Biology subjects

Ngo, J. T.

Publications and source records attributed to Ngo, J. T..

17 recordsLinked to original sources

bioRxiv2026

ProNotch converts extracellular protease activity into programmable transcriptional outputs

Synthetic receptors that convert extracellular protease activity into programmable transcriptional outputs would expand the toolkit of mammalian cell biology and cell engineering, yet modular platforms for directly coupling extracellular proteolysis to gene expression remain limited. Here we introduce ProNotch, a receptor architecture that harnesses protease-gated derepression of a mutant Notch1 negative regulatory region (NRR) to drive user-defined gene expression. ProNotch tethers destabilized NRR mutants to inhibitory anti-NRR single-chain variable fragments (scFvs) via protease-cleavable linkers. NRR engagement by high-affinity scFvs simultaneously rescues surface trafficking of mutant receptors and suppresses basal signaling until linker cleavage releases the inhibitory scFv module, permitting the destabilized NRR to initiate ligand-independent signaling. Linker substitution reprogrammed protease specificity across diverse enzymes, and tandem substrate repeats enhanced sensitivity without increasing basal activity. Single-chain receptor designs enabled OR and AND logic gates, allowing integration of multi-protease inputs into a single transcriptional output. ProNotch detected endogenous MMP-14 activity from cancer cell lines in cis and in trans and drove protease-dependent cell-state transitions in C3H/10T1/2 fibroblasts. The scFv-NRR module also functioned as a protease-activated pro-antibody that conditionally inhibited DLL4-dependent signaling and ligand-independent activation of mutant NOTCH1 in the T cell acute lymphoblastic leukemia cell line HPB-ALL. Together, these results establish ProNotch as a modular platform for engineering protease-responsive cells and demonstrate that its regulatory module can be extended to a soluble, conditionally activated inhibitor of NOTCH1 signaling.

synthetic biology↗

bioRxiv2026

Engineered Allosteric RNA Editors Enable Compact, Stimulus-Responsive Post-Transcriptional Circuits

Translational regulation offers a powerful biological control axis with the potential to enable programmable control over synthetic mRNAs. Here, we introduce inducible Deaminases Acting on RNA (iDARs): deaminase domains (DDs) with conditional RNA-editing activities. Using a domain-insertion strategy, we designed autoinhibited enzymes that can be converted into active RNA editors in response to triggers based on small molecules (chemiDARs), intracellular antigens (antiDARs), protease cleavage (lysiDAR), and optical excitation (optiDAR). Coupling these domains with novel stop codon containing RNA substrates enabled conditional protein translation or transcript degradation. Mutational tuning of inositol hexaphosphate (IP6)-binding pockets produced tightly regulated deaminases with minimal basal activity, facilitating dose-dependent readthrough translation in response to low-nanomolar drug concentration, with dynamic ranges exceeding 100-fold. By encoding iDARs alongside their substrates, we developed "self-editing" polycistronic transcripts capable of directing translation of encoded proteins in a trigger-dependent manner following delivery to cells as in vitro transcribed mRNAs. Overall, iDARs provide a generalizable framework for generating controllable deaminases, enabling the design of post-transcriptional circuits that link biochemical sensing to readouts based on de novo translation or mRNA decay.

synthetic biology↗

bioRxiv2025

Superresolution imaging of live samples by centroid reassignment microscopy

Superresolution imaging has become one of the most important recent advances in microscopy development. However, most superresolution methods are ill-adapted for live-sample imaging because they are unacceptably slow, susceptible to artifacts, or require the use of specialized fluorophores and labeling protocols. We introduce a superresolution method called centroid reassignment microscopy (CRM) that overcomes these limitations. CRM is a simple variation on confocal microscopy wherein the single-element detector and small pinhole are replaced by a centroid detector and larger pinhole. Superresolution is obtained by reassigning the detected fluorescence to its centroid location of as a function of the scanning excitation focus location. Our method bears resemblance to the method of image scanning microscopy, which involves the use of an array detector, with the advantage that CRM provides improved resolution for the same number of detected photons while being simpler to implement and less sensitive to noise. CRM is light-efficient, fast (single-frame), robust to defocus aberrations, and requires no changes whatsoever in standard fluorescence imaging protocols, making it uniquely attractive for superresolution imaging of live, dynamic samples.

bioengineering↗

bioRxiv2025

Protease-Containing Nanobodies for Detecting and Manipulating Intracellular Antigens Using Antiviral Drugs

