Optimizing the strain engineering process for industrial-scale production of bio-based molecules.

Biomanufacturing could contribute as much as ${\$}$30 trillion to the global economy by 2030. However, the success of the growing bioeconomy depends on our ability to manufacture high-performing strains in a time- and cost-effective manner. The Design-Build-Test-Learn (DBTL) framework has proven to be an effective strain engineering approach.

3D RNA-scaffolded wireframe origami.

Hybrid RNA:DNA origami, in which a long RNA scaffold strand folds into a target nanostructure via thermal annealing with complementary DNA oligos, has only been explored to a limited extent despite its unique potential for biomedical delivery of mRNA, tertiary structure characterization of long RNAs, and fabrication of artificial ribozymes. Here, we investigate design principles of three-dimensional wireframe RNA-scaffolded origami rendered as polyhedra composed of dual-duplex edges. We computationally design, fabricate, and characterize tetrahedra folded from an EGFP-encoding messenger RNA and de Bruijn sequences, an octahedron folded with M13 transcript RNA, and an octahedron and pentagonal bipyramids folded with 23S ribosomal RNA, demonstrating the ability to make diverse polyhedral shapes with distinct structural and functional RNA scaffolds.

Disrupting the ArcA Regulatory Network Amplifies the Fitness Cost of Tetracycline Resistance in Escherichia coli.

There is an urgent need for strategies to discover secondary drugs to prevent or disrupt antimicrobial resistance (AMR), which is causing >700,000 deaths annually. Here, we demonstrate that tetracycline-resistant (Tet) Escherichia coli undergoes global transcriptional and metabolic remodeling, including downregulation of tricarboxylic acid cycle and disruption of redox homeostasis, to support consumption of the proton motive force for tetracycline efflux. Using a pooled genome-wide library of single-gene deletion strains, at least 308 genes, including four transcriptional regulators identified by our network analysis, were confirmed as essential for restoring the fitness of Tet E.

Random access DNA memory using Boolean search in an archival file storage system.

DNA is an ultrahigh-density storage medium that could meet exponentially growing worldwide demand for archival data storage if DNA synthesis costs declined sufficiently and if random access of files within exabyte-to-yottabyte-scale DNA data pools were feasible. Here, we demonstrate a path to overcome the second barrier by encapsulating data-encoding DNA file sequences within impervious silica capsules that are surface labelled with single-stranded DNA barcodes. Barcodes are chosen to represent file metadata, enabling selection of sets of files with Boolean logic directly, without use of amplification.

Role of nanoscale antigen organization on B-cell activation probed using DNA origami.

Vaccine efficacy can be increased by arraying immunogens in multivalent form on virus-like nanoparticles to enhance B-cell activation. However, the effects of antigen copy number, spacing and affinity, as well as the dimensionality and rigidity of scaffold presentation on B-cell activation remain poorly understood. Here, we display the clinical vaccine immunogen eOD-GT8, an engineered outer domain of the HIV-1 glycoprotein-120, on DNA origami nanoparticles to systematically interrogate the impact of these nanoscale parameters on B-cell activation in vitro.

Conformational Dynamics and Cooperativity Drive the Specificity of a Protein-Ligand Interaction.

Molecular recognition is critical for the fidelity of signal transduction in biology. Conversely, the disruption of protein-protein interactions can lead to disease. Thus, comprehension of the molecular determinants of specificity is essential for understanding normal biological signaling processes and for the development of precise therapeutics.

Programming Structured DNA Assemblies to Probe Biophysical Processes.

Structural DNA nanotechnology is beginning to emerge as a widely accessible research tool to mechanistically study diverse biophysical processes. Enabled by scaffolded DNA origami in which a long single strand of DNA is weaved throughout an entire target nucleic acid assembly to ensure its proper folding, assemblies of nearly any geometric shape can now be programmed in a fully automatic manner to interface with biology on the 1-100-nm scale. Here, we review the major design and synthesis principles that have enabled the fabrication of a specific subclass of scaffolded DNA origami objects called wireframe assemblies.

Bioproduction of pure, kilobase-scale single-stranded DNA.

Scalable production of kilobase single-stranded DNA (ssDNA) with sequence control has applications in therapeutics, gene synthesis and sequencing, scaffolded DNA origami, and archival DNA memory storage. Biological production of circular ssDNA (cssDNA) using M13 addresses these needs at low cost. However, one unmet goal is to minimize the essential protein coding regions of the exported DNA while maintaining its infectivity and production purity to produce sequences less than 3,000 nt in length, relevant to therapeutic and materials science applications.

