Recruitment of HAB1 and SnRK2.2 by C2-domain protein CAR1 in plasma membrane ABA signaling.

Plasma membrane (PM)-associated abscisic acid (ABA) signal transduction is an important component of ABA signaling. The C2-domain ABA-related (CAR) proteins have been reported to play a crucial role in recruiting ABA receptor PYR1/PYL/RCAR (PYLs) to the PM. However, the molecular details of the involvement of CAR proteins in membrane-delimited ABA signal transduction remain unclear. For instance, where this response process takes place and whether any additional members besides PYL are taking part in this signaling process. Here, the GUS-tagged materials for all Arabidopsis CAR members were used to comprehensively visualize the extensive expression patterns of the CAR family genes. Based on the representativeness of CAR1 in response to ABA, we determined to use it as a target to study the function of CAR proteins in PM-associated ABA signaling. Single-particle tracking showed that ABA affected the spatiotemporal dynamics of CAR1. The presence of ABA prolonged the dwell time of CAR1 on the membrane and showed faster lateral mobility. Surprisingly, we verified that CAR1 could directly recruit hypersensitive to ABA1 (HAB1) and SNF1-related protein kinase 2.2 (SnRK2.2) to the PM at both the bulk and single-molecule levels. Furthermore, PM localization of CAR1 was demonstrated to be related to membrane microdomains. Collectively, our study revealed that CARs recruited the three main components of ABA signaling to the PM to respond positively to ABA. This study deepens our understanding of ABA signal transduction.

Transition-Metal-Catalyzed Dehydrogenative (3 + 2) Annulation of Aromatic Compounds: Synthesis of Indenes and Indanes via Dual Functionalization of Benzylic and ortho C-H Bonds.

Intermolecular (3 + 2) annulation emerges as a potent approach for constructing 5-membered carbocycles through the fusion of two distinct components. This synopsis encapsulates recent strides in the realm of transition-metal-catalyzed dehydrogenative (3 + 2) annulation of aromatic hydrocarbons, achieved through the dual functionalization of benzylic and ortho C-H bonds. Encompassing three pivotal strategies, namely, (i) C-H bond activation, (ii) benzylic oxidation, and (iii) π-coordination activation, this review offers an overview of the field's recent developments.

Catalytic Dehydrogenative (3 + 2) Cycloaddition of Alkylbenzenes via π-Coordination.

Given the wide availability and low cost of alkylbenzenes, direct C-H functionalization of these aromatic hydrocarbons to afford structurally complex building blocks has long been of interest in organic synthesis. Herein we describe a method for rhodium-catalyzed dehydrogenative (3 + 2) cycloaddition reactions of alkylbenzenes with 1,1-bis(phenylsulfonyl)ethylene. The π-coordination with a rhodium catalyst facilitates the benzylic deprotonation, allowing for the subsequent (3 + 2) cycloaddition in which the metal-complexed carbanion serves as a unique all-carbon 1,3-dipole equivalent. We demonstrated the generality of this catalytic method by carrying out reactions of a large array of alkylbenzenes to generate dihydroindene derivatives bearing two synthetically versatile sulfonyl groups. Quantum-chemical calculations revealed details of the reaction process.

Asymmetric hydrogenation of 1,1-diarylethylenes and benzophenones through a relay strategy.

Homogenous transition-metal catalysts bearing a chiral ligand are widely used for asymmetric hydrogenation of unsaturated compounds such as olefins and ketones, providing efficient concise access to products with chiral carbon centers. However, distinguishing the re and si prochiral faces of a double bond bearing two substituents that are sterically and electronically similar is challenging for these catalysts. Herein, we report a relay strategy for constructing compounds with a chiral gem-diaryl carbon center by means of a combination of selective arene exchange between 1,1-diarylethylenes or benzophenones with (naphthalene)Cr(CO)3 and subsequent asymmetric hydrogenation. During the hydrogenation, the Cr(CO)3 unit facilitate differentiation of the two prochiral faces of the substrate double bond via formation of a three-dimensional complex with one of the aromatic rings by selective arene exchange. Density functional theory calculations reveal that during the hydrogenation, chromium coordination affected π-π stacking of the substrate and the catalyst ligand, leading to differentiation of the prochiral faces.

