Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis.

The rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range-determining regions (HRDRs) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity while minimizing disruptions to the overall tail fiber structure, resulting in synthetic "phagebodies." We showed that mutating HRDRs yields phagebodies with altered host-ranges, and select phagebodies enable long-term suppression of bacterial growth in vitro, by preventing resistance appearance, and are functional in vivo using a murine model. We anticipate that this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.

Fingerprinting DNAzyme Cross-Reactivity for Pattern-Based Detection of Heavy Metals.

Heavy metal contamination in food and water is a major public health concern because heavy metals are toxic in minute amounts. DNAzyme sensors are emerging as a promising tool for rapid onsite detection of heavy metals, which can aid in minimizing exposure. However, DNAzyme activity toward its target metal is not absolute and has cross-reactivity with similar metals, which is a major challenge in the wide-scale application of DNAzyme sensors for environmental monitoring. To address this, we constructed a four DNAzyme array (17E, GR-5, EtNA, and NaA43) and used a pattern-based readout to improve sensor accuracy. We measured cross-reactivity between three metal cofactors (Pb2+, Ca2+, and Na+) and common interferents (Mg2+, Zn2+, Mn2+, UO22+, Li+, K+, and Ag+) and then used t-SNE analysis to identify and quantify the metal ion. We further showed that this method can be used for distinguishing mixtures of metals and detecting Pb2+ in environmental soil samples at micromolar concentrations.

Hydrogel-based biocontainment of bacteria for continuous sensing and computation.

Genetically modified microorganisms (GMMs) can enable a wide range of important applications including environmental sensing and responsive engineered living materials. However, containment of GMMs to prevent environmental escape and satisfy regulatory requirements is a bottleneck for real-world use. While current biochemical strategies restrict unwanted growth of GMMs in the environment, there is a need for deployable physical containment technologies to achieve redundant, multi-layered and robust containment. We developed a hydrogel-based encapsulation system that incorporates a biocompatible multilayer tough shell and an alginate-based core. This deployable physical containment strategy (DEPCOS) allows no detectable GMM escape, bacteria to be protected against environmental insults including antibiotics and low pH, controllable lifespan and easy retrieval of genomically recoded bacteria. To highlight the versatility of DEPCOS, we demonstrated that robustly encapsulated cells can execute useful functions, including performing cell-cell communication with other encapsulated bacteria and sensing heavy metals in water samples from the Charles River.

Highly Polyvalent DNA Motors Generate 100+ pN of Force via Autochemophoresis.

Motor proteins such as myosin, kinesin, and dynein are essential to eukaryotic life and power countless processes including muscle contraction, wound closure, cargo transport, and cell division. The design of synthetic nanomachines that can reproduce the functions of these motors is a longstanding goal in the field of nanotechnology. DNA walkers, which are programmed to "walk" along defined tracks via the burnt bridge Brownian ratchet mechanism, are among the most promising synthetic mimics of these motor proteins. While these DNA-based motors can perform useful tasks such as cargo transport, they have not been shown to be capable of cooperating to generate large collective forces for tasks akin to muscle contraction. In this work, we demonstrate that highly polyvalent DNA motors (HPDMs), which can be viewed as cooperative teams of thousands of DNA walkers attached to a microsphere, can generate and sustain substantial forces in the 100+ pN regime. Specifically, we show that HPDMs can generate forces that can unzip and shear DNA duplexes (∼12 and ∼50 pN, respectively) and rupture biotin-streptavidin bonds (∼100-150 pN). To help explain these results, we present a variant of the burnt-bridge Brownian ratchet mechanism that we term autochemophoresis, wherein many individual force generating units generate a self-propagating chemomechanical gradient that produces large collective forces. In addition, we demonstrate the potential of this work to impact future engineering applications by harnessing HPDM autochemophoresis to deposit "molecular ink" via mechanical bond rupture. This work expands the capabilities of synthetic DNA motors to mimic the force-generating functions of biological motors. Our work also builds upon previous observations of autochemophoresis in bacterial transport processes, indicating that autochemophoresis may be a fundamental mechanism of pN-scale force generation in living systems.

Nanoscale optomechanical actuators for controlling mechanotransduction in living cells.

To control receptor tension optically at the cell surface, we developed an approach involving optomechanical actuator nanoparticles that are controlled with near-infrared light. Illumination leads to particle collapse, delivering piconewton forces to specific cell surface receptors with high spatial and temporal resolution. We demonstrate optomechanical actuation by controlling integrin-based focal adhesion formation, cell protrusion and migration, and T cell receptor activation.

