Spatiotemporal Control Research Articles

Spatiotemporal controls on organic matter sourcing to minerogenic salt marshes

AbstractSalt marshes are important carbon sinks; however, identification of the drivers of carbon burial rates is challenging because estuaries sit at terrestrial and marine interfaces. Here, we address the questions: what are the sources of organic matter sequestered in minerogenic salt marshes, and how do sources change through time? We characterized down‐core sediment biogeochemistry (C : N, δ13C, δ15N) for the last century in seven Oregon USA high marshes and used a mixing model to elucidate differences in organic matter sources across estuaries and through time. Autochthonous biomass production consistently accounted for only half of overall organic matter accumulation, and the remainder was allochthonous, originating from a combination of estuarine and terrestrial sources. Salt marshes with high sediment loads buried more terrestrial organic matter, whereas those with low loads and substantial subtidal habitat buried more estuarine‐derived organic matter. When assessed through depth/time, stable isotope trends indicated that decomposition is not the primary control. Instead, organic matter became increasingly more terrestrial in recent decades, especially in salt marshes with low fluvial loads. We hypothesize that salt marshes with lower loads took longer to regain lost elevation following coseismic subsidence of the 1700 ce Cascadia earthquake. This result magnifies the role of river floods in sediment and carbon accumulation on the marsh surface. Ultimately, the highest carbon burial rates coincided with highest fractions of terrestrially sourced organic matter, though spatiotemporal complexities obscured any potentially significant trends. While the term “blue” typically excludes terrestrial carbon, minerogenic salt marshes still perform the important ecosystem function of burying organic matter.

Image-guided optogenetic spatiotemporal tissue patterning using μPatternScope

In the field of tissue engineering, achieving precise spatiotemporal control over engineered cells is critical for sculpting functional 2D cell cultures into intricate morphological shapes. In this study, we engineer light-responsive mammalian cells and target them with dynamic light patterns to realize 2D cell culture patterning control. To achieve this, we developed μPatternScope (μPS), a modular framework for software-controlled projection of high-resolution light patterns onto microscope samples. μPS comprises hardware and software suite governing pattern projection and microscope maneuvers. Together with a 2D culture of the engineered cells, we utilize μPS for controlled spatiotemporal induction of apoptosis to generate desired 2D shapes. Furthermore, we introduce interactive closed-loop patterning, enabling a dynamic feedback mechanism between the measured cell culture patterns and the light illumination profiles to achieve the desired target patterning trends. Our work offers innovative tools for advanced tissue engineering applications through seamless fusion of optogenetics, optical engineering, and cybernetics.

Polymorphing Hydrogels Regulated by Photo‐reactive DNA Cross‐links

AbstractPolymorphing hydrogels can morph into another structure on demand with reprogrammable features. This concept extends the degree of morphing beyond that of traditional shape‐morphing hydrogels, which predetermine their morphing capabilities at the fabrication stage. However, current polymorphing hydrogels face limitations due to the need for complex, non‐sustained responsiveness or additional chemical steps for reconfigurable morphing. Here, photo‐reactive DNA‐cross‐linked polymorphing hydrogels are presented that enable polymorphing under patterned light. The photo‐reactive DNA cross‐links act as a regulator in changing the shapes of the hydrogels by adjusting the lengths of the cross‐links depending on the wavelength of light, which allows precise and dynamic morphing in a programmable way through a one‐pot reaction. The high programmability involving spatiotemporal controllability and reprogrammable features offers advanced solutions for multifunctional soft machines and applications requiring complex processes.

