Biology subjects

Ting, A. Y.

Publications and source records attributed to Ting, A. Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Computational design of a versatile, zero-radius proximity labeling enzyme

The ability to map protein interactomes and organelle proteomes is foundational for achieving a molecular understanding of living cells. Proximity labeling (PL) provides a powerful strategy for this, but existing enzymes and photocatalysts are limited by their spatial resolution, reliance on biotin, and/or in vivo compatibility. Here we report FlexID, an engineered promiscuous ligase that catalyzes the rapid attachment of diverse small-molecule probes to proximal endogenous proteins. Critically, FlexID operates through a zero-radius, direct-contact mechanism, offering superior spatial precision compared to existing PL tools. We engineered FlexID by combining the strengths of sequence- and structure-trained computational models to enhance its catalytic activity and structural stability. Biophysical analysis revealed that specific conformational changes in FlexID improve its ability to recognize diverse target proteins while simultaneously preventing the premature release of the reactive intermediate. We demonstrate FlexID's versatility through in vivo proximity labeling, comprehensive organelle proteome mapping, and a high-throughput, fluorescence-based screen for molecular glues. Our work shows that computational methods can be harnessed to create mechanistically distinct PL enzymes and establishes FlexID as a flexible, high-resolution tool for mapping protein interactions and proteomes in living cells.

bioengineering↗

bioRxiv2026

Cell-Based Sensor for Extracellular DNA

Detection of molecules with cell-based sensors allows for conversion of binding events into gene expression outputs. Here, we present a cell-based sensor that can detect extracellular double-stranded DNA. This sensor is based on an engineered receptor which we call Luminescent Ultrasensitive Nucleic Acid Reporter, or LUNAR. LUNAR is based on a recently developed Programmable Antigen-gated G-protein-coupled Engineered Receptor (PAGER). PAGERs are a genetic fusion of an auto-inhibitory peptide, a protein-binding domain, and a modified kappa opioid receptor. PAGERs are gated by two binding events. First, a protein ligand displaces an intramolecular inhibitor, Arodyn, then a second ligand activates the receptor. By replacing the protein-binding domain with a DNA binding zinc finger protein (ZFP) we could detect extracellular DNA in a dose-dependent fashion. Here, we show that first-generation LUNAR constructs can detect both oligonucleotides and plasmid double-stranded DNA with nanomolar sensitivity in mammalian cells. Future work will focus on improving sensitivity, fold-change, and multiplexing capabilities for sequence-specific DNA detection.

synthetic biology↗

bioRxiv2026

Programming cell behavior with synthetic protease-activated receptors

Extracellular proteases are important signaling molecules in coagulation, inflammation, cell migration, and pain. Dysregulation of extracellular protease activity is common in diseases that perturb these critical functions. Engineering cells to sense and respond programmatically to protease activity has applications in biosensing, cell-based screening for protease activity, and therapeutics. Here we report synthetic protease-activated receptors (SynPARs) based on engineered, auto-inhibited G protein-coupled receptors (GPCRs). Relief of autoinhibition by proteolysis enables receptor activation by an exogenous or tethered agonist to generate transgene expression, real-time fluorescence, or endogenous G-protein signaling. We demonstrate SynPAR modularity with diverse secreted proteases, establish a cell-based SynPAR library selection to optimize protease recognition sequences, and control neuronal activity in response to protease activity. Finally, we use SynPARs in the dorsal root ganglion of mice to counteract hyperalgesia produced by trypsin activity, rewiring neurons to produce an analgesic response to a pain-inducing stimulus. Our study establishes SynPAR as a versatile and modular platform for recording, sensing, and responding to pericellular proteolysis. This fills a critical gap in protease-sensing tools and lays the groundwork for protease-activated genetic and cell-based medicines.

bioengineering↗

bioRxiv2026

Subcellular transcriptome sequencing with single cell APEX-seq identifies regulators of cell-cell interactions

Single-cell RNA sequencing has transformed our understanding of tissue complexity and heterogeneous cell states, yet provides little information about the subcellular organization of transcriptomes - despite the central role of RNA localization in splicing, translation, and function. Here we introduce single-cell APEX-seq (scAPEX-seq), a proximity labeling-based method for mapping subcellular transcriptomes at single-cell resolution. Improvements in probe design and RNA recovery enable APEX integration with droplet-based RNA-seq to capture endoplasmic reticulum-associated transcripts from thousands of individual cells. Applied to tumor-macrophage co-cultures, ER-targeted scAPEX-seq revealed interaction-dependent cell states and transcriptomic signatures by enriching for cell surface and secretory transcripts that are poorly resolved by conventional scRNA-seq. We further applied scAPEX-seq to short- and long-term co-cultures of HER2+ tumor cells with human chimeric antigen receptor (CAR) T cells, resolving distinct activated CAR T cell states, including populations characterized by upregulated NT5E or CTSW expression. We showed that overexpression of CTSW, a cathepsin protease, in CAR T cells promotes stem-like phenotypes, long-term proliferation, and sustained tumor cell killing. scAPEX-seq provides a powerful and scalable approach for profiling subcellular RNA populations, enabling the discovery of cell-cell interaction regulators missed by conventional approaches.