ZYG11B potentiates the antiviral innate immune response by enhancing cGAS-DNA binding and condensation.

As a key dsDNA recognition receptor, cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) plays a vital role in innate immune responses. Activated cGAS, by sensing DNA, catalyzes the synthesis of the secondary messenger cyclic GMP-AMP (cGAMP), which subsequently activates downstream signaling to induce production of interferons and inflammatory cytokines. Here, we report Zyg-11 family member B (ZYG11B) as a potent amplifier in cGAS-mediated immune responses. Knockdown of ZYG11B impairs production of cGAMP and subsequent transcription of interferon and inflammatory cytokines. Mechanistically, ZYG11B enhances cGAS-DNA binding affinity, potentiates cGAS-DNA condensation, and stabilizes the cGAS-DNA condensed complex. Moreover, herpes simplex virus 1 (HSV-1) infection induces ZYG11B degradation in a cGAS-unrelated manner. Our findings not only reveal an important role of ZYG11B in the early stage of DNA-induced cGAS activation but also indicate a viral strategy to dampen the innate immune response.

Comprehensive insights into the structures and dynamics of plant telomeric G-quadruplexes.

Telomeres, which are located at the ends of eukaryotic chromosomes, are crucial for genomic maintenance. Most telomeric DNA is composed of tandemly repeated guanine (G)-rich sequences, which form G-quadruplexes (G4s). The structures and dynamics of telomeric G4s are essential for telomere functioning and helpful for G4-based biosensing. However, they are far from being understood, especially for plants. In this contribution, the folding, environment-induced G4 dynamics, and protein-catalyzed unfolding of plant telomeric G4s were comprehensively studied. It was found that diverse plant telomeric sequences from land plants to green algae could fold into G4 structures. In addition, 5'-proximal ssDNA but not 3'-proximal ssDNA drove conversion of anti-parallel G4 structures to parallel structures, and both 5' and 3' ssDNA decreased the stability of G4s in dilute solution. Furthermore, molecular crowding promoted the formation of parallel structures for three-layer but not for two-layer G4s, and increased the stability of all selected G4s. Finally, AtRecQ2 helicase resolved the stable parallel structure of typical plant telomeric G4 in crowded solution, but ssDNA binding protein AtRPA did not. Furthermore, AtRecQ2 unwound the structure more efficiently in the presence of AtRPA. The results may expand our understanding on the structures and dynamics of plant telomeric G4s.

5-Methyl-cytosine stabilizes DNA but hinders DNA hybridization revealed by magnetic tweezers and simulations.

5-Methyl-cytosine (5mC) is one of the most important DNA modifications and plays versatile biological roles. It is well known that 5mC stabilizes DNA duplexes. However, it remains unclear how 5mC affects the kinetics of DNA melting and hybridization. Here, we studied the kinetics of unzipping and rezipping using a 502-bp DNA hairpin by single-molecule magnetic tweezers. Under constant loading rates, 5mC increases the unzipping force but counterintuitively decreases the rezipping force at various salt and temperature conditions. Under constant forces, the non-methylated DNA hops between metastable states during unzipping and rezipping, which implies low energy barriers. Surprisingly, the 5mC DNA can't rezip after fully unzipping unless much lower forces are applied, where it rezips stochastically in a one-step manner, which implies 5mC kinetically hinders DNA hybridization and high energy barriers in DNA hybridization. All-atom molecular dynamics simulations reveal that the 5mC kinetically hinders DNA hybridization due to steric effects rather than electrostatic effects caused by the additional methyl groups of cytosines. Considering the possible high speed of DNA unzipping and zipping during replication and transcription, our findings provide new insights into the biological roles of 5mC.

Mechanistic insights into poly(C)-binding protein hnRNP K resolving i-motif DNA secondary structures.

