", "clpid": "Rothemund-P-W-K" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "clpid": "Winfree-E" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "clpid": "Rothemund-P-W-K" }, { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Fygenson", "given_name": "Deborah K.", "clpid": "Fygenson-D-K" }, { "family_name": "Andersen", "given_name": "Ebbe S.", "clpid": "Andersen-E-S" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Life is the result of the execution of molecular programs: like how an embryo is fated to become a human or a whale, or how a person\u2019s appearance is inherited from their parents, many biological phenomena are governed by genetic programs written in DNA molecules. At the core of such programs is the highly reliable base pairing interaction between nucleic acids. DNA nanotechnology exploits the programming power of DNA to build artificial nanostructures, molecular computers, and nanomachines. In particular, DNA origami\u2014which is a simple yet versatile technique that allows one to create various nanoscale shapes and patterns\u2014is at the heart of the technology. In this thesis, I describe the development of programmable self-assembly and reconfiguration of DNA origami nanostructures based on a unique strategy: rather than relying on Watson-Crick base pairing, we developed programmable bonds via the geometric arrangement of stacking interactions, which we termed stacking bonds. We further demonstrated that such bonds can be dynamically reconfigurable.
\r\n\r\nThe first part of this thesis describes the design and implementation of stacking bonds. Our work addresses the fundamental question of whether one can create diverse bond types out of a single kind of attractive interaction\u2014a question first posed implicitly by Francis Crick while seeking a deeper understanding of the origin of life and primitive genetic code. For the creation of multiple specific bonds, we used two different approaches: binary coding and shape coding of geometric arrangement of stacking interaction units, which are called blunt ends. To construct a bond space for each approach, we performed a systematic search using a computer algorithm. We used orthogonal bonds to experimentally implement the connection of five distinct DNA origami nanostructures. We also programmed the bonds to control cis/trans configuration between asymmetric nanostructures.
\r\n\r\nThe second part of this thesis describes the large-scale self-assembly of DNA origami into two-dimensional checkerboard-pattern crystals via surface diffusion. We developed a protocol where the diffusion of DNA origami occurs on a substrate and is dynamically controlled by changing the cationic condition of the system. We used stacking interactions to mediate connections between the origami, because of their potential for reconfiguring during the assembly process. Assembling DNA nanostructures directly on substrate surfaces can benefit nano/microfabrication processes by eliminating a pattern transfer step. At the same time, the use of DNA origami allows high complexity and unique addressability with six-nanometer resolution within each structural unit.
\r\n\r\nThe third part of this thesis describes the use of stacking bonds as dynamically breakable bonds. To break the bonds, we used biological machinery called the ParMRC system extracted from bacteria. The system ensures that, when a cell divides, each daughter cell gets one copy of the cell\u2019s DNA by actively pushing each copy to the opposite poles of the cell. We demonstrate dynamically expandable nanostructures, which makes stacking bonds a promising candidate for reconfigurable connectors for nanoscale machine parts.
", "doi": "10.7907/4NZK-XK58", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" } ]