cell biology↗

bioRxiv2025

Towards CRISPR-based editing of the mitochondrial genome in yeast

Mitochondria, which evolved from symbiotic bacteria, possess their own genomes (mtDNA) and support independent transcription and translation within the organelle. Given the essential role of mtDNA in energy production, metabolism, as well as cellular homeostasis, and the high density of confirmed pathogenic mutations that map to mtDNA, there is a pressing need for versatile methods to study and manipulate this genome. Although CRISPR technology has revolutionized the editing of nuclear genomes, it has not been successfully extended to mtDNA, primarily due to the challenge of delivering single guide RNAs (sgRNAs) across both outer and inner mitochondrial membranes. Here we develop a survival-based reporter in Saccharomyces cerevisiae to screen for potential RNA import motifs. We identify a 40-nucleotide aptamer (IM83) that facilitates sgRNA entry into the mitochondrial matrix, enabling CRISPR editing by a mitochondrially-localized adenine base editor. We show that mitochondrial import of IM83 is ATP-dependent and enhanced by the tRNA synthetase Msk1. Further investigations identify barriers to efficient CRISPR editing of mtDNA, including loss of membrane potential associated with mitochondrial targeting of the base editor. These insights lay the groundwork for future improvements in CRISPR-based editing of mtDNA in eukaryotes.

genetics↗

bioRxiv2025

Endocytome profiling uncovers cell-surface protein dynamics underlying neuronal connectivity

Endocytosis actively remodels the neuronal surface proteome to drive diverse cellular processes, yet its global extent and circuit-level consequences have defied comprehensive interrogation. Here, we introduce endocytome profiling: a systematic, cell-type-specific approach for mapping cell-surface protein (CSP) dynamics in situ. Quantitative proteomic analysis of developing olfactory receptor neuron (ORN) axons generated an endocytic atlas comprising over 1,100 proteins and revealed the extent to which the surface proteome is remodeled to meet distinct developmental demands. Targeted interrogation of a junctional CSP showed that its endosome-to-surface ratio is precisely balanced to enable developmental axon pruning while preserving mature axon integrity. Multi-omic integration uncovered wide-spread transcellular signaling and identified a growth factor secreted by neighboring neurons to direct ORN axon targeting via endocytic regulation of its receptor. Endocytome profiling thus provides unprecedented access to cell-surface proteome dynamics and offers a powerful platform for dissecting proteome remodeling across diverse cell types and contexts.

neuroscience↗

bioRxiv2025

Multi-omic mapping of Drosophila protein secretomes reveals tissue-specific origins and inter-organ trafficking

Secreted proteins regulate many aspects of animal biology and are attractive targets for biomarkers and therapeutics. However, comprehensively identifying the "secretome", along with their tissues of origin, remains extremely challenging. To address this, we employed multiple omics methods to define a tissue-secretome map of 535 blood plasma proteins derived from specific cell-types and organs in Drosophila melanogaster. This map was enabled by methodological improvements including a collection of transgenic flies to label endogenous secreted proteins in 10 major tissue types, large-scale blood isolation, whole animal snRNA-seq, and a collection of 40 knock-in strains. Using this map, we discover surprising findings about circulating proteins: most originate from specific tissues including unusual sources (e.g. glia), many are uncharacterized, and some are shed ectodomains of transmembrane proteins. In addition, in vivo experiments revealed circulating proteins with remarkably tissue-specific expression, as well as proteins that are deposited in a different tissue from where they are synthesized, suggesting potential inter-organ functions. Our secretome map will serve as a resource to investigate blood protein function, discover novel tissue-tissue communication signals, and mine for homologues of human biomarkers.

genomics↗

bioRxiv2025

Computational design of conformation-biasing mutations to alter protein functions