Titin-Based Nanoparticle Tension Sensors Map High-Magnitude Integrin Forces within Focal Adhesions.

Mechanical forces transmitted through integrin transmembrane receptors play important roles in a variety of cellular processes ranging from cell development to tumorigenesis. Despite the importance of mechanics in integrin function, the magnitude of integrin forces within adhesions remains unclear. Literature suggests a range from 1 to 50 pN, but the upper limit of integrin forces remains unknown. Herein we challenge integrins with the most mechanically stable molecular tension probe, which is comprised of the immunoglobulin 27th (I27) domain of cardiac titin flanked with a fluorophore and gold nanoparticle. Cell experiments show that integrin forces unfold the I27 domain, suggesting that integrin forces exceed ∼30-40 pN. The addition of a disulfide bridge within I27 "clamps" the probe and resists mechanical unfolding. Importantly, incubation with a reducing agent initiates SH exchange, thus unclamping I27 at a rate that is dependent on the applied force. By recording the rate of S-S reduction in clamped I27, we infer that integrins apply 110 ± 9 pN within focal adhesions of rat embryonic fibroblasts. The rates of S-S exchange are heterogeneous and integrin subtype-dependent. Nanoparticle titin tension sensors along with kinetic analysis of unfolding demonstrate that a subset of integrins apply tension many fold greater than previously reported.

Mechanically Induced Catalytic Amplification Reaction for Readout of Receptor-Mediated Cellular Forces.

Mechanics play a fundamental role in cell biology, but detecting piconewton (pN) forces is challenging because of a lack of accessible and high throughput assays. A mechanically induced catalytic amplification reaction (MCR) for readout of receptor-mediated forces in cells is described. Mechanically labile DNA duplexes presenting ligands are surface immobilized such that specific receptor forces denature the duplex and thus expose a blocked primer. Amplification of primers is achieved using an isothermal polymerization reaction and quantified by fluorescence readout. As a proof of concept, the assay was used to test the activity of a mechanomodulatory drug and integrin adhesion receptor antibodies. To the best of our knowledge, this is the first example of a catalytic reaction triggered in response to molecular piconewton forces. The MCR may transform the field of mechanobiology by providing a new facile tool to detect receptor specific mechanics with the convenience of the polymerase chain reaction (PCR).

Nanoparticle tension probes patterned at the nanoscale: impact of integrin clustering on force transmission.

Herein we aimed to understand how nanoscale clustering of RGD ligands alters the mechano-regulation of their integrin receptors. We combined molecular tension fluorescence microscopy with block copolymer micelle nanolithography to fabricate substrates with arrays of precisely spaced probes that can generate a 10-fold fluorescence response to pN-forces. We found that the mechanism of sensing ligand spacing is force-mediated. This strategy is broadly applicable to investigating receptor clustering and its role in mechanotransduction pathways.

Quantum dots encapsulated within phospholipid membranes: phase-dependent structure, photostability, and site-selective functionalization.

Lipid vesicle encapsulation is an efficient approach to transfer quantum dots (QDs) into aqueous solutions, which is important for renewable energy applications and biological imaging. However, little is known about the molecular organization at the interface between a QD and lipid membrane. To address this issue, we investigated the properties of 3.0 nm CdSe QDs encapsulated within phospholipid membranes displaying a range of phase transition temperatures (Tm). Theoretical and experimental results indicate that the QD locally alters membrane structure, and in turn, the physical state (phase) of the membrane controls the optical and chemical properties of the QDs. Using photoluminescence, ICP-MS, optical microscopy, and ligand exchange studies, we found that the Tm of the membrane controls optical and chemical properties of lipid vesicle-embedded QDs. Importantly, QDs encapsulated within gel-phase membranes were ultrastable, providing the most photostable non-core/shell QDs in aqueous solution reported to date. Atomistic molecular dynamics simulations support these observations and indicate that membranes are locally disordered displaying greater disordered organization near the particle-solution interface. Using this asymmetry in membrane organization near the particle, we identify a new approach for site-selective modification of QDs by specifically functionalizing the QD surface facing the outer lipid leaflet to generate gold nanoparticle-QD assemblies programmed by Watson-Crick base-pairing.

Phage-layer interferometry: a companion diagnostic for phage therapy and a bacterial testing platform.