Ultrasound Control of Genomic Regulatory Toolboxes for Cancer Immunotherapy

There remains a critical need for the precise control of CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies. Here, we engineer a set of inducible CRISPR-based tools controllable by focused ultrasound (FUS), which can penetrate deep and induce localized hyperthermia for transgene activation. We demonstrate the capabilities of FUS-inducible CRISPR, CRISPR activation (CRISPRa), and CRISPR epigenetic editor (CRISPRee) in modulating the genome and epigenome. We show that FUS-CRISPR-mediated telomere disruption primes solid tumours for chimeric antigen receptor (CAR)-T cell therapy. We further deliver FUS-CRISPR in vivo using adeno-associated viruses (AAVs), followed by FUS-induced telomere disruption and the expression of a clinically validated antigen in a subpopulation of tumour cells, functioning as “training centers” to activate synthetic Notch (synNotch) CAR-T cells to produce CARs against a universal tumour antigen to exterminate neighboring tumour cells. The FUS-CRISPR(a/ee) toolbox hence allows the noninvasive and spatiotemporal control of genomic/epigenomic reprogramming for cancer treatment.

Vector-Free Deep Tissue Targeting of DNA/RNA Therapeutics via Single Capacitive Discharge Conductivity-Clamped Gene Electrotransfer.

Viral vector and lipid nanoparticle based gene delivery have limitations around spatiotemporal control, transgene packaging size, and vector immune reactivity, compromising translation of nucleic acid (NA) therapeutics. In the emerging field of DNA and particularly RNA-based gene therapies, vector-free delivery platforms are identified as a key unmet need. Here, this work addresses these challenges through gene electrotransfer (GET) of "naked" polyanionic DNA/mRNA using a single needle form-factor which supports "electro-lens" based compression of the local electric field, and local control of tissue conductivity, enabling single capacitive discharge minimal charge gene delivery. Proof-of-concept studies for "single capacitive discharge conductivity-clamped gene electrotransfer" (SCD-CC-GET) deep tissue delivery of naked DNA and mRNA in the mouse hindlimb skeletal muscle achieve stable (>18 month) expression of luciferase reporter synthetic DNA, and mRNA encoding the reporter yield rapid onset (<3 h) high transient expression for several weeks. Delivery of DNAs encoding secreted alkaline phosphatase and Cal/09 influenza virus hemagglutinin antigen generate high systemic circulating recombinant protein levels and antibody titres. The findings support adoption of SCD-CC-GET for vaccines and immunotherapies, and extend the utility of this technology to meet the demand for efficient vector-free, precision, deep tissue delivery of NA therapeutics.

Femtosecond laser ablation of nitrocellulose for spatio-temporal flow control in µPADs

Microfluidic paper-based analytical devices (μPADs) are gaining popularity due to their low cost and ease of use, but controlling fluid flow for more complex biochemical assays within these devices remains challenging. This study investigates femtosecond laser ablation of nitrocellulose (NC), a preferred material for µPADs, to create mechanically switchable barriers and flow controllers. We investigated NC ablation using single laser pulses and spatially overlapping pulses that generate lines. Single pulse ablation thresholds were determined for wavelengths of 1,030 nm, 515 nm and 343 nm. Line ablation characteristics were investigated as a function of the temporal and spatial pulse separation and laser wavelength. High aspect ratio grooves (up to 4.26) were achieved under specific conditions. These grooves can be used to define the spatial separation of the flow in separated microchannels or to form a barrier line perpendicular to the microchannel that can modulate the temporal behavior of the fluid flow. This barrier introduced an additional high flow resistance slowing down the flow or, if it was designed to cut through nitrocellulose at the entire depth, completely stopped the liquid flow. It was further shown that a barrier formed in this way could be switched by mechanically bending the µPAD at the barrier position. The femtosecond laser patterning method presented here provides precise spatio-temporal control not only for flow branching and multiplexing, but also for controlling flow speed and switching flow on and off within the same manufacturing process. Our results open up new possibilities for complex, multi-step assays on µPADs.

Multifunctional hydrogel electronics for closed-loop antiepileptic treatment.

