Single-cell multi-omics analysis identifies context-specific gene regulatory gates and mechanisms.

There is a growing interest in inferring context specific gene regulatory networks from single-cell RNA sequencing (scRNA-seq) data. This involves identifying the regulatory relationships between transcription factors (TFs) and genes in individual cells, and then characterizing these relationships at the level of specific cell types or cell states. In this study, we introduce scGATE (single-cell gene regulatory gate) as a novel computational tool for inferring TF-gene interaction networks and reconstructing Boolean logic gates involving regulatory TFs using scRNA-seq data. In contrast to current Boolean models, scGATE eliminates the need for individual formulations and likelihood calculations for each Boolean rule (e.g. AND, OR, XOR). By employing a Bayesian framework, scGATE infers the Boolean rule after fitting the model to the data, resulting in significant reductions in time-complexities for logic-based studies. We have applied assay for transposase-accessible chromatin with sequencing (scATAC-seq) data and TF DNA binding motifs to filter out non-relevant TFs in gene regulations. By integrating single-cell clustering with these external cues, scGATE is able to infer context specific networks. The performance of scGATE is evaluated using synthetic and real single-cell multi-omics data from mouse tissues and human blood, demonstrating its superiority over existing tools for reconstructing TF-gene networks. Additionally, scGATE provides a flexible framework for understanding the complex combinatorial and cooperative relationships among TFs regulating target genes by inferring Boolean logic gates among them.

Dual-inhibitory domain iCARs improve the efficiency of the AND-NOT gate CAR T strategy.

CAR (chimeric antigen receptor) T cell therapy has shown clinical success in treating hematological malignancies, but its treatment of solid tumors has been limited. One major challenge is on-target, off-tumor toxicity, where CAR T cells also damage normal tissues that express the targeted antigen. To reduce this detrimental side-effect, Boolean-logic gates like AND-NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected nonmalignant tissue sites. However, the strategy seems inefficient, requiring high levels of iCAR and its target antigen for inhibition. Using a TROP2-targeting iCAR with a single PD1 inhibitory domain to inhibit a CEACAM5-targeting CAR (CEACAR), we observed that the inefficiency was due to a kinetic delay in iCAR inhibition of cytotoxicity. To improve iCAR efficiency, we modified three features of the iCAR-the avidity, the affinity, and the intracellular signaling domains. Increasing the avidity but not the affinity of the iCAR led to significant reductions in the delay. iCARs containing twelve different inhibitory signaling domains were screened for improved inhibition, and three domains (BTLA, LAIR-1, and SIGLEC-9) each suppressed CAR T function but did not enhance inhibitory kinetics. When inhibitory domains of LAIR-1 or SIGLEC-9 were combined with PD-1 into a single dual-inhibitory domain iCAR (DiCARs) and tested with the CEACAR, inhibition efficiency improved as evidenced by a significant reduction in the inhibitory delay. These data indicate that a delicate balance between CAR and iCAR signaling strength and kinetics must be achieved to regulate AND-NOT gate CAR T cell selectivity.

TALEN-edited allogeneic inducible dual CAR T cells enable effective targeting of solid tumors while mitigating off-tumor toxicity.

Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has been limited in solid tumors. One key factor for this is cancer-associated fibroblasts (CAFs) that modulate the tumor microenvironment (TME) to inhibit T cell infiltration and induce "T cell dysfunction." Additionally, the sparsity of tumor-specific antigens (TSA) and expression of CAR-directed tumor-associated antigens (TAA) on normal tissues often results in "on-target off-tumor" cytotoxicity, raising safety concerns. Using TALEN-mediated gene editing, we present here an innovative CAR T cell engineering strategy to overcome these challenges. Our allogeneic "Smart CAR T cells" are designed to express a constitutive CAR, targeting FAP+ CAFs in solid tumors. Additionally, a second CAR targeting a TAA such as mesothelin is specifically integrated at a TCR signaling-inducible locus like PDCD1. FAPCAR-mediated CAF targeting induces expression of the mesothelin CAR, establishing an IF/THEN-gated circuit sensitive to dual antigen sensing. Using this approach, we observe enhanced anti-tumor cytotoxicity, while limiting "on-target off-tumor" toxicity. Our study thus demonstrates TALEN-mediated gene editing capabilities for design of allogeneic IF/THEN-gated dual CAR T cells that efficiently target immunotherapy-recalcitrant solid tumors while mitigating potential safety risks, encouraging clinical development of this strategy.