Tools to induce the formation of protein-protein interactions (PPIs) via small molecules are essential for investigating and engineering biological systems. Here, we introduce a protease-based strategy for controlling the preservation of otherwise self-cleaving nanobodies. By inserting the Hepatitis C Virus NS3 cis-protease into the nanobody scaffold, we showed that the antigen-binding ability of these chimeric nanobodies can be controlled in a dose-dependent manner using NS3 inhibitors. We demonstrated the generalizability of this approach by designing and validating drug-controllable nanobodies targeting mCherry (LaM4), eGFP (LaG16), and the ALFA peptide tag. Additionally, we showed that an NS3-containing version of a nanobody targeting the {beta}2-adrenergic receptor can control endogenous G-protein-mediated signaling activity. Overall, we introduce new chemogenetic components for controlling intracellular PPIs using clinically approved antiviral drugs.

synthetic biology↗

bioRxiv2024

Immunofocusing on the conserved fusion peptide of HIV envelope glycoprotein in rhesus macaques

SummaryDuring infection, the fusion peptide (FP) of HIV envelope glycoprotein (Env) serves a central role in viral fusion with the host cell. As such, the FP is highly conserved and therefore an attractive epitope for vaccine design. Here, we describe a vaccination study in non-human primates (NHPs) where glycan deletions were made on soluble HIV Env to increase FP epitope exposure. When delivered via implantable osmotic pumps, this immunogen primed immune responses against the FP, which were then boosted with heterologous trimers resulting in a focused immune response targeting the conserved FP epitope. Although autologous immunizations did not elicit high affinity FP-targeting antibodies, the conserved FP epitope on a heterologous trimer further matured the lower affinity, FP-targeting B cells. This study suggests using epitope conservation strategies on distinct Env trimer immunogens can focus humoral responses on desired neutralizing epitopes and suppress immune-distracting antibody responses against non-neutralizing epitopes.

immunology↗

bioRxiv2024

RNA-Stabilized Coat Proteins for Sensitive and Simultaneous Imaging of Distinct Single mRNAs in Live Cells

RNA localization and regulation are critical for cellular function, yet many live RNA imaging tools suffer from limited sensitivity due to background emissions from unbound probes. Here, we introduce conditionally stable variants of MS2 and PP7 coat proteins (which we name dMCP and dPCP) designed to decrease background in live-cell RNA imaging. Using a protein engineering approach that combines circular permutation and degron masking, we generated dMCP and dPCP variants that rapidly degrade except when bound to cognate RNA ligands. These enhancements enabled the sensitive visualization of single mRNA molecules undergoing differential regulation within various sub-compartments of live cells. We further demonstrate dual-color imaging with orthogonal MS2 and PP7 motifs, allowing simultaneous low-background visualization of distinct RNA species within the same cell. Overall, this work provides versatile, low-background probes for RNA imaging, which should have broad utility in the imaging and biotechnological utilization of MS2- and PP7-containing RNAs.

bioengineering↗

bioRxiv2024

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues

We introduce an adaptor-based strategy for regulating fluorescein-binding synthetic Notch (SynNotch) receptors using ligands based on conjugates of fluorescein isomers and analogs. To develop a versatile system, we evaluated the surface expression and activities of multiple constructs containing distinct extracellular fluorescein-binding domains. Using an optimized receptor, we devised ways to regulate signaling via fluorescein-based chemical transformations, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of an o-nitrobenzyl-caged fluorescein conjugate. We further demonstrate that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can regulate SynNotch activity depending on the folding state of collagen-based ECM networks. Treatment with these conjugates enabled cells to distinguish between folded versus denatured collagen proteins and enact dose-dependent gene expression responses depending on the nature of the signaling adaptors presented. To demonstrate the utility of these tools, we applied them to control the myogenic conversion of fibroblasts into myocytes with spatial and temporal precision and in response to denatured collagen-I, a biomarker of multiple pathological states. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to extracellular ligands based on the widely used and clinically-approved fluorescein dye.

synthetic biology↗

bioRxiv2023

A novel HIV triple broadly neutralizing antibody (bNAb) combination-based passive immunization of infant rhesus macaques achieves durable protective plasma neutralization levels and mediates anti-viral effector functions.

