Downstream Reactions Research Articles

STK19 facilitates the clearance of lesion-stalled RNAPII during transcription-coupled DNA repair.

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and transcription factor IIH (TFIIH) around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryoelectron microscopy (cryo-EM) and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

Intramolecular Photochemical C−H Insertions of α‐Diazocarbonyl Compounds

AbstractIntramolecular photochemical C−H insertion reactions of α‐diazocarbonyl compounds are powerful synthetic transformations leading to carbon‐carbon bond formation. While there are limitations in the scope of C−H bonds amenable to C−H insertion under photochemical conditions relative to metal catalysed C−H insertions, the benefits of conducting these synthetically powerful transformations in a metal‐free approach offers “green” advantages. Recent advances in technology have revolutionised the potential for use of photochemical transformations in synthesis, including the wavelength control enabled by LEDs and the benefits associated with continuous flow processing which overcome many of the traditional challenges associated with synthetic photochemistry including scale‐up. The potential for telescoping the synthesis of α‐diazocarbonyl compounds and downstream photochemical reactions is clear. This review summarises the development of intramolecular photochemical C−H insertions and highlights the benefits of recent technological advances.

New insights on zero-valent iron permeable reactive barrier for Cr(VI) removal: The function of FeS reaction zone downstream in-situ generated by sulfate-reducing bacteria

The biogeochemical behavior downstream of the zero-valent iron permeable reactive barrier (ZVI-PRB) plays an enormous positive role in the remediation of contaminated-groundwater, but has been completely neglected for a long time. Therefore, this study conducted a 240-day SRB-enhanced ZVI-PRB column experiment, focusing on what exactly happens downstream of ZVI-PRB. Results show that biosulfidation of SRB inside ZVI-PRB prolonged the complete Cr(VI) removal longevity of ZVI-PRB from 38 days to at least 240 days. More importantly, unlike previous studies that focused on improving the performance of ZVI-PRB itself, this study found an in-situ generated FeS reduction reaction zone downstream of the ZVI-PRB. When the ZVI-PRB fails, the downstream reaction zone can continue to play a role in Cr(VI) removal. The maximum Cr(VI) removal capacity of the aquifer media from the reaction zone reached 155.1mg/kg, which was 39.7% of commercial ZVI capacity. The reduction zone was further confirmed to be predominantly FeS rather than FeS2. Biogeochemistry occurring within and downstream of ZVI-PRB leads to the formation of FeS. Gene sequencing revealed significantly higher SRB abundance downstream of ZVI-PRB than within the ZVI-PRB. The understanding of the downstream FeS reaction zone provides new insights for more effective remediation using ZVI-PRB.

Effect of plasma-induced oxidation on NK cell immune checkpoint ligands: A computational-experimental approach

Non-thermal plasma (NTP) shows promise as a potent anti-cancer therapy with both cytotoxic and immunomodulatory effects. In this study, we investigate the chemical and biological effects of NTP-induced oxidation on several key, determinant immune checkpoints of natural killer (NK) cell function. We used molecular dynamics (MD) and umbrella sampling simulations to investigate the effect of NTP-induced oxidative changes on the MHC-I complexes HLA-Cw4 and HLA-E. Our simulations indicate that these chemical alterations do not significantly affect the binding affinity of these markers to their corresponding NK cell receptor, which is supported with experimental read-outs of ligand expression on human head and neck squamous cell carcinoma cells after NTP application. Broadening our scope to other key ligands for NK cell reactivity, we demonstrate rapid reduction in CD155 and CD112, target ligands of the inhibitory TIGIT axis, and in immune checkpoint CD73 immediately after treatment. Besides these transient chemical alterations, the reactive species in NTP cause a cascade of downstream cellular reactions. This is underlined by the upregulation of the stress proteins MICA/B, potent ligands for NK cell activation, 24 h post treatment. Taken together, this work corroborates the immunomodulatory potential of NTP, and sheds light on the interaction mechanisms between NTP and cancer cells.

Annotation of Metabolites in Stable Isotope Tracing Untargeted Metabolomics via Khipu-web.

Stable isotope tracing is a crucial technique for understanding the metabolic wiring of biological systems, determining metabolic flux through pathways of interest, and detecting novel metabolites and pathways. Despite the potential insights provided by this technique, its application remains limited to a small number of targeted molecules and pathways. Because previous software tools usually require chemical formulas to find relevant features, and the data are highly complex, especially in untargeted metabolomics and when the downstream reactions and metabolites are poorly characterized. We report here Khipu version 2 and its new user-friendly web application. New functions are added to enhance analyzing stable isotope tracing data including metrics that evaluate peak enrichment in labeled samples, scoring methods to facilitate robust detection of intensity patterns and integrated natural abundance correction. We demonstrate that this approach can be applied to untargeted metabolomics to systematically extract isotope-labeled compounds and annotate the unidentified metabolites.