Securing LYTAC with Logic-Identification System for Cancer Cell-Selective Membrane Protein Degradation.

Lysosome-targeting chimera (LYTAC) links proteins of interest (POIs) with lysosome-targeting receptors (LTRs) to achieve membrane protein degradation, which is becoming a promising therapeutic modality. However, cancer cell-selective membrane protein degradation remains a big challenge considering expressions of POIs in both cancer cells and normal cells, as well as broad tissue distribution of LTRs. Here a logic-identification system is designed, termed Logic-TAC, based on cell membrane-guided DNA calculations to secure LYTAC selectively for cancer cells. Logic-TAC is designed as a duplex DNA structure, with both POI and LTR recognition regions sealed to avoid systematic toxicity during administration. MCF-7 and MCF-10A are chosen as sample cancer cell and normal cell respectively. As input 1 for logic-identification, membrane proteins EpCAM, which is highly expressed by MCF-7 but barely by MCF-10A, reacts with Logic-TAC to expose POI recognition region. As input 2 for logic-identification, Logic-TAC binds to POI, membrane protein MUC1, to expose LTR recognition region. As output, MUC1 is connected to LTR and degraded via lysosome pathway selectively for cancer cell MCF-7 with little side effect on normal cell MCF-10A. The logic-identification system also demonstrated satisfactory in vivo therapeutic results, indicating its promising potential in precise targeted therapy.

Split Proteins and Reassembly Modules for Biological Applications.

Split systems, modular entities enabling controlled biological processes, have become instrumental in biological research. This review highlights their utility across applications like gene regulation, protein interaction identification, and biosensor development. Covering significant progress over the last decade, it revisits traditional split proteins such as GFP, luciferase, and inteins, and explores advancements in technologies like Cas proteins and base editors. We also examine reassembly modules and their applications in diverse fields, from gene regulation to therapeutic innovation. This review offers a comprehensive perspective on the recent evolution of split systems in biological research.

High-throughput screen to identify and optimize NOT gate receptors for cell therapy.

Logic-gated engineered cells are an emerging therapeutic modality that can take advantage of molecular profiles to focus medical interventions on specific tissues in the body. However, the increased complexity of these engineered systems may pose a challenge for prediction and optimization of their behavior. Here we describe the design and testing of a flow cytometry-based screening system to rapidly select functional inhibitory receptors from a pooled library of candidate constructs. In proof-of-concept experiments, this approach identifies inhibitory receptors that can operate as NOT gates when paired with activating receptors. The method may be used to generate large datasets to train machine learning models to better predict and optimize the function of logic-gated cell therapeutics.

Boolean Logic Gate Operation in Bacteriorhodopsin of Purple Membrane Based on a Molten Globule-like State.

Bacteriorhodopsin (bR) of purple membrane (PM) has increasing technical interests, particularly in photonic devices and bioelectronics. The present work has concerned with monitoring the temperature dependence of passive electric responses in-plane and out-of-plane of the membranes. Based on thermal properties observed orthogonally here for PM, a high-temperature intermediate of bR has been suggested to populate at around 60 °C, which may be ascribed to a molten globule-like state. This intermediate has been found to be enclosed between two reversible thermal transitions for PM. Large-scale turnover in the energy of activation, for these two thermal transitions, occurs steeply at such state at 60 °C, above which does bR reverse the sign of dielectric anisotropy (i.e. crossover) provided the operating frequency should be above the crossover frequency, at which the reversal occurs. No such crossover was found to occur below the crossover frequency, even above the crossover temperature (i.e. 60 °C). Likewise, no such crossover was found to occur below the crossover temperature, even above the crossover frequency. Relying on this reasoning, a logic gate operation may be declared implicating bR for bioelectronics and sense technological relevance. In addition, the results specify "dual frequency" as well as "dual temperature" characteristics to bacteriorhodopsin.

