Spatial engineering of E. coli with addressable phase-separated RNAs.

Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E. coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.

RNA-Dependent Epigenetic Silencing Directs Transcriptional Downregulation Caused by Intronic Repeat Expansions.

Transcriptional downregulation caused by intronic triplet repeat expansions underlies diseases such as Friedreich's ataxia. This downregulation of gene expression is coupled with epigenetic changes, but the underlying mechanisms are unknown. Here, we show that an intronic GAA/TTC triplet expansion within the IIL1 gene of Arabidopsis thaliana results in accumulation of 24-nt short interfering RNAs (siRNAs) and repressive histone marks at the IIL1 locus, which in turn causes its transcriptional downregulation and an associated phenotype. Knocking down DICER LIKE-3 (DCL3), which produces 24-nt siRNAs, suppressed transcriptional downregulation of IIL1 and the triplet expansion-associated phenotype. Furthermore, knocking down additional components of the RNA-dependent DNA methylation (RdDM) pathway also suppressed both transcriptional downregulation of IIL1 and the repeat expansion-associated phenotype. Thus, our results show that triplet repeat expansions can lead to local siRNA biogenesis, which in turn downregulates transcription through an RdDM-dependent epigenetic modification.

DNA triplet repeat expansion and mismatch repair.

DNA mismatch repair is a conserved antimutagenic pathway that maintains genomic stability through rectification of DNA replication errors and attenuation of chromosomal rearrangements. Paradoxically, mutagenic action of mismatch repair has been implicated as a cause of triplet repeat expansions that cause neurological diseases such as Huntington disease and myotonic dystrophy. This mutagenic process requires the mismatch recognition factor MutSß and the MutLα (and/or possibly MutLγ) endonuclease, and is thought to be triggered by the transient formation of unusual DNA structures within the expanded triplet repeat element. This review summarizes the current knowledge of DNA mismatch repair involvement in triplet repeat expansion, which encompasses in vitro biochemical findings, cellular studies, and various in vivo transgenic animal model experiments. We present current mechanistic hypotheses regarding mismatch repair protein function in mediating triplet repeat expansions and discuss potential therapeutic approaches targeting the mismatch repair pathway.

Enhanced triplet superconductivity in next-generation ultraclean UTe2.

The unconventional superconductor UTe[Formula: see text] exhibits numerous signatures of spin-triplet superconductivity-a rare state of matter which could enable quantum computation protected against decoherence. UTe[Formula: see text] possesses a complex phase landscape comprising two magnetic field-induced superconducting phases, a metamagnetic transition to a field-polarized state, along with pair- and charge-density wave orders. However, contradictory reports between studies performed on UTe[Formula: see text] specimens of varying quality have severely impeded theoretical efforts to understand the microscopic origins of the exotic superconductivity. Here, we report a comprehensive suite of high magnetic field measurements on a generation of pristine quality UTe[Formula: see text] crystals. Our experiments reveal a significantly revised high magnetic field superconducting phase diagram in the ultraclean limit, showing a pronounced sensitivity of field-induced superconductivity to the presence of crystalline disorder. We employ a Ginzburg-Landau model that excellently captures this acute dependence on sample quality. Our results suggest that in close proximity to a field-induced metamagnetic transition the enhanced role of magnetic fluctuations-that are strongly suppressed by disorder-is likely responsible for tuning UTe[Formula: see text] between two distinct spin-triplet superconducting phases.

Reaction amplification with a gain: Triplet exciton-mediated quantum chain using mixed crystals with a tailor-made triplet sensitizer.