To eliminate vertical HIV transmission and achieve therapy-free viral suppression among children living with HIV, novel strategies beyond antiretroviral therapy (ART) are necessary. Our group previously identified a triple bNAb combination comprising of 3BNC117, PGDM1400 and PGT151 that mediates robust in vitro neutralization and non-neutralizing effector functions against a cross-clade panel of simian human immunodeficiency viruses (SHIVs). In this study, we evaluated the safety, pharmacokinetics, and antiviral potency of this bNAb combination in infant rhesus macaques (RMs). We demonstrate that subcutaneous infusion of the triple bNAb regimen was well tolerated in pediatric monkeys and resulted in durable systemic and mucosal distribution. Plasma obtained from passively-immunized RMs demonstrated potent HIV-neutralizing and Fc-mediated antiviral effector functions. Finally, using the predicted serum neutralization 80% inhibitory dilution titer (PT80) biomarker, which was recently identified as a surrogate endpoint for evaluation of the preventative efficacy (PE) of bNAbs against mucosal viral acquisition in human clinical trials, we demonstrated that our regimen has a predicted PE[≥]90% against a large panel of plasma and breast milk-derived HIV strains and cross-clade SHIV variants. This data will guide the development of combination bNAbs for eliminating vertical HIV transmission and for achieving ART-free viral suppression among children living with HIV.

immunology↗

bioRxiv2023

Fe-TAMLs as a new class of small molecule peroxidase probes for correlated light and electron microscopy

We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAMLs catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.

cell biology↗

bioRxiv2023

Focusing antibody responses to the fusion peptide in rhesusmacaques

Immunodominance of antibodies targeting non-neutralizing epitopes and the high level of somatic hypermutation within germinal centers (GCs) required for most HIV broadly neutralizing antibodies (bnAbs) are major impediments to the development of an effective HIV vaccine. Rational protein vaccine design and non-conventional immunization strategies are potential avenues to overcome these hurdles. Here, we report using implantable osmotic pumps to continuously deliver a series of epitope-targeted immunogens to rhesus macaques over the course of six months to elicit immune responses against the conserved fusion peptide. Antibody specificities and GC responses were tracked longitudinally using electron microscopy polyclonal epitope mapping (EMPEM) and lymph node fine-needle aspirates, respectively. Application of cryoEMPEM delineated key residues for on-target and off-target responses that can drive the next round of structure-based vaccine design.

immunology↗

bioRxiv2023

Controlled protein activities with viral proteases, antiviral peptides, and antiviral drugs

Chemical control of protein activity is a powerful tool for scientific study, synthetic biology, and cell therapy; however, for broad use, effective chemical inducer systems must minimally crosstalk with endogenous processes and exhibit desirable drug delivery properties. Accordingly, the drug-controllable proteolytic activity of hepatitis C cis-protease NS3 and its associated antiviral drugs have been used to regulate protein activity and gene modulation. These tools advantageously exploit non-eukaryotic/prokaryotic proteins and clinically approved inhibitors. Here we expand the toolkit by utilizing catalytically inactive NS3 protease as a high affinity binder to genetically encoded, antiviral peptides. Through our approach, we create NS3-peptide complexes that can be displaced by FDA-approved drugs to modulate transcription, cell signaling, split-protein complementation. With our developed system, we discover a new mechanism to allosterically regulate Cre recombinase. Allosteric Cre regulation with NS3 ligands enables orthogonal recombination tools in eukaryotic cells and functions in divergent organisms to control prokaryotic recombinase activity.

synthetic biology↗

bioRxiv2022

SNAP-tag and HaloTag fused proteins for HaSX8-inducible control over synthetic biological functions in engineered mammalian cells

Drug-inducible systems allow biological processes to be regulated through the administration of exogenous chemical inducers. Such methods can be used to study native biological activities, or to control synthetically engineered ones, with temporal and dose-dependent control. However, the number of existing drug-inducible systems is limited, and there remains a need for synthetic biology components that can be combined with the existing toolset and regulated with independent and orthogonal control. Here, we describe new cell engineering components that can be regulated via a heterodimerization of SNAP-tag and HaloTag domains using a selective small molecule crosslinker termed "HaXS8." The construction and validation of multiple HaXS8-sensitive components are described, including systems for regulating transcription, Cre recombinase activity, and caspase-9 activity in mammalian cells. The systems elaborate the ability to control gene expression, DNA recombination, and apoptosis in cell engineered systems.

synthetic biology↗

bioRxiv2022

A Genetically Encodable and Chemically Disruptable System for Synthetic Post-Translational Modification Dependent SynNotch Signaling