The continuing and rapid emergence of antibiotic resistance (AMR) calls for innovations in antimicrobial therapies. A promising, 're-emerging' approach is the application of bacteriophage viruses to selectively infect and kill pathogenic bacteria, referred to as phage therapy. In practice, phage therapy is personalized and requires companion diagnostics to identify efficacious phages, which are then formulated into a therapeutic cocktail. The predominant means for phage screening involves optical-based assays, but these methods cannot be carried out in complex media, such as colored solutions, inhomogeneous mixtures, or high-viscosity samples, which are often conditions encountered in vivo. Moreover, these assays cannot distinguish phage binding and lysis parameters, which are important for standardizing phage cocktail formulation. To address these challenges, we developed Phage-layer Interferometry (PLI) as a companion diagnostic. Herein, PLI is assessed as a quantitative phage screening method and prototyped as a bacterial detection platform. Importantly, PLI is amenable to automation and is functional in complex, opaque media, such as baby formula. Due to these newfound capabilities, we foresee immediate and broad impact of PLI for combating AMR and protecting against foodborne illnesses.

IbpAB small heat shock proteins are not host factors for bacteriophage ϕX174 replication.

Bacteriophage ϕX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ϕX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ϕX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ϕX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ϕX174 replication; moreover, their absence has no discernible effect on ϕX174 fecundity. These results suggest IbpA/B upregulation is a response to ϕX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.

Tension sensing nanoparticles for mechano-imaging at the living/nonliving interface.

Studying chemomechanical coupling at interfaces is important for fields ranging from lubrication and tribology to microfluidics and cell biology. Several polymeric macro- and microscopic systems and cantilevers have been developed to image forces at interfaces, but few materials are amenable for molecular tension sensing. To address this issue, we have developed a gold nanoparticle sensor for molecular tension-based fluorescence microscopy. As a proof of concept, we imaged the tension exerted by integrin receptors at the interface between living cells and a substrate with high spatial (<1 µm) resolution, at 100 ms acquisition times and with molecular specificity. We report integrin tension values ranging from 1 to 15 pN and a mean of ~1 pN within focal adhesions. Through the use of a conventional fluorescence microscope, this method demonstrates a force sensitivity that is 3 orders of magnitude greater than is achievable by traction force microscopy or polydimethylsiloxane micropost arrays, which are the standard in cellular biomechanics.

PET-RAFT Increases Uniformity in Polymer Networks.

Photoinduced electron/energy transfer (PET)-reversible addition-fragmentation chain transfer polymerization (RAFT) and conventional photoinitiated RAFT were used to synthesize polymer networks. In this study, two different metal catalysts, namely, tris[2-phenylpyridinato-C2,N]iridium(III) (Ir(ppy)3) and zinc tetraphenylporphyrin (ZnTPP), were selected to generate two different catalytic pathways, one with Ir(ppy)3 proceeding through an energy-transfer pathway and one with ZnTPP proceeding through an electron-transfer pathway. These PET-RAFT systems were contrasted against a conventional photoinitated RAFT process. Mechanically robust materials were generated. Using bulk swelling ratios and degradable cross-linkers, the homogeneity of the networks was evaluated. Especially at high primary chain length and cross-link density, the PET-RAFT systems generated more uniform networks than those made by conventional RAFT, with the electron transfer-based ZnTPP giving superior results to those of Ir(ppy)3. The ability to deactivate radicals either by RAFT exchange or reversible coupling in PET RAFT was proposed as the mechanism that gave better control in PET-RAFT systems.

Phage-Based Applications in Synthetic Biology.

Bacteriophage research has been instrumental to advancing many fields of biology, such as genetics, molecular biology, and synthetic biology. Many phage-derived technologies have been adapted for building gene circuits to program biological systems. Phages also exhibit significant medical potential as antibacterial agents and bacterial diagnostics due to their extreme specificity for their host, and our growing ability to engineer them further enhances this potential. Phages have also been used as scaffolds for genetically programmable biomaterials that have highly tunable properties. Furthermore, phages are central to powerful directed evolution platforms, which are being leveraged to enhance existing biological functions and even produce new ones. In this review, we discuss recent examples of how phage research is influencing these next-generation biotechnologies.

Site-Selective RNA Splicing Nanozyme: DNAzyme and RtcB Conjugates on a Gold Nanoparticle.