Most natural proteins alternate between distinct conformational states, each associated with specific functions. Intentional manipulation of conformational equilibria could lead to improved or altered protein properties. Here we develop Conformational Biasing (CB), a rapid and streamlined computational method that utilizes contrastive scoring by inverse folding models to predict variants biased towards desired conformational states. We validated CB across seven diverse deep mutational scanning datasets, successfully predicting variants of K-Ras, SARS-CoV-2 spike, {beta}2 adrenergic receptor, and Src kinase with improved conformation-specific functions including enhanced effector binding or enzymatic activity. Furthermore, applying CB to lipoic acid ligase, a conformation-switching bacterial enzyme that has been used for the development of protein labeling technologies, revealed a previously unknown mechanism for conformational gating of sequence-specificity. Variants biased toward the "open" conformation were highly promiscuous, while "closed" conformation-biased variants were even more specific than wild-type, enhancing the utility of LplA for site-specific protein labeling with fluorophores in living cells. The speed, simplicity, and versatility of CB (available at: https://github.com/alicetinglab/ConformationalBiasing/) suggest that it may be broadly applicable for understanding and engineering protein conformational dynamics, with implications for basic research, biotechnology, and medicine.

biophysics↗

bioRxiv2025

Light-activated tetanus neurotoxin for conditional proteolysis and inducible synaptic inhibition in vivo

The light chain of tetanus neurotoxin (TeNT) is a 52 kD metalloprotease that potently inhibits synaptic transmission by cleaving the endogenous vesicle fusion protein VAMP2. To mitigate the toxicity of TeNT and harness it as a conditional tool for neuroscience, we engineered Light-Activated TeNT (LATeNT) via insertion of the light-sensitive LOV domain into an allosteric site. LATeNT was optimized by directed evolution and shown to have undetectable activity in the dark mammalian brain. Following 30 seconds of weak blue light exposure, however, LATeNT potently inhibited synaptic transmission in multiple brain regions. The effect could be reversed over 24 hours. We used LATeNT to discover an interneuron population in hippocampus that controls anxiety-like behaviors in mouse, and to control the secretion of endogenous insulin from pancreatic beta cells. Synthetic circuits incorporating LATeNT converted drug, Ca2+, or receptor activation into transgene expression or reporter protein secretion. Due to its large dynamic range, rapid kinetics, and highly specific mechanism of action, LATeNT should be a robust tool for conditional proteolysis and spatiotemporal control of synaptic transmission in vivo.

biochemistry↗

bioRxiv2024

Electrical synapse molecular diversity revealed by proximity-based proteomic discovery

Neuronal circuits are composed of synapses that are either chemical, where signals are transmitted via neurotransmitter release and reception, or electrical, where signals pass directly through interneuronal gap junction channels. While the molecular complexity that controls chemical synapse structure and function is well appreciated, the proteins of electrical synapses beyond the gap-junction-forming Connexins are not well defined. Yet, electrical synapses are expected to be molecularly complex beyond the gap junctions. Connexins are integral membrane proteins requiring vesicular transport and membrane insertion/retrieval to achieve function, homeostasis, and plasticity. Additionally, electron microscopy of neuronal gap junctions reveals neighboring electron dense regions termed the electrical synapse density (ESD). To reveal the molecular complexity of the electrical synapse proteome, we used proximity-dependent biotinylation (TurboID) linked to neural Connexins in zebrafish. Proteomic analysis of developing and mature nervous systems identifies hundreds of Connexin-associated proteins, with overlapping and distinct representation during development and adulthood. The identified protein classes span cell adhesion molecules, cytoplasmic scaffolds, vesicular trafficking, and proteins usually associated with the post synaptic density (PSD) of chemical synapses. Using circuits with stereotyped electrical and chemical synapses, we define molecular sub-synaptic compartments of ESD localizing proteins, we find molecular heterogeneity amongst electrical synapse populations, and we examine the synaptic intermingling of electrical and chemical synapse proteins. Taken together, these results reveal a new complexity of electrical synapse molecular diversity and highlight a novel overlap between chemical and electrical synapse proteomes. Moreover, human homologs of the electrical synapse proteins are associated with autism, epilepsy, and other neurological disorders, providing a novel framework towards understanding neuro-atypical states.

neuroscience↗

bioRxiv2024

Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy

Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H2O2. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O2 instead of toxic H2O2, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.

biochemistry↗

bioRxiv2024

Directed evolution of a sequence-specific covalent protein tag for RNA labeling

Efficient methods for conjugating proteins to RNA are needed for RNA delivery, imaging, editing, interactome mapping, as well as for barcoding applications. Non-covalent coupling strategies using viral RNA binding proteins such as MCP have been applied extensively but are limited by tag size, sensitivity, and dissociation over time. We took inspiration from a sequence-specific, covalent protein-DNA conjugation method based on the Rep nickase of a porcine circovirus called "HUH tag". Though wild-type HUH protein has no detectable activity towards an RNA probe, we engineered an RNA-reactive variant, called rHUH, through 7 generations of yeast display-based directed evolution. Our 13.4 kD rHUH has 12 mutations relative to HUH, and forms a covalent tyrosine-phosphate ester linkage with a 10-nucleotide RNA recognition sequence ("rRS") within minutes. We engineered the sensitivity down to 1 nM of target RNA, shifted the metal ion requirement from Mn2+ towards Mg2+, and demonstrated efficient labeling in mammalian cell lysate. This work paves the way toward a new methodology for sequence-specific covalent protein-RNA conjugation in biological systems.