Selective Detection of Divalent Cations (Cu2+, Zn2+, Pb2+) and Anions (SO42-, S2-, CO32-) Using a pH-Sensitive Multi-functional Schiff Base in Neutral Medium.

A new Schiff base-based multi-cation/anion probe (L) has been synthesized and characterized using HR-MS, FT-IR, 1H, and 13C NMR techniques. The Schiff base motif provides specific binding sites that detect cations and anions by generating distinct optical output signals upon interaction. A noticeable color change of the probe solution was observed from pale yellow to various shades of yellow upon adding cations such as Cu2+, Zn2+, and Pb2+ and anions such as CO32⁻, S2⁻, and SO42⁻. This color change results from forming complexes like M3L2 with metal ions. Whereas origin of color in presence of anion were attributed due to the deprotonation of acidic proton in the ligand. Moreover, the complexes formed by Zn2+, S2/CO32⁻ ion with L are fluorescent, enabling the detection of Cu2+ and SO42⁻ using the Stern-Volmer plot, with a limit of detection (LODs) of 8.48 µM and 10.47 µM, respectively. Additionally, increasing the pH of the probe solution above 8 reveals a significant enhancement of fluorescence intensity due to the deprotonation of phenolic -OH and amide -NH in the presence of hydroxide ions. This emission in the basic medium is quenched by Cu2+ ions and restored when Cu2+ is complexed with EDTA. A logic gate has also been constructed for understanding the TURN-OFF-TURN-ON mechanism involving Cu2+ ions and EDTA. Overall, the versatile performance of a single probe L opens up new possibilities as a multifunctional sensor, making it highly suitable for practical applications.

Release and Transport of Nanomaterials from Hydrogels Controlled by Temperature.

Understanding the transport of nanoparticles from and within hydrogels is a key issue for the design of nanocomposite hydrogels for drug delivery systems and tissue engineering. To investigate the translocation of nanocarriers from and within hydrogel networks triggered by changes of temperature, ultrasmall (8 nm) and small (80 nm) silica nanocapsules are embedded in temperature-responsive hydrogels and non-responsive hydrogels. The ultrasmall silica nanocapsules are released from temperature-responsive hydrogels to water or transported to other hydrogels upon direct activation by heating or indirect activation by Joule heating; while, they are not released from non-responsive hydrogel. Programmable transport of nanocarriers from and in hydrogels provides insights for the development of complex biomedical devices and soft robotics.

Controllable and reusable seesaw circuit based on nicking endonucleases.

Seesaw circuits are essential for molecular computing and biosensing. However, a notable limitation of seesaw circuits lies in the irreversible depletion of components, precluding the attainment of system recovery and rendering nucleic acid circuits non-reusable. We developed a brand-new method for creating controllable and reusable seesaw circuits. By using the nicking endonucleases Nt.BbvCI and Nt.Alwi, we removed "functional components" while keeping the "skeletal components" for recurrent usage. T-inputs were introduced, increasing the signal-to-noise ratio of AND logic from 2.68 to 11.33 and demonstrating compatibility. We identified the logic switching feature and verified that it does not impair circuit performance. We also built intricate logic circuits, such as OR-AND gate, to demonstrate the versatility of our methodology. This controllable reusability extends the applications of nanotechnology and bioengineering, enhancing the practicality and efficiency of these circuits across various domains.

Ratiometric fluorescence probe based on boric acid-modified carbon dots and alizarin red for sensitive and rapid detection of glyphosate.