Photochemical valence bond isomerization of a crystalline Dewar benzene (DB) diacid monoanion salt with an acetophenone-linked piperazinium cation that serves as an intramolecular triplet energy sensitizer (DB-AcPh-Pz) exhibits a quantum chain reaction with as many as 450 product molecules per photon absorbed (Φ ≈ 450). By contrast, isomorphous crystals of the DB diacid monosalt of an ethylbenzene-linked piperazinium (DB-EtPh-Pz) lacking a triplet sensitizer showed a less impressive quantum yield of ca. Φ ≈ 22. To establish the critical importance of a triplet excited state carrier in the adiabatic photochemical reaction we prepared mixed crystals with DB-AcPh-Pz as a dilute triplet sensitizer guest in crystals of DB-EtPh-Pz. As expected from their high structural similarities, solid solutions were easily formed with the triplet sensitizer salt in the range of 0.1 to 10%. Experiments carried out under conditions where light is absorbed by the triplet sensitizer-linked DB-AcPh-Pz can be used to initiate a triplet state adiabatic reaction from 3DB-AcPh-Pz to 3HB*-AcPh-Pz, which can serve as a chain carrier and transfer energy to an unreacted DB-EtPh-Pz where exciton delocalization in the crystalline solid solution can help carry out an efficient energy transfer and enable a quantum chain employing the photoproduct as a triplet chain carrier. Excitation of mixed crystals with as little as 0.1% triplet sensitizer resulted in an extraordinarily high quantum yield Φ ≈ 517.

Polarizing agents beyond pentacene for efficient triplet dynamic nuclear polarization in glass matrices.

Triplet dynamic nuclear polarization (triplet-DNP) is a technique that can obtain high nuclear polarization under moderate conditions. However, in order to obtain practically useful polarization, large single crystals doped with a polarizing agent must be strictly oriented with respect to the magnetic field to sharpen the electron spin resonance (ESR) spectra, which is a fatal problem that prevents its application to truly useful biomolecular targets. Instead of this conventional physical approach of controlling crystal orientation, here, we propose a chemical approach, i.e., molecular design of polarizing agents; pentacene molecules, the most typical triplet-DNP polarizing agent, are modified so as to make the triplet electron distribution wider and more isotropic without loss of the triplet polarization. The thiophene-modified pentacene exhibits a sharper and stronger ESR spectrum than the parent pentacene, and state-of-the-art quantum chemical calculations revealed that the direction of the spin polarization is altered by the modification with thiophene moieties and the size of D and E parameters are reduced from parent pentacene due to the partial delocalization of spin densities on the thiophene moieties. The triplet-DNP with the new polarizing agent successfully exceeds the previous highest 1H polarization of glassy materials by a factor of 5. This demonstrates the feasibility of a polarizing agent that can surpass pentacene, the best polarizing agent for more than 30 y since triplet-DNP was first reported, in the unoriented state. This work provides a pathway toward practically useful high nuclear polarization of various biomolecules by triplet-DNP.

FAN1 removes triplet repeat extrusions via a PCNA- and RFC-dependent mechanism.

Human genome-wide association studies have identified FAN1 and several DNA mismatch repair (MMR) genes as modifiers of Huntington's disease age of onset. In animal models, FAN1 prevents somatic expansion of CAG triplet repeats, whereas MMR proteins promote this process. To understand the molecular basis of these opposing effects, we evaluated FAN1 nuclease function on DNA extrahelical extrusions that represent key intermediates in triplet repeat expansion. Here, we describe a strand-directed, extrusion-provoked nuclease function of FAN1 that is activated by RFC, PCNA, and ATP at physiological ionic strength. Activation of FAN1 in this manner results in DNA cleavage in the vicinity of triplet repeat extrahelical extrusions thereby leading to their removal in human cell extracts. The role of PCNA and RFC is to confer strand directionality to the FAN1 nuclease, and this reaction requires a physical interaction between PCNA and FAN1. Using cell extracts, we show that FAN1-dependent CAG extrusion removal relies on a very short patch excision-repair mechanism that competes with MutSß-dependent MMR which is characterized by longer excision tracts. These results provide a mechanistic basis for the role of FAN1 in preventing repeat expansion and could explain the antagonistic effects of MMR and FAN1 in disease onset/progression.

spaCI: deciphering spatial cellular communications through adaptive graph model.