Oxadiazolines as Photoreleasable Labels for Drug Target Identification.

Photoaffinity labeling is a widely used technique for studying ligand-protein and protein-protein interactions. Traditional photoaffinity labels utilize nonspecific C-H bond insertion reactions mediated by a highly reactive intermediate. Despite being the most widely used photoaffinity labels, diazirines exhibit limited compatibility with downstream organic reactions and suffer from storage stability concerns. This study introduces oxadiazolines as innovative and complementary photoactivatable labels for addition to the toolbox and demonstrates their application in vitro and through in cellulo labeling experiments. Oxadiazolines can be easily synthesized from ketone moieties and cleaved with 302-330 nm light to cleanly liberate a diazo reactive moiety that can covalently modify nucleophilic amino acid residues. Notably, oxadiazolines are compatible with various organic reaction conditions and functional groups, allowing for the exploration of a large chemical space. Several known inhibitors featuring the oxadiazoline functionality were prepared without affecting their binding affinity. Furthermore, we confirmed the ability of oxadiazolines to form covalent bonds with proteins upon UV-irradiation, both in vitro and in cellulo, yielding comparable results to those of the matched diazirine compounds.

Identification of Kibdelomycin and Related Biosynthetic Gene Clusters and Characterization of the C‐Branching of Amycolose

AbstractMany bacterial natural products contain C‐branched sugars, including components from the outer cell wall or antibiotically active metabolites. The enzymatic C‐branching of keto sugars leading to longer side chains (≥C2) is catalyzed by thiamine diphosphate (ThDP)‐dependent enzymes. Chiral tertiary α‐hydroxy ketones are formed in this process. The ThDP‐dependent enzymes that catalyze C‐branching reactions belong to one of three enzymatic superfamilies: decarboxylases, transketolases, and α‐ketoacid dehydrogenases 2, but branching of keto sugars has only been demonstrated for decarboxylases. In this study, we showed that an α‐ketoacid dehydrogenase is responsible for C‐branching of the deoxyketo sugar amycolose in the biosynthesis of kibdelomycin in Kibdelosporangium sp. MA7385. In addition, we characterized an amino transferase in the same biosynthetic gene cluster (BGC) that accepts a sterically demanding tertiary α‐hydroxy ketone in a downstream reaction. Subsequently, we identified approximately 400 similar BGCs in silico, suggesting that there is a large diversity of possible ThDP‐dependent enzymes catalyzing the C‐branching of keto sugars and subsequent modifications.

Portable label-free electrochemical aptasensor for sensitive detection of paraquat herbicide mediated by oxygen reduction reaction

Paraquat (PQ) is a highly toxic herbicide that has been prohibited in almost 70 countries, but remains in use worldwide. Thus, routine on-site PQ monitoring is a key mechanism to ensure safety and efficiently enforce regulations. Herein, a label-free portable electrochemical aptasensor for the detection of PQ was developed by utilizing aptamer designed to specifically recognize PQ. The aptasensor employs square-wave voltammetry (SWV) to quantify PQ binding on the aptamer-functionalized electrode surface by tracking the downstream oxygen reduction reaction. It provided a detection range spanning from 0.01 to 100.0 μg mL−1 PQ with a limit of detection (LOD) of 8.9 ng mL−1. Validation against spiked tap water, pomegranate juice, and orange juice revealed recovery rate performances of 75 %–130 %. The aptasensor demonstrates promising feasibility for PQ detection in real-world applications, offering remarkable portability and operational simplicity. Notably, it can operate without supplementary redox agents, requiring only sample incubation and subsequent washing steps.

Characterization of 2-phenanthroate:CoA ligase from the sulfate-reducing, phenanthrene-degrading enrichment culture TRIP.

Polycyclic aromatic hydrocarbons (PAHs) constitute a class of very toxic and persistent pollutants in the environment. However, the anaerobic degradation of three-ring PAHs such as phenanthrene is barely investigated. The initial degradation step starts with a carboxylation followed by a CoA‑thioesterification reaction performed by an aryl-CoA ligase. The formation of a CoA-thioester is an important step in the degradation pathway of aromatic compounds because the CoA-ester is needed for all downstream biochemical reactions in the pathway. Furthermore, we provide biochemical proof for the identification of the first genes for anaerobic phenanthrene degradation. Results presented here provide information about the biochemical and structural properties of the purified 2‑phenanthroate:CoA ligase and expand our knowledge of aryl-CoA ligases.