We describe an engineered "writer/reader" framework for programming post-translational control into synthetic mammalian signaling proteins. In this approach, a bacterially-derived biotin protein ligase (BirA) was used as a "writer" element for the modification of artificial receptors and transcription factors containing a biotin acceptor peptide (AP) fusion tag. To enable modification events to transmit biochemical information, we designed encodable "reader" modules using sequences from a biotinamide-binding antibody. Proteins fused to reader domains were able to interact with AP-tagged polypeptides in a biotinylation-dependent manner, and control over the timing and extent of these interactions could be modulated through both genetic and chemically-based strategies. Genetic and cell-specific control over AP-reader module interactions was achieved via regulated BirA expression, and the interaction states of both intra-and inter-cellular complexes could be modulated with biotinamide-based and bioorthogonally-functionalized compounds. The utility of this approach was demonstrated by installing post-translational and chemogenetic control into synthetic Notch ("SynNotch")-based systems.

biochemistry↗

bioRxiv2022

Synthetic Mechanotransduction Using Engineered SynNotch Receptors

Cells can sense and interpret mechanical stimuli from their environments and neighbors, but the ability to engineer customized mechanosensing capabilities has remained a synthetic and mechanobiology challenge. Here, we introduce tension-tuned synthetic Notch (SynNotch) receptors that can be used to convert extracellular and intercellular forces into specifiable gene expression changes. By elevating the tension requirements of SynNotch activation, in combination with structure-guided mutagenesis, we designed a set of receptors with mechanical sensitivities spanning the physiologically relevant picoNewton (pN) range. Cells expressing these receptors can distinguish between varying tensile forces and respond by enacting customizable transcriptional programs. The synthetic utility of these tools is demonstrated by designing a decision-making circuit, through which fibroblasts can be made to differentiate into myoblasts upon stimulation with distinct tension magnitudes. Mechanobiological utility is also demonstrated by characterizing cell-generated forces transmitted between cells during Notch signaling. Overall, this work provides insight regarding how mechanically induced changes in protein structure can be used to transduce physical forces into biochemical signals. The system should facilitate the further programming and dissection of force-related phenomena in biological systems.

synthetic biology↗

bioRxiv2021

Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM

In BriefHerein, we evaluated the immunogenicity of several BG505 SOSIP-based HIV Env immunogens in the rhesus macaque animal model using a combination of serology and biophysical approaches. We applied electron cryo-microscopy for high-resolution mapping of elicited polyclonal antibody responses, which provided detailed insights into the binding modes of the most common classes of antibodies elicited by BG505 SOSIP immunogens as well as the critical differences in immunogenicity that can occur as a consequence of engineered stabilizing mutations and partial glycan occupancy at different sites. SummaryEngineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

immunology↗

bioRxiv2020

Engineering clinically-approved drug gated CAR circuits

Chimeric antigen receptor (CAR) T cell immunotherapy has the potential to revolutionize cancer medicine. However, excessive CAR activation, lack of tumor-specific surface markers, and antigen escape have limited the safety and efficacy of CAR T cell therapy. A multi-antigen targeting CAR system that is regulated by safe, clinically-approved pharmaceutical agents is urgently needed, yet only a few simple systems have been developed, and even fewer have been evaluated for efficacy in vivo. Here, we present NASCAR (NS3 ASsociated CAR), a collection of induc-ible ON and OFF switch CAR circuits engineered with a NS3 protease domain deriving from the Hepatitis C Virus (HCV). We establish their ability to regulate CAR activity using multiple FDA-approved antiviral protease inhibitors, including grazoprevir (GZV), both in vitro and in a xenograft tumor model. In addition, we have engineered several dual-gated NASCAR circuits, consisting of an AND logic gate CAR, universal ON-OFF CAR, and a switchboard CAR. These engineered receptors enhance control over T cell activity and tumor-targeting specificity. Together, our com-prehensive set of multiplex drug-gated CAR circuits represent a dynamic, tunable, and clinically-ready set of modules for enhancing the safety of CAR T cell therapy.

synthetic biology↗

bioRxiv2018

Split-miniSOG for detecting and localizing intracellular protein-protein interactions: application to correlated light and electron microscopy

A protein complementation assay (PCA) for detecting and localizing intracellular protein-protein interactions (PPIs) was built by bisection of miniSOG, a fluorescent flavoprotein derived from the light, oxygen, voltage (LOV)-2 domain of Arabidopsis phototropin. When brought together by interacting proteins, the fragments reconstitute a functional reporter that permits tagged protein complexes to be visualized by fluorescence light microscopy (LM), and then by standard as well as \"multicolor\" electron microscopy (EM) imaging methods via the photooxidation of 3-3-diaminobenzidine (DAB) and its lanthanide-conjugated derivatives.

cell biology↗