Closed-loop strategies offer advanced therapeutic potential through intelligent disease management. Here, we develop a hydrogel-based, single-component, organic electronic device for closed-loop neurotherapy. Fabricated out of conductive hydrogels, the device consists of a flexible array of microneedle electrodes, each of which can be individually addressed to perform electrical recording and control chemical release with sophisticated spatiotemporal control, thus pioneering a smart antiseizure therapeutic system by combining electrical and pharmacological interventions. The recorded neural signal acts as the trigger for a voltage-driven drug release in detected pathological conditions predicted by real-time electrophysiology analysis. When implanted into epileptic animals, the device enables autonomous antiseizure management, where the dosing of antiepileptic drug is controlled in a time-sensitive, region-selective, and dose-adaptive manner, allowing the inhibition of seizure outbursts through the delivery of just-necessary drug dosages. The side effects are minimized with dosages three orders of magnitude lower than the usage in approaches simulating existing clinical treatments.

Design Strategy, On-Demand Control, and Biomedical Engineering Applications of Wet Adhesion.

The adhesion of tissues to external devices is fundamental to numerous critical applications in biomedical engineering, including tissue and organ repair, bioelectronic interfaces, adhesive robotics, wearable electronics, biomedical sensing and actuation, as well as medical monitoring, treatment, and healthcare. A key challenge in this context is that tissues are typically situated in aqueous and dynamic environments, which poses a bottleneck to further advancements in these fields. Wet adhesion technology (WAT) presents an effective solution to this issue. In this review, we summarize the three major design strategies and control methods of wet adhesion, comprehensively and systematically introducing the latest applications and advancements of WAT in the field of biomedical engineering. First, single adhesion mechanism under the frameworks of the three design strategies is systematically introduced. Second, control methods for adhesion are comprehensively summarized, including spatiotemporal control, detachment control, and reversible adhesion control. Third, a systematic summary and discussion of the latest applications of WAT in biomedical engineering research and education were presented, with a particular focus on innovative applications such as tissue-electronic interface devices, ingestible devices, end-effector components, in vivo medical microrobots, and medical instruments and equipment. Finally, opportunities and challenges encountered in the design and development of wet adhesives with advanced adhesive performance and application prospects are discussed.

Red Light Responsive Cre Recombinase for Bacterial Optogenetics.

Optogenetic tools have been used in a wide range of microbial engineering applications that benefit from the tunable, spatiotemporal control that light affords. However, the majority of current optogenetic constructs for bacteria respond to blue light, limiting the potential for multichromatic control. In addition, other wavelengths offer potential benefits over blue light, including improved penetration of dense cultures and reduced potential for toxicity. In this study, we introduce OptoCre-REDMAP, a red light inducible Cre recombinase system in Escherichia coli. This system harnesses the plant photoreceptors PhyA and FHY1 and a split version of Cre recombinase to achieve precise control over gene expression and DNA excision. We optimized the design by modifying the start codon of Cre and characterized the impact of different levels of induction to find conditions that produced minimal basal expression in the dark and induced full activation within 4 h of red light exposure. We characterized the system's sensitivity to ambient light, red light intensity, and exposure time, finding OptoCre-REDMAP to be reliable and flexible across a range of conditions. In coculture experiments with OptoCre-REDMAP and the blue light responsive OptoCre-VVD, we found that the systems responded orthogonally to red and blue light inputs. Direct comparisons between red and blue light induction with OptoCre-REDMAP and OptoCre-VVD demonstrated the superior penetration properties of red light. OptoCre-REDMAP's robust and selective response to red light makes it suitable for advanced synthetic biology applications, particularly those requiring precise multichromatic control.

Pathophysiology-Driven Approaches for Overcoming Nanomedicine Resistance in Pancreatic Cancer.