By combining boric acid-modified carbon dots (p-CDs) and alizarin red (ARS), a double emission probe p-CDs@ARS with fluorescence at 410 nm and 600 nm is designed for the detection of glyphosate. When Cu2+ is added, it binds with ARS to cause ARS release from p-CDs@ARS, which decreases the fluorescence at 600 nm. However, in the presence of glyphosate, glyphosate competes to the binding of Cu2+, releasing ARS to bind with p-CDs again. Therefore, the fluorescence of 600 nm recovers. Based on this, the fluorescence of 410 nm and 600 nm act as the reference and response signal, respectively, achieving the ratiometric fluorescence detection of glyphosate. The linear range of glyphosate detection is 0.5-50 µM with a limit of detection at 0.37 µM which is well below the maximum residue limit for glyphosate in food. When the probe is used to detect the glyphosate residue in Pearl River water and cucumber, the detection results are well consistent with those detected by HPLC. The established method based on p-CDs@ARS has the advantages that the assembly of ratiometric fluorescence probe is simple, and the detection speed is fast. Additionally, a typical INHIBIT logical system has been successfully constructed based on glyphosate, Cu2+, and the fluorescence signal of p-CDs@ARS.

An Exo III-powered closed-loop DNA circuit architecture for biosensing/imaging.

With their regulated Boolean logic operations in vitro and in vivo, DNA logic circuits have shown great promise for target recognition and disease diagnosis. However, significant obstacles must be overcome to improve their operational efficiency and broaden their range of applications. In this study, we propose an Exo III-powered closed-loop DNA circuit (ECDC) architecture that integrates four highly efficient AND logic gates. The ECDC utilizes Exo III as the sole enzyme-activated actuator, simplifying the circuit design and ensuring optimal performance. Moreover, the use of Exo III enables a self-feedback (autocatalytic) mechanism in the dynamic switching between AND logic gates within this circulating logic circuit. After validating the signal flow and examining the impact of each AND logic gate on the regulation of the circuit, we demonstrate the intelligent determination of miR-21 using the carefully designed ECDC architecture in vitro. The proposed ECDC exhibits a linear detection range for miR-21 from 0 to 300 nM, with a limit of detection (LOD) of approximately 0.01 nM, surpassing most reported methods. It also shows excellent selectivity for miR-21 detection and holds potential for identifying and imaging live cancer cells. This study presents a practical and efficient strategy for monitoring various nucleic acid-based biomarkers in vitro and in vivo through specific sequence modifications, offering significant potential for early cancer diagnosis, bioanalysis, and prognostic clinical applications.

High-Performance Integrated Iontronic Circuits Based on Single Nano/Microchannels.

Recently, artificial channel-based ionic diodes and transistors are extensively studied to mimic biological systems. Most of them are constructed vertically and are challenging to be further integrated. Several examples of ionic circuits with horizontal ionic diodes are reported. However, they generally require nanoscale channel sizes to meet the demand for ion-selectivity, resulting in low current output and restricting potential applications. In this paper, a novel ionic diode is developed based on multiple-layer polyelectrolyte nanochannel network membranes. Both bipolar and unipolar ionic diodes can be achieved by simply switching the modification solution. Ionic diodes with a high rectification ratio of ≈226 are achieved in single channels with the largest channel size of 2.5 µm. This design can significantly reduce the channel size requirement and improve the output current level of ionic devices. The high-performance ionic diode with a horizontal structure enables the integration of advanced iontronic circuits. Ionic transistors, logic gates, and rectifiers are fabricated on a single chip and demonstrated for current rectification. Furthermore, the excellent current rectification ratio and the high output current of the on-chip ionic devices highlight the promise of the ionic diode as a component of complex iontronic systems for practical applications.

Sequence-Independent Logical Regulation of the Assembly and Disassembly of DNA Polyhedra.

Controlling the self-assembly of DNA nanostructures using rationally designed logic gates is a major goal of dynamic DNA nanotechnology, which could facilitate the development of biomedicine, molecular computation, et al. In previous works, the regulations mostly relied on either toehold-mediated strand displacement or stimuli-driven conformational switch, requiring elaborately-designed or specific DNA sequences. Herein, we reported a facile, base-sequence-independent strategy for logically controlling DNA self-assembly through external molecules. The INHIBIT and XOR logic controls over the assembly/disassembly of DNA polyhedra were realized through cystamine (Cyst) and ethylenediamine (EN) respectively, which were further integrated into a half subtractor circuit thanks to the sharing of the same inputs. Our work provides a sequence-independent strategy in logically controlling DNA self-assembly, which may open up new possibilities for dynamic DNA nanotechnology.