Coupling DNA Origami Filament Growth to an Autocatalytic Production of Fuel

AbstractIn this study, the hierarchical assembly of DNA origami filaments (DOF) initiated by an autocatalytic DNA reaction network (DRN) is investigated. The so‐formed filaments are subsequently disassembled by toehold‐mediated strand displacement mechanisms. Using fluorescence resonance energy transfer, the kinetics of DOF growth after direct addition of fuel and compared it to the polymerization process triggered by the release of fuel from the DRN is monitored. Optimization of design and experimental conditions enabled to fine‐tune the kinetics of the two processes, ensuring that the release of fuel from the DRN outpaced the consumption of fuel by the downstream polymerization reaction. This resulted in a sustained and controlled DOF growth leading to micrometer‐long filament structures. Finally, although the presence of a toehold in the fuel strand reduced the efficiency of monomer association in the polymerization process, a 10‐fold excess of the anti‐fuel strand is efficient in dissociating the filament structures, permitting a potential reset for new reactions. The study shows that the kinetics of DNA origami filaments growth can be finely manipulated by a cascade of upstream reactions, suggesting alternative approaches for the creation of programmable DNA‐based nanomaterials that can sense and respond to more complex and distant events.

The glycolytic reaction PGAM unexpectedly restrains Th17 pathogenicity and Th17-dependent autoimmunity.

Glucose metabolism is a critical regulator of T cell function, largely thought to support their activation and effector differentiation. Here, we investigate the relevance of individual glycolytic reactions in determining the pathogenicity of T helper 17 (Th17) cells using single-cell RNA-seq and Compass, an algorithm we previously developed for estimating metabolic flux from single-cell transcriptomes. Surprisingly, Compass predicted that the metabolic shunt between 3-phosphoglycerate (3PG) and 2-phosphoglycerate (2PG) is inversely correlated with pathogenicity in these cells, whereas both its upstream and downstream reactions were positively correlated. Perturbation of phosphoglycerate mutase (PGAM), an enzyme required for 3PG to 2PG conversion, resulted in an increase in protein expression of IL2, IL17, and TNFa, as well as induction of a pathogenic gene expression program. Consistent with PGAM playing a pro-regulatory role, inhibiting PGAM in Th17 cells resulted in exacerbated autoimmune responses in the adoptive transfer model of experimental autoimmune encephalomyelitis (EAE). Finally, we further investigated the effects of modulating glucose concentration on Th17 cells in culture. Th17 cells differentiated under high- and low-glucose conditions substantially differed in their metabolic and effector transcriptomic programs, both central to Th17 function. Importantly, the PGAM-dependent gene module marks the least pathogenic state of Th17 cells irrespective of glucose concentration. Overall, our study identifies PGAM, contrary to other glycolytic enzymes, as a negative regulator of Th17 pathogenicity.

(Invited) Understanding Photo-Driven Water Oxidation Mechanisms

Water oxidation is an important reaction that produces electrons and protons, which are important for downstream reactions such as hydrogen production and carbon dioxide reduction. Presently, the slow water oxidation is a key limiting factor for the development of hydrogen production and/or carbon dioxide reduction into practical solar energy storage technologies. An important reason for the slow progress in solve water oxidation problems is the lack of knowledge on the detailed mechanisms by which water is oxidized. As a multi-step process, the reaction involves at least 4 protons and 4 holes (when the desired product is oxygen gas). Intuitively, the coordination of charges and protons plays a critical role, but the details remain unknown. In this talk, we report our recent progress in understanding how water oxidation is influenced by surface hole behaviors. Using an Ir-based catalyst that features atomically defined structures, we were able to systematically vary a number of parameters, including the hole concentration, the density of active sites, as well as the properties of the supporting substrate that determines hole accumulation. It was found that matching the concentrations of surface holes and active site densities is critical. It was also revealed that the support substrate could have a profound influence on the reaction kinetics, even though they do not directly participate in the reactions. This knowledge is expected to propel the development of catalysts for improved water oxidation under photochemical conditions.

Automated Droplet Ejection from a Digital Microfluidics Sample Pretreatment Device Enables Batch-Mode Chemiluminescence Immunoassay.

