Biology subjects

Stephanopoulos, N.

Publications and source records attributed to Stephanopoulos, N..

8 recordsLinked to original sources

bioRxiv2026

Engineered mRNA nanostructures expand the design space of mRNA therapeutics through programmable protein expression and immune stimulation

Messenger RNA (mRNA) therapeutics have transformed vaccination and protein replacement strategies, yet efforts to improve their performance have focused largely on sequence engineering, nucleotide modification, and delivery vehicles. Here we show that mRNA function can be controlled by rational design of higher-order RNA architectures. We develop self-assembling mRNA origami (mRNA-OG), a class of unimolecular RNA nanostructures that encode protein-coding sequences within higher-order, programmable, and compact nucleic acid architectures. Using computational design and experimental validation, we demonstrate that mRNA-OG folds into well-defined nanostructures while remaining translationally competent in mammalian cells. Although folded mRNA-OG recruits ribosomes comparably to unfolded constructs, it produces lower protein output, indicating that RNA architecture can directly influence translational efficiency. The compact geometry of mRNA-OG also enhances encapsulation by cationic lipid delivery systems, suggesting a structural route to improved cargo packaging. Beyond its effects on translation, mRNA architecture modulates innate immune recognition. In primary human dendritic cells, folded and unfolded mRNA-OG elicit distinct cytokine programs and differential activation of stress-response pathways, including a modest induction of the integrated stress response that is not fully explained by canonical PKR signaling. Our results establish programmable structure as a new design parameter for mRNA therapeutics that can affect its functionality, delivery properties, and immune sensing.

bioengineering↗

bioRxiv2023

Site-specific arrangement and structure determination of minor groove binding molecules in self-assembled three-dimensional DNA crystals

The structural analysis of guest molecules in rationally designed and self-assembling DNA crystals has proven elusive since its conception. Oligonucleotide frameworks provide an especially attractive route towards studying DNA-binding molecules by using three-dimensional lattices with defined sequence and structure. In this work, we site-specifically position a suite of minor groove binding molecules, and solve their structures via x-ray crystallography, as a proof-of-principle towards scaffolding larger guest species. Two crystal motifs were used to precisely immobilize the molecules DAPI, Hoechst, and netropsin at defined positions in the lattice, allowing us to control occupancy within the crystal. We also solved the structure of a three-ring imidazole-pyrrole-pyrrole polyamide molecule, which sequence-specifically packs in an anti-parallel dimeric arrangement within the minor groove. Finally, we engineered a crystal designed to position both netropsin and the polyamide at two distinct locations within the same lattice. Our work elucidates the design principles for the spatial arrangement of functional guests within lattices and opens new potential opportunities for the use of DNA crystals to display and structurally characterize small molecules, peptides, and ultimately proteins of unknown structure.

bioengineering↗

bioRxiv2023

Construction and reconfiguration of dynamic DNA origami assemblies with coiled-coil patches and patterns

DNA origami nanodevices achieve programmable structure and tunable mechanical and dynamic properties by leveraging the sequence specific interactions of nucleic acids. Previous advances have also established DNA origami as a useful building block to make well-defined micron-scale structures through hierarchical self-assembly, but these efforts have largely leveraged the structural features of DNA origami. The tunable dynamic and mechanical properties also provide an opportunity to make assemblies with adaptive structure and properties. Here we report the integration of DNA origami hinge nanodevices and coiled-coil peptides into hybrid reconfigurable assemblies. With the same dynamic device and peptide interaction, we make multiple higher order assemblies by organizing clusters of peptides (i.e. patches) or arranging single peptides (i.e. patterns) on the surfaces of DNA origami to control the relative orientation of devices. We use coiled-coil interactions to construct circular and linear assemblies whose structure and mechanical properties can be modulated with DNA-based actuation. Actuation of linear assemblies leads to micron scale motions and [~]2.5-10-fold increase in bending stiffness. Our results provide a foundation for stimulus responsive hybrid assemblies that can adapt their structure and properties in response to nucleic acid, peptide, protein, or other triggers.

bioengineering↗

bioRxiv2023

High-affinity binding to the SARS-CoV-2 spike trimer by a nanostructured, trivalent protein-DNA synthetic antibody