I-motifs are four-strand noncanonical secondary structures formed by cytosine (C)-rich sequences in living cells. The structural dynamics of i-motifs play essential roles in many cellular processes, such as telomerase inhibition, DNA replication, and transcriptional regulation. In cells, the structural dynamics of the i-motif can be modulated by the interaction of poly(C)-binding proteins (PCBPs), and the interaction is closely related to human health, through modulating the transcription of oncogenes and telomere stability. Therefore, the mechanisms of how PCBPs interact with i-motif structures are fundamentally important. However, the underlying mechanisms remain elusive. I-motif structures in the promoter of the c-MYC oncogene can be unfolded by heterogeneous nuclear ribonucleoprotein K (hnRNP K), a PCBP, to activate its transcription. Here, we selected this system as an example to comprehensively study the unfolding mechanisms. We found that the promoter sequence containing 5 C-runs preferred folding into type-1245 to type-1234 i-motif structures based on their folding stability, which was further confirmed by single-molecule FRET. In addition, we first revealed that the c-MYC i-motif structure was discretely resolved by hnRNP K through two intermediate states, which were assigned to the opposite hairpin and neighboring hairpin, as further confirmed by site mutations. Furthermore, we found all three KH (hnRNP K homology) domains of hnRNP K could unfold the c-MYC i-motif structure, and KH2 and KH3 were more active than KH1. In conclusion, this study may deepen our understanding of the interactions between i-motifs and PCBPs and may be helpful for drug development.

Insights into the structural dynamics and helicase-catalyzed unfolding of plant RNA G-quadruplexes.

RNA G-quadruplexes (rG4s) are noncanonical RNA secondary structures formed by guanine (G)-rich sequences. These complexes play important regulatory roles in both animals and plants through their structural dynamics and are closely related to human diseases and plant growth, development, and adaption. Thus, studying the structural dynamics of rG4s is fundamentally important; however, their folding pathways and their unfolding by specialized helicases are not well understood. In addition, no plant rG4-specialized helicases have been identified. Here, using single-molecule FRET, we experimentally elucidated for the first time the folding pathway and intermediates, including a G-hairpin and G-triplex. In addition, using proteomics screening and microscale thermophoresis, we identified and validated five rG4-specialized helicases in Arabidopsis thaliana. Furthermore, DExH1, the ortholog of the famous human rG4 helicase RHAU/DHX36, stood out for its robust rG4 unwinding ability. Taken together, these results shed light on the structural dynamics of plant rG4s.

Rhodium-Catalyzed Benzylic Addition Reactions of Alkylarenes to Michael Acceptors.

The use of alkylarenes as nucleophile precursors in benzylic addition is challenging because the benzylic hydrogen atoms of these compounds are inert to deprotonation. Herein, we report Rh-catalyzed benzylic addition of alkylarenes to Michael acceptors for the formation of C(sp3 )-C(sp3 ) bonds. The catalyst is proposed to activate the aromatic ring via η6 -coordination, dramatically facilitating deprotonation of the unactivated benzylic C-H bond and addition of the resulting carbanion to the α,ß-unsaturated double bond in the absence of bases. Notably, this byproduct-free method provides an access to all-carbon quaternary centers through the development of ligands.

Catalytic Amination of Phenols with Amines.

Given the wide prevalence and ready availability of both phenols and amines, aniline synthesis through direct coupling between these starting materials would be extremely attractive. Herein, we describe a rhodium-catalyzed amination of phenols, which provides concise access to diverse anilines, with water as the sole byproduct. The arenophilic rhodium catalyst facilitates the inherently difficult keto-enol tautomerization of phenols by means of π-coordination, allowing for the subsequent dehydrative condensation with amines. We demonstrate the generality of this redox-neutral catalysis by carrying out reactions of a large array of phenols with various electronic properties and a wide variety of primary and secondary amines. Several examples of late-stage functionalization of structurally complex bioactive molecules, including pharmaceuticals, further illustrate the potential broad utility of the method.

Rhodium-Catalyzed Stereoselective Deuteration of Benzylic C-H Bonds via Reversible η6 -Coordination.

We report a convenient method for benzylic H/D exchange of a wide variety of substrates bearing primary, secondary, or tertiary C-H bonds via a reversible η6 -coordination strategy. A doubly cationic [CpCF3 RhIII ]2+ catalyst that serves as an arenophile facilitates deprotonation of inert benzylic hydrogen atoms (pKa >40 in DMSO) without affecting other hydrogen atoms, such as those on aromatic rings or in α-positions of carboxylate groups. Notably, the H/D exchange reactions feature high stereoretention. We demonstrated the potential utility of this method by using it for deuterium labeling of ten pharmaceuticals and their analogues.