Cell-cell communications are vital for biological signalling and play important roles in complex diseases. Recent advances in single-cell spatial transcriptomics (SCST) technologies allow examining the spatial cell communication landscapes and hold the promise for disentangling the complex ligand-receptor (L-R) interactions across cells. However, due to frequent dropout events and noisy signals in SCST data, it is challenging and lack of effective and tailored methods to accurately infer cellular communications. Herein, to decipher the cell-to-cell communications from SCST profiles, we propose a novel adaptive graph model with attention mechanisms named spaCI. spaCI incorporates both spatial locations and gene expression profiles of cells to identify the active L-R signalling axis across neighbouring cells. Through benchmarking with currently available methods, spaCI shows superior performance on both simulation data and real SCST datasets. Furthermore, spaCI is able to identify the upstream transcriptional factors mediating the active L-R interactions. For biological insights, we have applied spaCI to the seqFISH+ data of mouse cortex and the NanoString CosMx Spatial Molecular Imager (SMI) data of non-small cell lung cancer samples. spaCI reveals the hidden L-R interactions from the sparse seqFISH+ data, meanwhile identifies the inconspicuous L-R interactions including THBS1-ITGB1 between fibroblast and tumours in NanoString CosMx SMI data. spaCI further reveals that SMAD3 plays an important role in regulating the crosstalk between fibroblasts and tumours, which contributes to the prognosis of lung cancer patients. Collectively, spaCI addresses the challenges in interrogating SCST data for gaining insights into the underlying cellular communications, thus facilitates the discoveries of disease mechanisms, effective biomarkers and therapeutic targets.

Photon Upconversion at Organic-Inorganic Interfaces.

Photon upconversion is a process that combines low-energy photons to form useful high-energy photons. There are potential applications in photovoltaics, photocatalysis, biological imaging, etc. Semiconductor quantum dots (QDs) are promising for the absorption of these low-energy photons due to the high extinction coefficient of QDs, especially in the near infrared (NIR). This allows the intriguing use of diffuse light sources such as solar irradiation. In this review, we describe the development of this organic-QD upconversion platform based on triplet-triplet annihilation, focusing on the dark exciton in QDs with triplet character. Then we introduce the underlying energy transfer steps, starting from QD triplet photosensitization, triplet exciton transport, triplet-triplet annihilation, and ending with the upconverted emission. Design principles to improve the total upconversion efficiency are presented. We end with limitations in current reports and proposed future directions. This review provides a guide for designing efficient organic-QD upconversion platforms for future applications, including overcoming the Shockley-Queisser limit for more efficient solar energy conversion, NIR-based phototherapy, and diagnostics in vivo.

Triplet-pair spin signatures from macroscopically aligned heteroacenes in an oriented single crystal.

The photo-driven process of singlet fission generates coupled triplet pairs (TT) with fundamentally intriguing and potentially useful properties. The quintet 5TT0 sublevel is particularly interesting for quantum information because it is highly entangled, is addressable with microwave pulses, and could be detected using optical techniques. Previous theoretical work on a model Hamiltonian and nonadiabatic transition theory, called the JDE model, has determined that this sublevel can be selectively populated if certain conditions are met. Among the most challenging, the molecules within the dimer undergoing singlet fission must have their principal magnetic axes parallel to one another and to an applied Zeeman field. Here, we present time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy of a single crystal sample of a tetracenethiophene compound featuring arrays of dimers aligned in this manner, which were mounted so that the orientation of the field relative to the molecular axes could be controlled. The observed spin sublevel populations in the paired TT and unpaired (T+T) triplets are consistent with predictions from the JDE model, including preferential 5TT0 formation at z ‖ B0, with one caveat-two 5TT spin sublevels have little to no population. This may be due to crossings between the 5TT and 3TT manifolds in the field range investigated by TR-EPR, consistent with the intertriplet exchange energy determined by monitoring photoluminescence at varying magnetic fields.