Multivalency enables nanostructures to bind molecular targets with high affinity. Although IgG antibodies can be generated against a wide range of antigens, their shape and size cannot be tuned to match a given target. DNA nanotechnology provides an attractive approach for designing customized multivalent scaffolds due to the addressability and programmability of the nanostructure shape and size. Here, we use computational simulation to guide the design and synthesis of a DNA nanostructure-based synthetic antibody ("nano-synbody"). The nano-synbody is comprised of a three-helix bundle DNA nanostructure with three identical arms terminating in a mini-binder protein that targets the SARS-CoV-2 spike protein. The structure was designed to match the valence and distance between the three receptor binding domains (RBDs) in the spike trimer, in order to enhance binding through avidity effects. Moreover, the design allowed for the display of one, two, or three protein-displaying arms, thereby systematically probing the effect of multivalency on binding affinity. The binding strength of the nano-synbody increased with the increasing number of arms, yielding 11.2 pM affinity ([~]100-fold enhancement over monovalent binding) for the wild-type spike protein for the three-arm structure. Moreover, the multivalency was able to yield a 95 pM affinity for the Omicron variant, a mutant against which the monovalent protein was ineffective. The nano-synbody could also block infection of a spike protein-bearing pseudovirus, and similarly demonstrated effective inhibition of the Omicron variant when trimerized. The structure of the three-arm nano-synbody bound to the Omicron variant spike trimer was solved by negative-stain transmission electron microscopy reconstruction, and shows the protein-DNA nanostructure with all three arms bound to the RBD domains, confirming the intended trivalent attachment. Finally, nano-synbody binding could be reversed by removing one, two, or three arms in a programmable fashion, via toehold-mediated strand displacement. The ability to tune the size and shape of the nano-synbody, as well as its potential ability to attach (and then remove) two or more different binding ligands, will enable the high-affinity binding of a range of proteins, and pave the way towards their manipulation using DNA-based nano-robotic devices.

bioengineering↗

bioRxiv2023

DNA Nanostructure-Guided Assembly of Proteins into Programmable Shapes

The development of methods to synthesize protein oligomers with precisely controlled number and configuration of components will enable artificial protein complexes and nanostructures with diverse biological and medical applications. Using DNA hybridization to link protein-DNA conjugates provides for a programmability that can be difficult to achieve with other methods, such as engineering multiple orthogonal protein-protein interfaces. However, it is still difficult to construct well-defined protein assemblies, and the challenge is magnified if only a single type of building block is used. To overcome this hurdle, we use a DNA origami as an "assembler" to guide the linking of protein-DNA conjugates using a series of oligonucleotide hybridization and displacement operations. We constructed several isomeric protein nanostructures on a DNA origami platform by using a C3-symmetric building block comprised of a protein trimer modified with DNA handles. By changing the number of protein-DNA building blocks attached to the origami, and the sequence of the linking and displacement strands added, we were able to produce dimers, two types of trimer structures, and three types of tetramer assemblies. Our approach expands the scope for the precise design and assembly of protein-based nanostructures, and will enable the formulation of functional protein complexes with stoichiometric and geometric control.

bioengineering↗

bioRxiv2023

Using dynamic biomaterials to study the temporal role of osteogenic growth peptide during osteogenesis

The extracellular matrix is a highly dynamic environment, and the precise temporal presentation of biochemical signals is critical for regulating cell behavior during development, healing, and disease progression. To mimic this behavior, we developed a modular DNA-based hydrogel platform to enable independent and reversible control over the immobilization of multiple biomolecules during in vitro cell culture. We combined reversible DNA handles with a norbornene-modified hyaluronic acid hydrogel to orthogonally add and remove multiple biomolecule-DNA conjugates at user-defined timepoints. We demonstrated that the persistent presentation of the cell adhesion peptide RGD was required to maintain cell spreading on hyaluronic acid hydrogels. Further, we discovered the delayed presentation of osteogenic growth peptide (OGP) increased alkaline phosphatase activity compared to other temporal variations. This finding is critically important when considering the design of OGP delivery approaches for bone repair. More broadly, this platform provides a unique approach to tease apart the temporal role of multiple biomolecules during development, regeneration, and disease progression.

bioengineering↗

bioRxiv2023

Calcium-triggered DNA-mediated membrane fusion in synthetic cells

In cells, membrane fusion is mediated by SNARE proteins, whose activities are calcium-dependent. While several non-native membrane fusion mechanisms have been demonstrated, few can respond to external stimuli. Here, we develop a calcium-triggered DNA-mediated membrane fusion strategy where fusion is regulated using surface-bound PEG chains that are cleavable by the calcium-activated protease calpain-1.

synthetic biology↗

bioRxiv2020

Uncovering temporally sensitive targeting motifs for traumatic brain injury via phage display

The heterogeneous injury pathophysiology of traumatic brain injury (TBI) is a barrier to developing highly sensitive and specific diagnostic tools. Embracing neural injury complexity is critical for the development and advancement of diagnostics and therapeutics. The current study employs a unique discovery pipeline to identify targeting motifs that recognize specific phases of TBI pathology. This pipeline entails in vivo biopanning with a domain antibody (dAb) phage display library, next generation sequencing (NGS) analysis, and peptide synthesis. Here, we identify targeting motifs based on the HCDR3 structure of dAbs for acute (1 day) and subacute (7 days) post-injury timepoints using a mouse controlled cortical impact model. Their bioreactivity was validated using immunohistochemistry and candidate target epitopes were identified via immunoprecipitation-mass spectrometry. The acute targeting motif recognizes targets associated with metabolic and mitochondrial dysfunction whereas the subacute motif was largely associated with neurodegenerative processes. This phage display biomarker discovery pipeline for TBI successfully achieved discovery of temporally specific TBI targeting motif/epitope pairs that will advance the TBI diagnostics and therapeutics.

bioengineering↗