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Open Access Publications from the University of California

Scholarly Works (18 results)

  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2017)
A molecular origami method uses double-stranded DNA scaffolds and protein staples to create hybrid nanostructures
    Cover page: Bringing proteins into the fold
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2018)
    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.
      Cover page: Gelbox — An Interactive Simulation Tool for Gel ElectrophoresisCreative Commons 'BY' version 4.0 license
      • Thesis
      • Peer Reviewed
      UC San Francisco Electronic Theses and Dissertations (2018)

      DNA origami is a powerful tool for the production of self-assembled nanostructures and has shown promising applications in a wide range of fields. These structures are typically assembled through a reaction between one long single-stranded DNA (ssDNA) scaffold and many short oligonucleotide staples. In our efforts towards developing applications of DNA origami, we determined that two important properties for scaffold production include the ability to customize its size and sequence, and the ability to produce scaffolds cost effectively at scale. Several in vitro methods exist for producing custom scaffolds. However, because they rely on enzymatic processes, these methods are unreliable at large scale. On the other hand, scalable production of scaffolds based on the M13 bacteriophage genome has been demonstrated. However, the M13 genome will only accommodate insertions up to 2.5 kilobases (kb) reliably, limiting customizability. Likewise, phagemid vectors allow for the production of phage-based scaffolds with larger custom regions, but they still require 2.9 kb of fixed sequence. To overcome these issues, we developed a modified expression system using a novel phagemid, which we call pScaf. pScaf includes a standard M13 origin of replication (M13 ori) as well as a modified M13 ori which behaves as a terminator of ssDNA synthesis, allowing for packaging and export of the intervening sequence as a custom phage particle, while reducing the fixed-sequence region to only 381 bases.

      Here, I discuss the steps taken to develop this novel phagemid vector. I also describe the resulting custom scaffolds which we produced using this vector and demonstrate the usefulness of these scaffolds for applications in DNA origami.

        Cover page: Improving the production of highly custom single-stranded DNA sequences for applications in DNA nanotechnology through the development of novel bacteriophage-based DNA-production methods
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2005)
        We have developed PubNet, a web-based tool that extracts several types of relationships returned by PubMed queries and maps them into networks, allowing for graphical visualization, textual navigation, and topological analysis. PubNet supports the creation of complex networks derived from the contents of individual citations, such as genes, proteins, Protein Data Bank (PDB) IDs, Medical Subject Headings (MeSH) terms, and authors. This feature allows one to, for example, examine a literature derived network of genes based on functional similarity.
          Cover page: PubNet: a flexible system for visualizing literature derived networks
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2018)
          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 those sequences 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 393 bases. We used pScaf to generate new scaffolds ranging in size from 1512 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.
            Cover page: Construction of a novel phagemid to produce custom DNA origami scaffolds.
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2021)
            Correct reconstruction of macromolecular structure by cryo-electron microscopy (cryo-EM) 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. We apply DNA origami to construct molecular goniometers-instruments that precisely orient objects-and use them to dock a DNA-binding protein on a double-helix stage that has user-programmable tilt and rotation angles. We construct goniometers with 14 different stage configurations to orient and visualize the protein just above the cryo-EM grid surface. Each goniometer has a distinct barcode pattern that we use during particle classification to assign angle priors to the bound protein. We use goniometers to obtain a 6.5-Å structure of BurrH, an 82-kDa DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using traditional cryo-EM. Our approach should be adaptable to other DNA-binding proteins as well as small proteins fused to DNA-binding domains.
              Cover page: Molecular goniometers for single-particle cryo-electron microscopy of DNA-binding proteins
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2018)
              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.
                Cover page: Construction of a novel phagemid to produce custom DNA origami scaffoldsCreative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2018)
                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 those sequences 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 393 bases. We used pScaf to generate new scaffolds ranging in size from 1512 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.
                  Cover page: Construction of a novel phagemid to produce custom DNA origami scaffolds
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2005)
                  A direct impediment to the optimal use of online databases is the increasing prevalence, severity, and toll of computer and network security incidents. Funding agencies should set up working groups that can provide essential services such as universal backup, archival storage, and mirroring of community resources, consistent with the key goal of security in academia: to preserve data and results for posterity.
                    Cover page: Network security and data integrity in academia: an assessment and a proposal for large-scale archiving
                    • Article
                    • Peer Reviewed
                    UC San Francisco Previously Published Works (2021)
                    Macrophages destroy pathogens and diseased cells through Fcγ receptor (FcγR)-driven phagocytosis of antibody-opsonized targets. Phagocytosis requires activation of multiple FcγRs, 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.
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