Modifying RNA through either splicing or editing is a fundamental biological process for creating protein diversity from the same genetic code. Developing novel chemical biology tools for RNA editing has potential to transiently edit genes and to provide a better understanding of RNA biochemistry. Current techniques used to modify RNA include the use of ribozymes, adenosine deaminase, and tRNA endonucleases. Herein, we report a nanozyme that is capable of splicing virtually any RNA stem-loop. This nanozyme is comprised of a gold nanoparticle functionalized with three enzymes: two catalytic DNA strands with ribonuclease function and an RNA ligase. The nanozyme cleaves and then ligates RNA targets, performing a splicing reaction that is akin to the function of the spliceosome. Our results show that the three-enzyme reaction can remove a 19 nt segment from a 67 nt RNA loop with up to 66% efficiency. The complete nanozyme can perform the same splice reaction at 10% efficiency. These splicing nanozymes represent a new promising approach for gene manipulation that has potential for applications in living cells.

Scaling Computation and Memory in Living Cells.

The semiconductor revolution that began in the 20th century has transformed society. Key to this revolution has been the integrated circuit, which enabled exponential scaling of computing devices using silicon-based transistors over many decades. Analogously, decreasing costs in DNA sequencing and synthesis, along with the development of robust genetic circuits, are enabling a "biocomputing revolution". First-generation gene circuits largely relied on assembling various transcriptional regulatory elements to execute digital and analog computing functions in living cells. Basic design rules and computational tools have since been derived so that such circuits can be scaled in order to implement complex computations. In the past five years, great strides have been made in expanding the biological programming toolkit to include recombinase- and CRISPR-based gene circuits that execute complex cellular logic and memory. Recent advances have enabled increasingly dense computing and memory circuits to function in living cells while expanding the application of these circuits from bacteria to eukaryotes, including human cells, for a wide range of uses.

High-speed DNA-based rolling motors powered by RNase H.

DNA-based machines that walk by converting chemical energy into controlled motion could be of use in applications such as next-generation sensors, drug-delivery platforms and biological computing. Despite their exquisite programmability, DNA-based walkers are challenging to work with because of their low fidelity and slow rates (∼1 nm min(-1)). Here we report DNA-based machines that roll rather than walk, and consequently have a maximum speed and processivity that is three orders of magnitude greater than the maximum for conventional DNA motors. The motors are made from DNA-coated spherical particles that hybridize to a surface modified with complementary RNA; the motion is achieved through the addition of RNase H, which selectively hydrolyses the hybridized RNA. The spherical motors can move in a self-avoiding manner, and anisotropic particles, such as dimerized or rod-shaped particles, can travel linearly without a track or external force. We also show that the motors can be used to detect single nucleotide polymorphism by measuring particle displacement using a smartphone camera.

Knockdown of TNF-α by DNAzyme gold nanoparticles as an anti-inflammatory therapy for myocardial infarction.

In this study, we used deoxyribozyme (DNAzyme) functionalized gold nanoparticles (AuNPs) to catalytically silence tumor necrosis factor-α (TNF-α) in vivo as a potential therapeutic for myocardial infarction (MI). Using primary macrophages as a model, we demonstrated 50% knockdown of TNF-α, which was not attainable using Lipofectamine-based approaches. Local injection of DNAzyme conjugated to gold particles (AuNPs) in the rat myocardium yielded TNF-α knockdown efficiencies of 50%, which resulted in significant anti-inflammatory effects and improvement in acute cardiac function following MI. Our results represent the first example showing the use of DNAzyme AuNP conjugates in vivo for viable delivery and gene regulation. This is significant as TNF-α is a multibillion dollar drug target implicated in many inflammatory-mediated disorders, thus underscoring the potential impact of DNAzyme-conjugated AuNPs.

Catalytic deoxyribozyme-modified nanoparticles for RNAi-independent gene regulation.

DNAzymes are catalytic oligonucleotides with important applications in gene regulation, DNA computing, responsive soft materials, and ultrasensitive metal-ion sensing. The most significant challenge for using DNAzymes in vivo pertains to nontoxic delivery and maintaining function inside cells. We synthesized multivalent deoxyribozyme "10-23" gold nanoparticle (DzNP) conjugates, varying DNA density, linker length, enzyme orientation, and linker composition in order to study the role of the steric environment and gold surface chemistry on catalysis. DNAzyme catalytic efficiency was modulated by steric packing and proximity of the active loop to the gold surface. Importantly, the 10-23 DNAzyme was asymmetrically sensitive to the gold surface and when anchored through the 5' terminus was inhibited 32-fold. This property was used to generate DNAzymes whose catalytic activity is triggered by thiol displacement reactions or by photoexcitation at λ = 532 nm. Importantly, cell studies revealed that DzNPs are less susceptible to nuclease degradation, readily enter mammalian cells, and catalytically down-regulate GDF15 gene expression levels in breast cancer cells, thus addressing some of the key limitations in the adoption of DNAzymes for in vivo work.