Safety and efficacy of thalidomide in patients with transfusion-dependent ß-thalassemia: a randomized clinical trial.

Thalidomide induces γ-globin expression in erythroid progenitor cells, but its efficacy on patients with transfusion-dependent ß-thalassemia (TDT) remains unclear. In this phase 2, multi-center, randomized, double-blind clinical trial, we aimed to determine the safety and efficacy of thalidomide in TDT patients. A hundred patients of 14 years or older were randomly assigned to receive placebo or thalidomide for 12 weeks, followed by an extension phase of at least 36 weeks. The primary endpoint was the change of hemoglobin (Hb) level in the patients. The secondary endpoints included the red blood cell (RBC) units transfused and adverse effects. In the placebo-controlled period, Hb concentrations in patients treated with thalidomide achieved a median elevation of 14.0 (range, 2.5 to 37.5) g/L, whereas Hb in patients treated with placebo did not significantly change. Within the 12 weeks, the mean RBC transfusion volume for patients treated with thalidomide and placebo was 5.4 ± 5.0 U and 10.3 ± 6.4 U, respectively (P < 0.001). Adverse events of drowsiness, dizziness, fatigue, pyrexia, sore throat, and rash were more common with thalidomide than placebo. In the extension phase, treatment with thalidomide for 24 weeks resulted in a sustainable increase in Hb concentrations which reached 104.9 ± 19.0 g/L, without blood transfusion. Significant increase in Hb concentration and reduction in RBC transfusions were associated with non ß0/ß0 and HBS1L-MYB (rs9399137 C/T, C/C; rs4895441 A/G, G/G) genotypes. These results demonstrated that thalidomide is effective in patients with TDT.

Ni-catalyzed hydroalkylation of olefins with N-sulfonyl amines.

Hydroalkylation, the direct addition of a C(sp3)-H bond across an olefin, is a desirable strategy to produce valuable, complex structural motifs in functional materials, pharmaceuticals, and natural products. Herein, we report a reliable method for accessing α-branched amines via nickel-catalyzed hydroalkylation reactions. Specifically, by using bis(cyclooctadiene)nickel (Ni(cod)2) together with a phosphine ligand, we achieved a formal C(sp3)-H bond insertion reaction between olefins and N-sulfonyl amines without the need for an external hydride source. The amine not only provides the alkyl motif but also delivers hydride to the olefin by means of a nickel-engaged ß-hydride elimination/reductive elimination process. This method provides a platform for constructing chiral α-branched amines by using a P-chiral ligand, demonstrating its potential utility in organic synthesis. Notably, a sulfonamidyl boronate complex formed in situ under basic conditions promotes ring-opening of the azanickellacycle reaction intermediate, leading to a significant improvement of the catalytic efficiency.

Single-Molecule Imaging in Living Plant Cells: A Methodological Review.

Single-molecule imaging is emerging as a revolutionary approach to studying fundamental questions in plants. However, compared with its use in animals, the application of single-molecule imaging in plants is still underexplored. Here, we review the applications, advantages, and challenges of single-molecule fluorescence imaging in plant systems from the perspective of methodology. Firstly, we provide a general overview of single-molecule imaging methods and their principles. Next, we summarize the unprecedented quantitative details that can be obtained using single-molecule techniques compared to bulk assays. Finally, we discuss the main problems encountered at this stage and provide possible solutions.

Proximal Single-Stranded RNA Destabilizes Human Telomerase RNA G-Quadruplex and Induces Its Distinct Conformers.

Single-stranded guanine-rich RNA sequences have a propensity to fold into compact G-quadruplexes (RG4s). The conformational transitions of these molecules provide an important way to regulate their biological functions. Here, we examined the stability and conformation of an RG4-forming sequence identified near the end of human telomerase RNA. We found that a proximal single-stranded (ss) RNA significantly impairs RG4 stability at physiological K+ concentrations, resulting in a reduced RG4 rupture force of ∼ 24.4 pN and easier accessibility of the G-rich sequence. The destabilizing effect requires a minimum of six nucleotides of ssRNA and is effective at either end of RG4. Remarkably, this RG4-forming sequence, under the influence of such a proximal ssRNA, exhibits interconversions between at least three less stable RG4 conformers that might represent potential intermediates along its folding/unfolding pathway. This work provides insights into the stability and folding dynamics of RG4 that are essential for understanding its biological functions.