Dual Roles of the Photooxidation of Organic Amines for Enhanced Triplet-Triplet Annihilation Upconversion in Nanoparticles.

Oxygen-mediated triplet-triplet annihilation upconversion (TTA-UC) quenching limits the application of such organic upconversion materials. Here, we report that the photooxidation of organic amines is an effective and versatile strategy to suppress oxygen-mediated upconversion quenching in both organic solvents and aqueous solutions. The strategy is based on the dual role of organic amines in photooxidation, i.e., as singlet oxygen scavengers and electron donors. Under photoexcitation, the photosensitizer sensitizes oxygen to produce singlet oxygen for the oxidation of alkylamine, reducing the oxygen concentration. However, photoinduced electron transfer among photosensitizers, organic amines, and oxygen leads to the production of superoxide anions that suppress TTA-UC. To observe oxygen-tolerating TTA-UC, we find that alkyl secondary amines can balance the production of singlet oxygen and superoxide anions. We then utilize polyethyleneimine (PEI) to synthesize amphiphilic polymers to encapsulate TTA-UC pairs for the formation of water-dispersible, ultrasmall, and multicolor-emitting TTA-UC nanoparticles.

Quantum Beats between Spin-Singlet and Spin-Triplet Interlayer Exciton Transitions in WSe2-MoSe2 Heterobilayers.

The long-lived interlayer excitons (IXs) of semiconducting transition metal dichalcogenide heterobilayers are prime candidates for developing various optoelectronic and valleytronic devices. Their photophysical properties, including fine structure, have been the focus of recent studies, and the presence of two spin states, namely, spin-singlet and spin-triplet, has been experimentally confirmed. However, the existence of the interaction between these states and their nature remains unknown to date. Here, we demonstrate the presence of coherent coupling between the spin-singlet and spin-triplet IXs of a WSe2-MoSe2 heterobilayer utilizing quantum beat spectroscopy via a home-built Michelson interferometer. As a clear signature of coherent coupling, the quantum beat signal has been observed for the first time between closely spaced transitions of IXs. The observed strong damping of the quantum beat signals with fast dephasing times of 270-400 fs indicates that fluctuations giving rise to inhomogeneous broadening in the photoluminescence emission of these states are uncorrelated.

Quantum Interference and Coherent Population Trapping in a Double Quantum Dot.

Quantum interference is a natural consequence of wave-particle duality in quantum mechanics, and is widely observed at the atomic scale. One interesting manifestation of quantum interference is coherent population trapping (CPT), first proposed in three-level driven atomic systems and observed in quantum optical experiments. Here, we demonstrate CPT in a gate-defined semiconductor double quantum dot (DQD), with some unique twists as compared to the atomic systems. Specifically, we observe CPT in both driven and nondriven situations. We further show that CPT in a driven DQD could be used to generate adiabatic state transfer. Moreover, our experiment reveals a nontrivial modulation to the CPT caused by the longitudinal driving field, yielding an odd-even effect and a tunable CPT. Our results broaden the field of CPT, and open up the possibility of quantum simulation and quantum computation based on adiabatic passage in quantum dot systems.

Simulating the full spin manifold of triplet-pair states in a series of covalently linked TIPS-pentacenes.

Combined density functional theory and multireference configuration interaction methods have been used to elucidate singlet fission (SF) pathways and mechanisms in three regioisomers of side-on linked pentacene dimers. In addition to the optically bright singlets (S 1 $$ {}_1 $$ and S 2 $$ {}_2 $$ ) and singly excited triplets (T 1 $$ {}_1 $$ and T 2 $$ {}_2 $$ ), the full spin manifold of multiexcitonic triplet-pair states ( 1 $$ {}^1 $$ ME, 3 $$ {}^3 $$ ME, 5 $$ {}^5 $$ ME) has been considered. In the ortho- and para-regioisomers, the 1 $$ {}^1 $$ ME and S 1 $$ {}_1 $$ potentials intersect upon geometry relaxation of the S 1 $$ {}_1 $$ excitation. In the meta-regioisomer, the crossing occurs upon delocalization of the optically bright excitation. The energetic accessibility of these conical intersections and the absence of low-lying charge-transfer states suggests a direct SF mechanism, assisted by charge-resonance effects in the 1 $$ {}^1 $$ ME state. While the 5 $$ {}^5 $$ ME state does not appear to play a role in the SF mechanism of the ortho- and para-regioisomers, its participation in the disentanglement of the triplet pair is conceivable in the meta-regioisomer.