Autonomously designed free-form 2D DNA origami.

Scaffolded DNA origami offers the unique ability to organize molecules in nearly arbitrary spatial patterns at the nanometer scale, with wireframe designs further enabling complex 2D and 3D geometries with irregular boundaries and internal structures. The sequence design of the DNA staple strands needed to fold the long scaffold strand to the target geometry is typically performed manually, limiting the broad application of this materials design paradigm. Here, we present a fully autonomous procedure to design all DNA staple sequences needed to fold any free-form 2D scaffolded DNA origami wireframe object.

Automated Sequence Design of 3D Polyhedral Wireframe DNA Origami with Honeycomb Edges.

3D polyhedral wireframe DNA nanoparticles (DNA-NPs) fabricated using scaffolded DNA origami offer complete and independent control over NP size, structure, and asymmetric functionalization on the 10-100 nm scale. However, the complex DNA sequence design needed for the synthesis of these versatile DNA-NPs has limited their widespread use to date. While the automated sequence design algorithms DAEDALUS and vHelix-BSCOR apply to DNA-NPs synthesized using either uniformly dual or hybrid single-dual duplex edges, respectively, these DNA-NPs are relatively compliant mechanically and are therefore of limited utility for some applications.

In vitro synthesis of gene-length single-stranded DNA.

Single-stranded DNA (ssDNA) increases the likelihood of homology directed repair with reduced cellular toxicity. However, ssDNA synthesis strategies are limited by the maximum length attainable, ranging from a few hundred nucleotides for chemical synthesis to a few thousand nucleotides for enzymatic synthesis, as well as limited control over nucleotide composition. Here, we apply purely enzymatic synthesis to generate ssDNA greater than 15 kilobases (kb) using asymmetric PCR, and illustrate the incorporation of diverse modified nucleotides for therapeutic and theranostic applications.

De novo design and synthesis of a 30-cistron translation-factor module.

Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA.

Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ.

RlmJ catalyzes the m(6)A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain.

A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress.

Hemodynamic forces regulate embryonic organ development, hematopoiesis, vascular remodeling, and atherogenesis. The mechanosensory stimulus of blood flow initiates a complex network of intracellular pathways, including activation of Rac1 GTPase, establishment of endothelial cell (EC) polarity, and redox signaling. The activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be modulated by the GTP/GDP state of Rac1; however, the molecular mechanisms of Rac1 activation by flow are poorly understood.

The structure of the Tiam1 PDZ domain/ phospho-syndecan1 complex reveals a ligand conformation that modulates protein dynamics.

PDZ (PSD-95/Dlg/ZO-1) domains are protein-protein interaction modules often regulated by ligand phosphorylation. Here, we investigated the specificity, structure, and dynamics of Tiam1 PDZ domain/ligand interactions. We show that the PDZ domain specifically binds syndecan1 (SDC1), phosphorylated SDC1 (pSDC1), and SDC3 but not other syndecan isoforms.

Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA.

RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2'O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V.

Structural and thermodynamic analysis of PDZ-ligand interactions.

Tiam-family guanine exchange proteins are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These modular proteins contain a variety of signaling domains, including a single postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Here, we show how structural and thermodynamic approaches applied to the Tiam1 PDZ domain can be used to gain unique insights into the affinity and specificity of PDZ-ligand interactions with peptides derived from Syndecan1 and Caspr4 proteins.

Distinct ligand specificity of the Tiam1 and Tiam2 PDZ domains.

Guanine nucleotide exchange factor proteins of the Tiam family are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These proteins are modular and contain a variety of interaction domains, including a single post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Previous studies suggest that the specificities of the Tiam1 and Tiam2 PDZ domains are distinct.

The Tiam1 PDZ domain couples to Syndecan1 and promotes cell-matrix adhesion.

The T-cell lymphoma invasion and metastasis gene 1 (Tiam1) is a guanine exchange factor (GEF) for the Rho-family GTPase Rac1 that is crucial for the integrity of adherens junctions, tight junctions, and cell-matrix interactions. This GEF contains several protein-protein interaction domains, including a PDZ domain. Earlier studies identified a consensus PDZ-binding motif and a synthetic peptide capable of binding to the Tiam1 PDZ domain, but little is known about its ligand specificity and physiological role in cells.