Quantitative and real-time measurement of helicase-mediated intra-stranded G4 unfolding in bulk fluorescence stopped-flow assays.

G-Quadruplexes (G4s) are thermodynamically stable, compact, and poorly hydrated structures that pose a potent obstacle for chromosome replication and gene expression, and requiring resolution by helicases in a cell. Bulk stopped-flow fluorescence assays have provided many mechanistic insights into helicase-mediated duplex DNA unwinding. However, to date, detailed studies on intramolecular G-quadruplexes similar or comparable with those used for studying duplex DNA are still lacking. Here, we describe a method for the direct and quantitative measurement of helicase-mediated intramolecular G-quadruplex unfolding in real time. We designed a series of site-specific fluorescently double-labeled intramolecular G4s and screened appropriate substrates to characterize the helicase-mediated G4 unfolding. With the developed method, we determined, for the first time to our best knowledge, the unfolding and refolding constant of G4 (≈ 5 s-1), and other relative parameters under single-turnover experimental conditions in the presence of G4 traps. Our approach not only provides a new paradigm for characterizing helicase-mediated intramolecular G4 unfolding using stopped-flow assays but also offers a way to screen for inhibitors of G4 unfolding helicases as therapeutic drug targets. Graphical abstract.

Single-molecule probing the duplex and G4 unwinding patterns of a RecD family helicase.

RecD family helicases play an important role in prokaryotic genome stability and serve as the structural models for studying superfamily 1B (SF1B) helicases. However, RecD-catalyzed duplex DNA unwinding behavior and the underlying mechanism are still elusive. RecD family helicases share a common proto-helicase with eukaryotic Pif1 family helicases, which are well known for their outstanding G-quadruplex (G4) unwinding ability. However, there are still controversial points as to whether and how RecD helicases unfold G4 structures. Here, single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers (MT) were used to study Deinococcus radiodurans RecD2 (DrRecD2)-mediated duplex DNA unwinding and resolution of G4 structures. A symmetric, repetitive unwinding phenomenon was observed on duplex DNA, revealed from the strand switch and translocation of one monomer. Furthermore, we found that DrRecD2 was able to unwind both parallel and antiparallel G4 structures without obvious topological preferences. Surprisingly, the unwinding properties of RecD on duplex and G4 DNA are different from those of Pif1. The findings provide an example, in which the patterns of two molecules derived from a common ancestor deviate during evolution, and they are of significance for understanding the unwinding mechanism and function of SF1B helicases.

A comprehensive evaluation of a typical plant telomeric G-quadruplex (G4) DNA reveals the dynamics of G4 formation, rearrangement, and unfolding.

Telomeres are specific nucleoprotein structures that are located at the ends of linear eukaryotic chromosomes and play crucial roles in genomic stability. Telomere DNA consists of simple repeats of a short G-rich sequence: TTAGGG in mammals and TTTAGGG in most plants. In recent years, the mammalian telomeric G-rich repeats have been shown to form G-quadruplex (G4) structures, which are crucial for modulating telomere functions. Surprisingly, even though plant telomeres are essential for plant growth, development, and environmental adaptions, only few reports exist on plant telomeric G4 DNA (pTG4). Here, using bulk and single-molecule assays, including CD spectroscopy, and single-molecule FRET approaches, we comprehensively characterized the structure and dynamics of a typical plant telomeric sequence, d[GGG(TTTAGGG)3]. We found that this sequence can fold into mixed G4s in potassium, including parallel and antiparallel structures. We also directly detected intermediate dynamic transitions, including G-hairpin, parallel G-triplex, and antiparallel G-triplex structures. Moreover, we observed that pTG4 is unfolded by the AtRecQ2 helicase but not by AtRecQ3. The results of our work shed light on our understanding about the existence, topological structures, stability, intermediates, unwinding, and functions of pTG4.