Biology subjects

Douglas, S. M.

Publications and source records attributed to Douglas, S. M..

9 recordsLinked to original sources

bioRxiv2026

Genomic and environmental determinants of circular single-stranded DNA production in engineered E. coli

Single-stranded DNA with hundreds to thousands of bases is a critical reagent in biotechnology. While traditional chemical synthesis is not suitable for efficient production of such long oligonucleotides, M13 bacteriophage offers a scalable pathway towards custom, long, single-stranded DNA production. However, conventional helper plasmid-phagemid systems suffer from batch-to-batch variability and off-target packaging. To address these limitations, we previously engineered eScaf, an E. coli strain with the M13 genome stably integrated into its chromosome. eScaf produces circular, single-stranded DNA with high purity, but yields of only 100 and 200 g of DNA per liter of culture, too low for cost-effective scale-up. Here, we systematically investigate the genomic and environmental determinants of cssDNA yield in eScaf, revealing that cssDNA yield depends on both the number and chromosomal loci of integrated M13 genomes. Combining these insights with optimized media, we achieve ~4 mg/L of cssDNA in shake flasks, a 40-fold improvement. We also evaluate fed-batch bioreactor feeding strategies for cssDNA production using eScaf. Our new strain, eScaf-IS3, enables cost-effective production of cssDNA for high-throughput therapeutic screening, operon-length knock-in donor production, and large-scale DNA origami assembly.

bioengineering↗

bioRxiv2024

Design principles for accurate folding of DNA origami

We describe design principles for accurate folding of three-dimensional DNA origami. To evaluate design rules, we reduced the problem of DNA strand routing to the known problem of shortest-path finding in a weighted graph. To score candidate DNA strand routes we used a thermodynamic model that accounts for enthalpic and entropic contributions of initial binding, hybridization, and DNA loop closure. We encoded and analyzed new and previously reported design heuristics. Using design principles emerging from this analysis, we redesigned and fabricated multiple shapes and compared their folding accuracy using electrophoretic mobility analysis and electron microscopy imaging. We demonstrate accurate folding can be achieved by optimizing staple routes using our model, and provide a computational framework for applying our methodology to any design.

biophysics↗

bioRxiv2024

Engineering an Escherichia coli strain for production of long single-stranded DNA

Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA- guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered E. coli "helper" strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20,724 nucleotides with titers up to 250 g/L following alkaline-lysis purification. The efficacy of our system was confirmed through its application in primary T cell genome modifications and DNA origami folding. The reliability, scalability, and ease of our approach promises to unlock new experimental applications requiring large quantities of long ssDNA.

bioengineering↗

bioRxiv2021

Tight nanoscale clustering of Fc gamma-receptors using DNA origami promotes phagocytosis

Macrophages destroy pathogens and diseased cells through Fc{gamma} receptor (Fc{gamma}R)-driven phagocytosis of antibody-opsonized targets. Phagocytosis requires activation of multiple Fc{gamma}Rs, but the mechanism controlling the threshold for response is unclear. We developed a DNA origami-based engulfment system that allows precise nanoscale control of the number and spacing of ligands. When the number of ligands remains constant, reducing ligand spacing from 17.5 nm to 7 nm potently enhances engulfment, primarily by increasing efficiency of the engulfment-initiation process. Tighter ligand clustering increases receptor phosphorylation, as well as proximal downstream signals. Increasing the number of signaling domains recruited to a single ligand-receptor complex was not sufficient to recapitulate this effect, indicating that clustering of multiple receptors is required. Our results suggest that macrophages use information about local ligand densities to make critical engulfment decisions, which has implications for the mechanism of antibody-mediated phagocytosis and the design of immunotherapies.

cell biology↗

bioRxiv2021

DNA origami patterning of synthetic T cell receptors reveals spatial control of the sensitivity and kinetics of signal activation

T cell receptor clustering plays a key role in triggering cell activation, but the relationship between the spatial configuration of clusters and elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami-based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by non-stimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands and translates that information into distinct signaling dynamics, and provide insights into engineering new immunotherapies.

cell biology↗

bioRxiv2020

Molecular goniometers for single-particle cryo-EM of DNA-binding proteins

Correct reconstruction of macromolecular structure by cryo-electron microscopy relies on accurate determination of the orientation of single-particle images. For small (<100 kDa) DNA-binding proteins, obtaining particle images with sufficiently asymmetric features to correctly guide alignment is challenging. DNA nanotechnology was conceived as a potential tool for building host nanostructures to prescribe the locations and orientations of docked proteins. We used DNA origami to construct molecular goniometers--instruments to precisely orient objects--to dock a DNA-binding protein on a double-helix stage that has user-programmable tilt and rotation angles. Each protein orientation maps to a distinct barcode pattern specifying particle classification and angle assignment. We used goniometers to obtain a 6.5 [A] structure of BurrH, an 82-kDa DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using classical cryo-EM. Our approach should be adaptable for other DNA-binding proteins, and a wide variety of other small proteins, by fusing DNA binding domains to them.

biophysics↗

bioRxiv2019

Amino and PEG-Amino Graphene Oxide Grids Enrich and Protect Samples for High-resolution Single Particle Cryo-electron Microscopy

Cryo-EM samples prepared using the traditional methods often suffer from too few particles, poor particle distribution, or strongly biased orientation, or damage from the air-water interface. Here we report that functionalization of graphene oxide (GO) coated grids with amino groups concentrates samples on the grid with improved distribution and orientation. By introducing a PEG spacer, particles are kept away from both the GO surface and the air-water interface, protecting them from potential denaturation.

biophysics↗

bioRxiv2018

Gelbox - An Interactive Simulation Tool for Gel Electrophoresis

Gel electrophoresis enables separation and visualization of biomolecules such as DNA, RNA, or proteins. Like many powerful tools, mastering the use of gels and making the information gained from them productive, can be dificult. Gelbox is a simulation tool that helps users understand how changing experimental input parameters can affect the data output from gel electrophoresis. Our simulation model handles a wide range of settings, including many \"suboptimal\" values that may be useful for troubleshooting common mistakes made by novices.

molecular biology↗

bioRxiv2018

Construction of a novel phagemid to produce custom DNA origami scaffolds

DNA origami, a method for constructing nanoscale objects, relies on a long single strand of DNA to act as the \"scaffold\" to template assembly of numerous short DNA oligonucleotide \"staples\". The ability to generate custom scaffold sequences can greatly benefit DNA origami design processes. Custom scaffold sequences can provide better control of the overall size of the final object and better control of low-level structural details, such as locations of specific base pairs within an object. Filamentous bacteriophages and related phagemids can work well as sources of custom scaffold DNA. However, scaffolds derived from phages require inclusion of multi-kilobase DNA sequences in order to grow in host bacteria, and thus cannot be altered or removed. These fixed-sequence regions constrain the design possibilities of DNA origami. Here we report the construction of a novel phagemid, pScaf, to produce scaffolds that have a custom sequence with a much smaller fixed region of only 381 bases. We used pScaf to generate new scaffolds ranging in size from 1,512 to 10,080 bases and demonstrated their use in various DNA origami shapes and assemblies. We anticipate our pScaf phagemid will enhance development of the DNA origami method and its future applications.

biophysics↗