Tumor heterogeneity poses a significant challenge in cancer therapy. To address this, we analyze pharmacotherapeutic challenges by categorizing them into static and dynamic barriers, reframing these challenges to improve drug delivery, efficacy, and the development of controlled-release nanomedicines (CRNMs). This pathophysiology-driven approach facilitates the design of novel therapeutics tailored to overcome obstacles in pancreatic ductal adenocarcinoma (PDAC) using nanotechnology. Advanced biomaterials in nanodrug delivery systems offer innovative solutions by combining controlled release, stimuli sensitivity, and smart design strategies. CRNMs are engineered to modulate spatiotemporal signaling and control drug release in PDAC, where resistance to conventional therapies is particularly high. This review explores pharmacokinetic considerations for nanomedicine design, RNA interference (RNAi) for stromal modulation, and the development of targeted nanomedicine strategies. Additionally, we highlight the limitations of current animal models in capturing the complexities of PDAC and discuss notable clinical failures, such as PEGylated hyaluronidase (Phase III HALO 109-301 trial) and evofosfamide (TH-302) with gemcitabine (MAESTRO trial), underscoring the need for improved models and treatment strategies. By targeting pathways like Notch and Hedgehog and incorporating stimuli-sensitive and pathway-modulating agents, CRNMs offer a promising avenue to enhance drug penetration and efficacy, reshaping the paradigm of pancreatic cancer treatment.

Abstract PR016: Early changes to the colon tumor microenvironment during benign-to- malignant transition

Abstract Colorectal cancer (CRC) is now the first and second leading cause of cancer-related deaths in men and women under 50, respectively, upending prior predictions. While many risk factors have been identified, the etiology of early onset disease is poorly understood, and it is unclear why some benign polyps progress to CRC while most do not. The genetic drivers of colon tumor initiation and progression to malignancy are not sufficient to drive disease on their own, as evidenced by the near ubiquitous presence of oncogenic mutations within morphologically “normal” adult tissues like the colon. This raises several fundamental questions: 1) what are the pioneering molecular and cellular events underlying benign-to-malignant transition following oncogenic mutation? 2) Is this sequence of events predetermined by cancer genetics or are additional (micro)environmental stimuli necessary? 3) How does the benign-to-malignant transition reshape tumor clonal architecture and is this immunogenic? We have begun to address these questions using a combination of rare human colon polyps with evidence of early malignant transition and innovative new mouse models that enable control of stepwise colon cancer progression. These autochthonous models combine in vivo CRISPR-Cas9 and an inducible “split-Cre” system to decouple focal tumor initiation in the distal colon (via Apc knockout) from spatiotemporal control of secondary driver events of malignant progression (e.g., Kras G12D, Trp53 knockout) in rare cells of benign adenomas. We have validated the in vivo functionality of these models, generating early “intramucosal carcinoma” and “adenocarcinoma-in-adenoma”—physiologically relevant and elusive stages of human colon cancer development that have never been reliably captured in model systems to date. Comparing these tumors with spatial transcriptomic data from human specimens, we report distinct transcriptional programs of early progression, a unique stromal reaction associated with early progression, and restructuring of glandular architecture and the stem cell niche in early carcinoma. By delineating the earliest events underlying cancer progression, this work promises to inform new strategies for early detection and prevention. We anticipate that the models will also be particularly valuable in functionally interrogating host and environmental factors that impact risk of early onset cancer. Citation Format: Peter M.K. Westcott, Yihan Qin, Daniel Zhang, Nikita Persaud, Zakeria Aminzada, Nischal Bhandari, Colin McLaughlin, William Rideout III, Santiago Naranjo, Song Han, Rodrigo Romero, Claire Regan, Jonathan Preall, Semir Beyaz, Tyler Jacks, Zhen Zhao, Sepideh Gholami. Early changes to the colon tumor microenvironment during benign-to- malignant transition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR016.

Supramolecular Systems Chemistry Based on the Interplay Between Peptides and Porphyrins

AbstractThe functions of living organisms are emergent from networks of biomolecules. In this review, we discuss the creation of synthetic life‐like systems based on the interplay of peptides and porphyrins in the supramolecular chemical systems. In particular, we focus on the spatiotemporal control of self‐assembly processes, which allows for the development of hierarchical structures for biomimetic catalysis and adaptive dynamics for stimulus‐responsive structural transformations. Notably, when operating in a nonequilibrium regime–characterized by kinetic traps, feedback loops, and dissipative conditions–the structural landscape expands and system‐level properties emerge, including transient catalysis, metabolic self‐replication, and Darwinian‐like evolution. Controlling these systems at the biointerface would facilitate intelligent therapeutic interventions in the anti‐tumor phototherapy. Supramolecular systems chemistry provides a valuable framework for exploring new physicochemical spaces of peptides and porphyrins, and may offer distinct advantages and extensive applications across diverse fields.