Digital microfluidics (DMF) features programmed manipulation of fluids in multiple steps, making it a valuable tool for sample pretreatment. However, the integration of sample pretreatment with its downstream reaction and detection requires transferring droplets from the DMF device to the outside world. To address this issue, the present study developed a modified DMF device that allows automated droplet ejection out of the chip, facilitated by a tailor-designed interface. A double-layered DMF microchip with an oil-filled medium was flipped over, with a liquid infusion port and a liquid expulsion port accommodated on the top working PCB plate and the bottom grounded ITO plate, respectively, to facilitate chip-to-world delivery of droplets. Using chemiluminescent immunoassay (CLIA) as an illustrative application, the sample pretreatment was programmed on the DMF device, and CLIA droplets were ejected from the chip for signal reading. In our workflow, CLIA droplets can be ejected from the DMF device through the chip-to-world interface, freeing up otherwise occupied electrodes for more sample pretreatment and enabling streamlined droplet microreactions and batch-mode operation for bioanalysis. Integrated with these interfacing portals, the DMF system achieved a single-channel throughput of 17 samples per hour, which can be further upscaled for more productive applications by parallelizing the DMF modules. The results of this study demonstrate that the droplet ejection function that is innovated in a DMF sample pretreatment microsystem can significantly improve analytical throughput, providing an approach to establishing an automated but decentralized biochemical sample preparation workstation for large-scale and continuous bioanalysis.

STK19 facilitates the clearance of lesion-stalled RNAPII during transcription-coupled DNA repair.

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

Identification of Kibdelomycin and Related Biosynthetic Gene Clusters and Characterization of the C-Branching of Amycolose.

Many bacterial natural products contain C-branched sugars, including components from the outer cell wall or antibiotically active metabolites. The enzymatic C-branching of keto sugars leading to longer side chains (≥C2) is catalyzed by thiamine diphosphate (ThDP)-dependent enzymes. Chiral tertiary α-hydroxy ketones are formed in this process. The ThDP-dependent enzymes that catalyze C-branching reactions belong to one of three enzymatic superfamilies: decarboxylases, transketolases, and α-ketoacid dehydrogenases 2, but branching of keto sugars has only been demonstrated for decarboxylases. In this study, we showed that an α-ketoacid dehydrogenase is responsible for C-branching of the deoxyketo sugar amycolose in the biosynthesis of kibdelomycin in Kibdelosporangium sp. MA7385. In addition, we characterized an amino transferase in the same biosynthetic gene cluster (BGC) that accepts a sterically demanding tertiary α-hydroxy ketone in a downstream reaction. Subsequently, we identified approximately 400 similar BGCs in silico, suggesting that there is a large diversity of possible ThDP-dependent enzymes catalyzing the C-branching of keto sugars and subsequent modifications.

Two-layer cascaded catalytic hairpin assemblies based on locked nucleic acids for one-step and highly sensitive ctDNA detection.

Enzyme-free signal amplification of catalytic hairpin assembly (CHA) has enabled sensitive detection of circulating tumor DNA (ctDNA) in early clinical diagnosis. Conventional CHA strategies are restrained by the limited amplification efficiency of the single-stage system, and signal leakage from "breathing" influence and nuclease degradation. Here, we introduced two-layer cascaded locked nucleic acid (LNA)-assisted CHA circuits with the intelligent incorporation of LNA in the hairpins and reporter for the highly sensitive one-step detection of scarce ctDNA. The target-triggered upstream CHA reaction continuously generates hybrid products to catalyze the downstream CHA reaction for transducing the primary sensing event, and the released target and the produced hybrid product trigger the next catalytic reaction round at the same time and finally cascade to amplify the target ctDNA fluorescence output signal. Meanwhile, the stronger binding affinity of the LNA-DNA duplex endows the two-layer LNA-assisted CHA system with thermodynamic stability and nuclease resistance, and thus our designed system exhibits an excellent detection performance for target ctDNA in the range from 2 pM to 5 nM with a low detection limit of 0.6 pM. Significantly, the two-layer LNA-assisted CHA circuits have been successfully implemented for the feasible analysis of clinical samples. This two-layer cascaded LNA-assisted CHA strategy provides a promising high sensitivity tool for one-step detection of scarce ctDNA from complex clinical samples and would facilitate the reconfiguration of DNA circuit-based DNA nanotechnology for the precise analysis of other biomarkers in clinical research fields.

Synthesis, Biological, and Computational Evaluations of Conformationally Restricted NAD-Mimics as Discriminant Inhibitors of Human NMN-Adenylyltransferase Isozymes.