bioengineering↗

bioRxiv2024

Synthetic G protein-coupled receptors for programmable sensing and control of cell behavior

Synthetic receptors that mediate antigen-dependent cell responses are transforming therapeutics, drug discovery, and basic research. However, established technologies such as chimeric antigen receptors (CARs) can only detect immobilized antigens, have limited output scope, and lack built-in drug control. Here, we engineer synthetic G protein-coupled receptors (GPCRs) capable of driving a wide range of native or nonnative cellular processes in response to user-defined antigen. We achieve modular antigen gating by engineering and fusing a conditional auto-inhibitory domain onto GPCR scaffolds. Antigen binding to a fused nanobody relieves auto-inhibition and enables receptor activation by drug, thus generating Programmable Antigen-gated G protein-coupled Engineered Receptors (PAGERs). We create PAGERs responsive to more than a dozen biologically and therapeutically important soluble and cell surface antigens, in a single step, from corresponding nanobody binders. Different PAGER scaffolds permit antigen binding to drive transgene expression, real-time fluorescence, or endogenous G protein activation, enabling control of cytosolic Ca2+, lipid signaling, cAMP, and neuronal activity. Due to its modular design and generalizability, we expect PAGER to have broad utility in discovery and translational science.

synthetic biology↗

bioRxiv2024

Molecular and Cellular Mechanisms of Teneurin Signaling in Synaptic Partner Matching

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionally conserved teneurins were the first identified transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching. HIGHLIGHTSO_LIIn situ spatial proteomics reveal the first intracellular interactome of teneurins C_LIO_LITen-m signals via a RhoGAP and Rac1 GTPase to regulate synaptic partner matching C_LIO_LISingle-axon analyses reveal a stabilization-upon-contact model for partner matching C_LIO_LITen-m signaling promotes F-actin in axon branches contacting partner dendrites C_LI

neuroscience↗

bioRxiv2023

Directed Evolution of Genetically Encoded LYTACs for Cell-Mediated Delivery

Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin like growth factor 2 (IGF2). After showing initial efficacy with wild type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially-selective targeted protein degradation. Significance StatementBetter therapeutic windows can be achieved by targeting therapeutics to their desired sites of action. For protein therapeutics, this might be achieved by engineering cell therapies that home to a tissue of interest and secrete the biologic drug locally. Here, we demonstrate that human primary T cells can be engineered to produce genetically encoded lysosome targeting chimeras (GELYTACs). These GELYTACs mediate the degradation of extracellular proteins associated with cancer progression. Thus, cells engineered to produce GELYTACs represent a potential new class of cancer therapeutics.

synthetic biology↗

bioRxiv2023

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions, and function with light. We integrated optogenetic control into proximity labeling (PL), a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the PL enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. "LOV-Turbo" works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by BRET from luciferase, enabling interaction-dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL, expanding the scope of experimental questions that can be addressed with PL.

molecular biology↗

bioRxiv2023

Cell type-specific intracellular protein delivery with inactivated botulinum toxin

The ability to deliver proteins and peptides across the plasma membrane into the cytosol of living mammalian cells would be highly impactful for both basic science and medicine. Natural cell-penetrating protein toxins have shown promise as protein delivery platforms but existing approaches are limited by immunogenicity, lack of cell type specificity, or their multicomponent nature. Here we explore inactivated botulinum toxin (BoNT) as a protein delivery platform. Using split luciferase reconstitution in the cytosol as a readout for endosomal escape and cytosolic delivery, we showed that BoNT chimeras with nanobodies replacing their natural receptor binding domain could be selectively targeted to cells expressing nanobody-matched surface markers. We used chimeric BoNTs to deliver a range of cargo from 1.3-55 kD, and demonstrated selective delivery of orthogonal cargoes to distinct cell populations within a heterogenous mixture. These explorations suggest that BoNT can be a versatile platform for targeted protein and peptide delivery into mammalian cells.

biochemistry↗

bioRxiv2023

Dynamic mapping of proteome trafficking within and between living cells by TransitID

The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules, uncovering a role for stress granules in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID introduces a powerful approach for distinguishing protein populations based on compartment or cell type of origin.

cell biology↗