Azobenzene-Tagged Photopeptides Exhibiting Excellent Selectivity and Light-Induced Cytotoxicity in MCF-7 Cells over HeLa and A549.

The precise regulation of proteasome activity has become a focal point in current research, particularly its implications in cancer treatment. Bortezomib is used for treating multiple myeloma and is found to be ineffective against solid tumors. A spatiotemporal control over the proteasome is one of the solutions to resolve these issues using external stimuli, such as light. Thus, we designed and synthesized azobenzene-containing tripeptide vinyl sulfones 1, 2, 3, and 4, as the azobenzene moiety can impart E↔Z isomerism upon exposure to UV light. Further, the hydrophobicity of these peptides was fine-tuned by systematically varying the size of hydrophobic amino acids at the P1, P2, and P3 positions. The light-induced Z isomers of these photopeptides showed excellent cellular potency in HeLa, MCF-7, and A549 cell lines. Photopeptide 4 with valine at the proximal position, phenylalanine at P2, and leucine at the P1 positions exhibited 19.3- and 6.6-fold cellular potency in MCF-7 and A549 cells, respectively.

Rational Design of Biocompatible Ir(III) Photosensitizer to Overcome Drug-Resistant Cancer via Oxidative Autophagy Inhibition.

Autophagy is a crucial quality control mechanism that degrades damaged cellular components through lysosomal fusion with autophagosomes. However, elevated autophagy levels can promote drug resistance in cancer cells, enhancing their survival. Downregulation of autophagy through oxidative stress is a clinically promising strategy to counteract drug resistance, yet precise control of oxidative stress in autophagic proteins remains challenging. Here, a molecular design strategy of biocompatible neutral Ir(III) photosensitizers is demonstrated, B2 and B4, for precise reactive oxygen species (ROS) control at lysosomes to inhibit autophagy. The underlying molecular mechanisms for the biocompatibility and lysosome selectivity of Ir(III) complexes are explored by comparing B2 with the cationic or the non-lysosome-targeting analogs. Also, the biological mechanisms for autophagy inhibition via lysosomal oxidation are explored. Proteome analyses reveal significant oxidation of proteins essential for autophagy, including lysosomal and fusion-mediator proteins. These findings are verified in vitro, using mass spectrometry, live cell imaging, and a model SNARE complex. The anti-tumor efficacy of the precise lysosomal oxidation strategy is further validated in vivo with B4, engineered for red light absorbance. This study is expected to inspire thetherapeutic use of spatiotemporal ROS control for sophisticated modulation of autophagy.

UV-induced coumarin-based spiropyran gradient photodimerization and photoisomerization to construct near-infrared responsive gradient hydrogel actuators in one-step

In recent years, the fabrication process of near-infrared (NIR) light-driven gradient hydrogels remains complex. Furthermore, NIR light-driven hydrogels based on the photothermal conversion mechanism have continued to face challenges in achieving spatio-temporal control of the internal structure due to the diffusive nature of heat. Therefore, it is sensible to develop NIR light-driven gradient hydrogels based on the near-infrared upconversion luminescence mechanism through a simple method. In this study, a novel photoresponsive coumarin-based spiropyran monomer (SPCMA) was designed and synthesized, followed by the fabrication of a series of double gradient hydrogels (MHMs) with rapid NIR light-driven capability and reusability through fast in situ photopolymerization of the precursor comprising SPCMA, upconversion nanoparticles (MUCNPs), and hydroxyethyl acrylate (HEA). MHMs exhibit excellent mechanical properties with fracture stress of 4.4 MPa and fracture elongation of 1282 %. Compared with hydrogel using N, N′-Methylenebis(2-propenamide) as a crosslinker, MHMs possess outstanding NIR light-driven capability and reversible cycling characteristics. MHMs rapidly transform from a three-dimensional spiral tubular to a sheet-like morphology within 30 s of NIR light irradiation and achieve a larger actuating angle of 450° within 120 s. Additionally, MHMs maintain similar driveability to the original samples after 10 cycles of NIR light-driven and UV light-recovery processes. More importantly, cargoes with seven times the mass of the "gripper" are firmly grasped and easily lifted by the "gripper" prepared using MHMs within 60 s of NIR light irradiation, demonstrating its powerful gripping ability. This work offers a new strategy for the development of biomimetic smart gradient materials.