Nicotinamide adenine dinucleotide (NAD) cofactor metabolism plays a significant role in cancer development. Tumor cells have an increased demand for NAD and ATP to support rapid growth and proliferation. Limiting the amount of available NAD by targeting critical NAD biosynthesis enzymes has emerged as a promising anticancer therapeutic approach. In mammals, the enzyme nicotinamide/nicotinic acid adenylyltransferase (NMNAT) catalyzes a crucial downstream reaction for all known NAD synthesis routes. Novel nicotinamide/nicotinic acid adenine dinucleotide (NAD/NaAD) analogues 1-4, containing a methyl group at the ribose 2'-C and 3'-C-position of the adenosine moiety, were synthesized as inhibitors of the three isoforms of human NMN-adenylyltransferase, named hNMNAT-1, hNMNAT-2, and hNMNAT-3. An NMR-based conformational analysis suggests that individual NAD-analogues (1-4) have distinct conformational preferences. Biological evaluation of dinucleotides 1-4 as inhibitors of hNMNAT isoforms revealed structural relationships between different conformations (North-anti and South-syn) and enzyme-inhibitory activity. Among the new series of NAD analogues synthesized and tested, the 2'-C-methyl-NAD analogue 1 (Ki = 15 and 21 µM towards NMN and ATP, respectively) emerged as the most potent and selective inhibitor of hNMNAT-2 reported so far. Finally, we rationalized the in vitro bioactivity and selectivity of methylated NAD analogues with in silico studies, helping to lay the groundwork for rational scaffold optimization.

Leveraging Language Model Multitasking To Predict C-H Borylation Selectivity.

C-H borylation is a high-value transformation in the synthesis of lead candidates for the pharmaceutical industry because a wide array of downstream coupling reactions is available. However, predicting its regioselectivity, especially in drug-like molecules that may contain multiple heterocycles, is not a trivial task. Using a data set of borylation reactions from Reaxys, we explored how a language model originally trained on USPTO_500_MT, a broad-scope set of patent data, can be used to predict the C-H borylation reaction product in different modes: product generation and site reactivity classification. Our fine-tuned T5Chem multitask language model can generate the correct product in 79% of cases. It can also classify the reactive aromatic C-H bonds with 95% accuracy and 88% positive predictive value, exceeding purpose-developed graph-based neural networks.

Electron-rich Ni2+ in Ni3S2 boosting electrocatalytic CO2 reduction to formate and syngas

Rationally constructed new catalyst can promote carbon dioxide reduction reaction (CO2RR) to valuable carbonaceous fuels such as formate and CO, providing a promising strategy for low CO2 emissions. Herein, the synthesized Ni3S2@C as a highly efficient electro-catalyst exhibits remarkable selectivity for formate with 73.9% faradaic efficiency (FE) at −0.7 V vs. RHE. At high applied potential, it shows a high syngas evolution with CO/H2 ratios (0.54–3.15) that are suitable for typical downstream thermochemical reactions. The experimental and theoretical analyses demonstrate that the electron-rich Ni2+ in Ni3S2 enhances the adsorption behavior of ∗OCHO intermediate, reduces the energy barrier of the formation of intermediates, and improves the selectivity of the formate product. Attenuated total reflection surface-enhanced infrared absorption spectra conducted in situ show that ∗OCHO intermediate is more likely to be generated and adsorbed on Ni3S2, enhancing the selectivity and activity of the formate product.

Polymerase-Based Signal Delay for Temporally Regulating DNA Involved Reactions, Programming Dynamic Molecular Systems, and Biomimetic Sensing.

Complex temporal molecular signals play a pivotal role in the intricate biological pathways of living organisms, and cells exhibit the ability to transmit and receive information by intricately managing the temporal dynamics of their signaling molecules. Although biomimetic molecular networks are successfully engineered outside of cells, the capacity to precisely manipulate temporal behaviors remains limited. In this study, the catalysis activity of isothermal DNA polymerase (DNAP) through combined use of molecular dynamics simulation analysis and fluorescence assays is first characterized. DNAP-driven delay in signal strand release ranged from 100 to 102min, which is achieved through new strategies including the introduction of primer overhangs, utilization of inhibitory reagents, and alteration of DNA template lengths. The results provide a deeper insight into the underlying mechanisms of temporal control DNAP-mediated primer extension and DNA strand displacement reactions. Then, the regulated DNAP catalysis reactions are applied in temporal modulation of downstream DNA-involved reactions, the establishment of dynamic molecular signals, and the generation of barcodes for multiplexed detection of target genes. The utility of DNAP-based signal delay as a dynamic DNA nanotechnology extends beyond theoretical concepts and achieves practical applications in the fields of cell-free synthetic biology and bionic sensing.