Dual Chemiexcitation by a Unique Dioxetane Scaffold Gated by an OR Logic Set of Triggers.

Chemiexcitation of phenoxy-1,2-dioxetane chemiluminescent luminophores is initiated by electron transfer from a meta-positioned phenolate ion to the peroxide-dioxetane bond. Here we report the development of a unique 1,2-dioxetane chemiluminescent scaffold with chemiexcitation gated by an OR logic dual-set of triggering events. This scaffold is composed of meta-dihydroxyphenyl-1,2-dioxetane-adamantyl molecules, equipped with acrylic acid and chlorine substituents, that chemiexcitation under physiological conditions. A dual-mode chemiluminescent probe, armed with two different triggering substrates designed for activation by the enzymes ß-galactosidase and alkaline phosphatase, was synthesized. The probe emitted intense light signals in the response to each enzyme, demonstrating its ability to serve as a single-component chemiluminescent sensor for dual-analyte detection. We also demonstrated the ability of the probe to detect ß-galactosidase and phosphatase activities in bacteria. This is the first 1,2-dioxetane scaffold capable of responding to two different chemiexcitation events from two different positions on the same dioxetane molecule. We anticipate that the OR-gated mode of chemiexcitation, described herein, will find utility in the preparation of chemiluminescent probes with a dual-analyte detection/imaging mode.

Improving the tumor selectivity of T cell engagers by logic-gated dual tumor-targeting.

Targeting single tumor antigens makes it difficult to provide sufficient tumor selectivity for T cell engagers (TCEs), leading to undesirable toxicity and even treatment failure, which is particularly serious in solid tumors. Here, we designed novel trispecific TCEs (TriTCEs) to improve the tumor selectivity of TCEs by logic-gated dual tumor-targeting. TriTCE can effectively redirect and activate T cells to kill tumor cells (∼18 pM EC50) by inducing the aggregation of dual tumor antigens, which was ∼70- or 750- fold more effective than the single tumor-targeted isotype controls, respectively. Further in vivo experiments indicated that TriTCE has the ability to accumulate in tumor tissue and can induce circulating T cells to infiltrate into tumor sites. Hence, TriTCE showed a stronger tumor growth inhibition ability and significantly prolonged the survival time of the mice. Finally, we revealed that this concept of logic-gated dual tumor-targeted TriTCE can be applied to target different tumor antigens. Cumulatively, we reported novel dual tumor-targeted TriTCEs that can mediate a robust T cell response by simultaneous recognition of dual tumor antigens at the same cell surface. TriTCEs allow better selective T cell activity on tumor cells, resulting in safer TCE treatment.

Anthracene scaffold as highly selective chemosensor for Al3+ and its AIEE activity.

Fluorescent chemosensor, 3-(Anthracen-2-yliminomethyl)-benzene-1,2-diol (ANB) has been synthesized by one-step condensation of 2-aminoanthracene and 2,3-dihydroxybenzaldehyde and characterized using 1H-NMR, FT-IR and Mass spectroscopic techniques. The probe ANB was found to be an efficient 'turn-on' fluorescence chemosensor for the selective detection of Al3+ ion over other metal ions in an aqueous solution. The chemosensor exhibits ~ 27-fold enhancement of emission intensity in presence of Al3+ ion. Fluorescence quantum values for ANB and (Al3+-ANB)-complex are 0.004 and 0.097, respectively. In addition, the binding constant and the limit of detection were found to be 1.22 × 104 M-1 and 0.391 µM, respectively. The chemosensor ANB binds to Al3+ ions in 2:1 stoichiometric ratio which was supported by Job's plot, 1H-NMR titration and florescence titration. Fluorescence reversibility of the sensor complex was well established by adding EDTA in the same condition and a molecular INHIBIT logic gate was fabricated using this reversible nature of the sensor complex. Additionally, the chemosensor ANB shows a novel aggregation-induced enhanced emission phenomenon, where the aggregate hydrosol of ANB shows enhance emission intensity.