Spatiotemporal control of subcellular O-GlcNAc signaling using Opto-OGT.

The post-translational modification of intracellular proteins through O-linked β-N-acetylglucosamine (O-GlcNAc) is a conserved regulatory mechanism in multicellular organisms. Catalyzed by O-GlcNAc transferase (OGT), this dynamic modification has an essential role in signal transduction, gene expression, organelle function and systemic physiology. Here, we present Opto-OGT, an optogenetic probe that allows for precise spatiotemporal control of OGT activity through light stimulation. By fusing a photosensitive cryptochrome protein to OGT, Opto-OGT can be robustly and reversibly activated with high temporal resolution by blue light and exhibits minimal background activity without illumination. Transient activation of Opto-OGT results in mTORC activation and AMPK suppression, which recapitulate nutrient-sensing signaling. Furthermore, Opto-OGT can be customized to localize to specific subcellular sites. By targeting OGT to the plasma membrane, we demonstrate the downregulation of site-specific AKT phosphorylation and signaling outputs in response to insulin stimulation. Thus, Opto-OGT is a powerful tool for defining the role of O-GlcNAcylation in cell signaling and physiology.

N-Cadherin — A Potential Target for Psychopharmacology

Glycoprotein N-cadherin (Neuronal cadherin) belongs to the family of calcium-dependent cell adhesion molecules, representing a key element that carries out intercellular contacts in brain neurons. However, it is involved not only in the mechanical connection of neurons, but also influences the specifics of the further development and functional state of the neuron. This is due to the active interaction of N-cadherin with many proteins at the pre- and post-synapse, initiating a cascade of reactions that provide such processes as long-term potentiation (underlying learning and memory), morphogenesis, neuronal recognition, activation of receptors (NMDA and AMPA types), regulation of cytoskeleton formation. This polyfunctionality is necessary for specific neurons to connect to each other in a certain way, and such adhesion leads to the coordination of cell behavior through intercellular signaling and spatio–temporal control of differential gene expression. Mutations in the genes responsible for the expression of N-cadherin lead to various disorders of the functional activity of the synapse and the processes of spatial orientation and memory. Thus, involvement in important neuroplastic processes regulating cognitive functions and behavior determines interest in studying the effect of drugs on N-cadherin. In particular, N-cadherin deserves closer consideration by pharmacologists as a potential target in the mechanism of action of various psychoactive substances.

EXTH-63. FOCUSED ULTRASOUND HYPERTHERMIA MEDIATED CONTROL OF THERMAL RESPONSIVE CAR T CELL ACTIVITY IN BREAST CANCER BRAIN METASTASIS