Recent Advances in Understanding of the Singlet Oxygen in Energy Storage and Conversion.

Singlet oxygen (term symbol 1Δg, hereafter 1O2), a reactive oxygen species, has recently attracted increasing interest in the field of rechargeable batteries and electrocatalysis and photocatalysis. These sustainable energy conversion and storage technologies are of vital significance to replace fossil fuels and promote carbon neutrality and finally tackle the energy crisis and climate change. Herein, the recent progresses of 1O2 for energy storage and conversion is summarized, including physical and chemical properties, formation mechanisms, detection technologies, side reactions in rechargeable batteries and corresponding inhibition strategies, and applications in electrocatalysis and photocatalysis. The formation mechanisms and inhibition strategies of 1O2 in particular aprotic lithium-oxygen (Li-O2) batteries are highlighted, and the applications of 1O2 in photocatalysis and electrocatalysis is also emphasized. Moreover, the confronting challenges and promising directions of 1O2 in energy conversion and storage systems are discussed.

Ultraviolet Circularly Polarized Luminescence in Chiral Perovskite Nanoplatelet-Molecular Hybrids: Direct Binding Versus Efficient Triplet Energy Transfer.

The development of ultraviolet circularly polarized light (UVCPL) sources has the potential to benefit plenty of practical applications but remains a challenge due to limitations in available material systems and a limited understanding of the excited state chirality transfer. Herein, by constructing hybrid structures of the chiral perovskite CsPbBr3 nanoplatelets and organic molecules, excited state chirality transfer is achieved, either via direct binding or triplet energy transfer, leading to efficient UVCPL emission. The underlying photophysical mechanisms of these two scenarios are clarified by comprehensive optical studies. Intriguingly, UVCPL realized via the triple energy transfer, followed by the triplet-triplet annihilation upconversion processes, demonstrates a 50-fold enhanced dissymmetry factor glum. Furthermore, stereoselective photopolymerization of diacetylene monomer is demonstrated by using such efficient UVCPL. This study provides both novel insights and a practical approach for realizing UVCPL, which can also be extended to other material systems and spectral regions, such as visible and near-infrared.

A phenotypically robust model of spinal and bulbar muscular atrophy in Drosophila.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked disorder that affects males who inherit the androgen receptor (AR) gene with an abnormal CAG triplet repeat expansion. The resulting protein contains an elongated polyglutamine (polyQ) tract and causes motor neuron degeneration in an androgen-dependent manner. The precise molecular sequelae of SBMA are unclear. To assist with its investigation and the identification of therapeutic options, we report here a new model of SBMA in Drosophila melanogaster. We generated transgenic flies that express the full-length, human AR with a wild-type or pathogenic polyQ repeat. Each transgene is inserted into the same safe harbor site on the third chromosome of the fly as a single copy and in the same orientation. Expression of pathogenic AR, but not of its wild-type variant, in neurons or muscles leads to consistent, progressive defects in longevity and motility that are concomitant with polyQ-expanded AR protein aggregation and reduced complexity in neuromuscular junctions. Additional assays show adult fly eye abnormalities associated with the pathogenic AR species. The detrimental effects of pathogenic AR are accentuated by feeding flies the androgen, dihydrotestosterone. This new, robust SBMA model can be a valuable tool toward future investigations of this incurable disease.

A crescendo of competent coding (c3) contains the Standard Genetic Code.