Metal ion-induced assembly of dipeptide-attached perylenediimide for fluorometric "turn on" detection of biologically important small molecule.

A dipeptide-appended perylenediimide (PDI-CFF) fluorescent molecule was designed, synthesized, and characterized. Though the molecule does not dissolve in any individual solvent, it dissolves well in an organic/water mixed solvent system such as tetrahydrofuran/water. This new fluorescent molecule was self-assembled in a tetrahydrofuran/water mixture to form both nanofibrous network structures and a nano ring structure. It has shown nanofibril morphology by the interactions with ferric ions (PDI-CFF/Fe3+ system) with diminishing fluorescent property. Interestingly, L-ascorbic acid (LAA) interacts with the PDI-CFF/Fe3+ system, showing turn-on fluorescence. Another interesting feature is that the minimum detection limits for Fe3+ ions and LAA are at the submicromolar levels of 6.2 × 10-8 and 3 × 10-8  M, respectively. Moreover, the fluorescent (10 µM) signals can be monitored by the naked eye under handheld UV lamp irradiation at 365 nm, and this is very convenient for the real application. In this study, the molecule offers the opportunity for processing these sequential fluorescence responses in order to fabricate a implication logic gate that includes NOT, AND, and OR simple logic gates using chemical stimuli (ferric ions and LAA) as inputs and fluorescence emission at 536 nm as output. The detailed mechanism of interactions of Fe3+ with PDI-CFF and LAA with the PDI-CFF/Fe3+ system is vividly studied by using Fourier transform infrared (FT-IR) analysis and fluorescence. Moreover, this new molecule was reusable for several times without significant loss of its activity. The construction of logic gates using biologically important molecules/ions holds future promise for the design and development of new bio-logic gates.

A Fast-Response, Phenanthroimidazole-Based Fluorescent Probe for Selective Detection of HClO.

A new phenanthroimidazole-based fluorescence probe for selective detection of HClO was synthesized and characterized using 1HNMR, 13CNMR, IR, and HRMS. With benzenesulfonohydrazide as the identification group, the probe demonstrated a fast fluorescence response from yellow-green to blue when the HC = N double bond was oxidized and broken into an aldehyde group by HClO. The probe showed high selectivity and sensitivity towards HClO with approximately 4.5-fold fluorescence enhancement and has been successfully applied in the molecular logic gate, determination of HClO in environmental water samples, and portable HClO detection.

A Novel Rhodamine Probe Acting as Chemosensor for Selective Recognition of Cu2+ and Hg2+ Ions: An Experimental and First Principle Studies.

Copper and Mercury ions have vital role to play in biological world as their excess or deficiency can cause different type of diseases in human being as well as biological species including plants and animals. Therefore, their detection at trace level becomes very important in term of biological. The current studies embody the fabrication, structural characterization and recognition behavior of a novel rhodamine B hydrazone formed when hydrazide of rhodamine B was condensed with 5-Allyl-3-methoxy salicylaldehyde (RBMA). RBMA was found to be responsive towards the very trace level of Cu2+ and Hg2+ among other tested cations so far. The sensing procedure is based on the classical opening of the spiroatom ring of rhodamine. The limit of detection (LOD) and binding constant is 5.35 ppm, 2.06 × 104 M-1 and 5.16 ppm, 1.26 × 104 M-1 for Cu2+ and Hg2+ ions respectively. The probable mechanism correlates the specific binding of RBMA with Cu2+ and Hg2+ ions. The 1:1 stoichiometry of RBMA with Cu2+ and Hg2+ ions have been supported by HRMS, FT-IR data, Job's plot, and binding constant data. Reversibility is well exhibited by RBMA by the involvement of CO32- ions via demetallation process. The real time application is well demonstrated by the use of paper strip test. The DFT study also carried out which agrees well with the experimental findings. The results displayed the novelty of this current work towards the trace level analysis of the Cu2+ and Hg2+ of the cations which are play the crucial role in industry.