Abstract HER2-targeted therapies are promising treatment options for metastatic breast cancer and have improved the median overall survival. However, breast cancer brain metastasis (BCBM), observed in up to 50% of HER2-positive breast cancer patients, remains a clinical challenge. While Chimeric Antigen Receptor (CAR) T cell therapy is promising therapeutic strategy to address the unmet need in treating BCBM, improving its effectiveness and safety is critical for clinical translation. We hypothesized that spatially and temporally controlled activity of CAR-T cells with a thermal gene switch (TS) under MRI-guided focused ultrasound (MRgFUS) would potentiate anti-tumor responses in difficult-to-treat BCBM. To test our hypothesis, we developed a closed-loop controlled MR-thermometry (MRTI) based FUS system (MRgFUS) and αHER2 CAR-T cells engineered with TS expressing the reporter gene firefly luciferase (TS.Fluc) or bi-specific engager on NKG2D ligands (TS.BTE) upon MRgFUS-hyperthermia (41.5°C). In vivo quantification of TS activity from intratumorally delivered TS.Fluc αHER2 CAR-T cells in HER2+ BCBM mice model (HER2+ MDA-MB-468 in NSG mice with primary human CAR-T cells) followed by pulsed MRgFUS-hyperthermia resulted in a ~10-fold increase in luminescent activity compared to unheated mice. To assess longitudinal activation, we quantified TS activity with a second sonication 4 days post the initial FUS treatment. Prior to the second sonication, TS.Fluc expression had returned to baseline levels, while following it robust TS.Fluc expression was observed again, demonstrating that MRgFUS can activate engineered T cells with TS in brain tumors with high spatiotemporal control. Subsequently we investigated the therapeutic efficacy of intratumorally delivered TS.BTE αHER2 CAR T cells in mixed tumor model of heterogeneous HER2 MDA-MB-468 (75% HER2+) and observed significant tumor regression of HER2- tumor cells in the treated group compared to controls, demonstrating the potential of the proposed strategy to noninvasively drive the local production of key transgenes and potentiate anti-tumor responses in difficult-to-treat BCBM.

A Rationally Designed Azobenzene Photoswitch for DNA G-Quadruplex Regulation in Live Cells.

G-quadruplex (G4) DNA structures are increasingly acknowledged as promising targets in cancer research, and the development of G4-specific stabilizing compounds may lay a fundamental foundation in precision medicine for cancer treatment. Here, we propose a light-responsive G4-binder for precise modulation of drug activation, providing dynamic and spatiotemporal control over G4-associated biological processes contributing to cancer cell death. We developed a specialized fluorinated azobenzene (AB) switch equipped with a quinoline unit and a positively charged carboxamide side chain, Q-Azo4F-C, designed for targeted binding to G4 structures within cells. Biophysical studies, combined with molecular dynamics simulations, provide insights into the unique coordination modes of the photoswitchable ligand in its trans and cis configurations when interacting with G4s. The observed variations in complexation processes between the two isomeric states in different cancer cell lines manifest in more than 25-fold reversible cytotoxic activity. Immunostaining conducted with the structure-specific G4 antibody (BG4), establishes a direct correlation between cytotoxicity and the varying extent of G4 induction regulated by the two isoforms. Finally, we demonstrate the photo-driven reversible regulation of G4 structures in lung cancer cells by Q-Azo4F-C. Our findings highlight the potential of light-responsive G4-binders in advancing precision cancer therapy through dynamic control of G4-mediated pathways.

A Rationally Designed Azobenzene Photoswitch for DNA G‐Quadruplex Regulation in Live Cells

AbstractG‐quadruplex (G4) DNA structures are increasingly acknowledged as promising targets in cancer research, and the development of G4‐specific stabilizing compounds may lay a fundamental foundation in precision medicine for cancer treatment. Here, we propose a light‐responsive G4‐binder for precise modulation of drug activation, providing dynamic and spatiotemporal control over G4‐associated biological processes contributing to cancer cell death. We developed a specialized fluorinated azobenzene (AB) switch equipped with a quinoline unit and a positively charged carboxamide side chain, Q‐Azo4F‐C, designed for targeted binding to G4 structures within cells. Biophysical studies, combined with molecular dynamics simulations, provide insights into the unique coordination modes of the photoswitchable ligand in its trans and cis configurations when interacting with G4s. The observed variations in complexation processes between the two isomeric states in different cancer cell lines manifest in more than 25‐fold reversible cytotoxic activity. Immunostaining conducted with the structure‐specific G4 antibody (BG4), establishes a direct correlation between cytotoxicity and the varying extent of G4 induction regulated by the two isoforms. Finally, we demonstrate the photo‐driven reversible regulation of G4 structures in lung cancer cells by Q‐Azo4F‐C. Our findings highlight the potential of light‐responsive G4‐binders in advancing precision cancer therapy through dynamic control of G4‐mediated pathways.