The Standard Genetic Code (SGC) can arise by fusion of partial codes evolved in different individuals, perhaps for differing prior tasks. Such code fragments can be unified into an SGC after later evolution of accurate third-position Crick wobble. Late wobble advent fills in the coding table, leaving only later development of translational initiation and termination to reach the SGC in separated domains of life. This code fusion mechanism is computationally implemented here. Late Crick wobble after C3 fusion (c3-lCw) is tested for its ability to evolve the SGC. Compared with previously studied isolated coding tables, or with increasing numbers of parallel, but nonfusing codes, c3-lCw reaches the SGC sooner, is successful in a smaller population, and presents accurate and complete codes more frequently. Notably, a long crescendo of SGC-like codes is exposed for selection of superior translation. c3-lCw also effectively suppresses varied disordered assignments, thus converging on a unified code. Such merged codes closely approach the SGC, making its selection plausible. For example: Under routine conditions, ≈1 of 22 c3-lCw environments evolves codes with ≥20 assignments and ≤3 differences from the SGC, notably including codes identical to the Standard Genetic Code.

Metric learning for comparing genomic data with triplet network.

Many biological applications are essentially pairwise comparison problems, such as evolutionary relationships on genomic sequences, contigs binning on metagenomic data, cell type identification on gene expression profiles of single-cells, etc. To make pair-wise comparison, it is necessary to adopt suitable dissimilarity metric. However, not all the metrics can be fully adapted to all possible biological applications. It is necessary to employ metric learning based on data adaptive to the application of interest. Therefore, in this study, we proposed MEtric Learning with Triplet network (MELT), which learns a nonlinear mapping from original space to the embedding space in order to keep similar data closer and dissimilar data far apart. MELT is a weakly supervised and data-driven comparison framework that offers more adaptive and accurate dissimilarity learned in the absence of the label information when the supervised methods are not applicable. We applied MELT in three typical applications of genomic data comparison, including hierarchical genomic sequences, longitudinal microbiome samples and longitudinal single-cell gene expression profiles, which have no distinctive grouping information. In the experiments, MELT demonstrated its empirical utility in comparison to many widely used dissimilarity metrics. And MELT is expected to accommodate a more extensive set of applications in large-scale genomic comparisons. MELT is available at https://github.com/Ying-Lab/MELT.

Excitation transfer and quenching in photosystem II, enlightened by carotenoid triplet state in leaves.

Accumulation of carotenoid (Car) triplet states was investigated by singlet-triplet annihilation, measured as chlorophyll (Chl) fluorescence quenching in sunflower and lettuce leaves. The leaves were illuminated by Xe flashes of 4 µs length at half-height and 525-565 or 410-490 nm spectral band, maximum intensity 2 mol quanta m-2 s-1, flash photon dose up to 10 µmol m-2 or 4-10 PSII excitations. Superimposed upon the non-photochemically unquenched Fmd state, fluorescence was strongly quenched near the flash maximum (minimum yield Fe), but returned to the Fmd level after 30-50 µs. The fraction of PSII containing a 3Car in equilibrium with singlet excitation was calculated as Te = (Fmd-Fe)/Fmd. Light dependence of Te was a rectangular hyperbola, whose initial slope and plateau were determined by the quantum yields of triplet formation and annihilation and by the triplet lifetime. The intrinsic lifetime was 9 µs, but it was strongly shortened by the presence of O2. The triplet yield was 0.66 without nonphotochemical quenching (NPQ) but approached zero when NP-Quenched fluorescence approached 0.2 Fmd. The results show that in the Fmd state a light-adapted charge-separated PSIIL state is formed (Sipka et al., The Plant Cell 33:1286-1302, 2021) in which Pheo-P680+ radical pair formation is hindered, and excitation is terminated in the antenna by 3Car formation. The results confirm that there is no excitonic connectivity between PSII units. In the PSIIL state each PSII is individually turned into the NPQ state, where excess excitation is quenched in the antenna without 3Car formation.