Backbone Sequences Research Articles

Molecular characterization of the tet (M)-carrying transposon Tn7124 and plasmids in Escherichia coli isolates recovered from swine.

Here, we report the genetic features and evolutionary mechanisms of two tet (M)-bearing plasmids (pTA2 and pTA7) recovered from swine Escherichia coli isolates. The genetic profiles of pTA2 and pTA7 and corresponding transconjugants were accessed by S1 nuclease pulsed-field gel electrophoresis and Southern hybridization, followed by whole genome sequencing and bioinformatics analysis. The biological influences of pTA2 and pTA7 were determined by stability and direct competition assays. Both pTA7 and pTA2 had the IncR backbone sequences but differed in the multidrug resistance region (MDR). The MDR of pTA2 consisted of sul3, tet (M), qnrS1, bleO, oqxAB, floR, aadA1, cmlA1, aadA2, and tet (A)-tetR (A) in addition to 22 insertion sequences. Notably, pTA2 carried the novel complex Tn7124 (IS26-ctp-lp-tet (M)-hp-IS406tnp-IntI4-IS26) harboring tet (M). The fragment carrying tet (M) (IS26-ctp-lp-tet (M)-IS406 tnp-ctp-aadA1-cmlA1-aadA2-dfrA12-IntI1), named Tn6942-like, and the two resistance modules ISVsa3-VirD2-floR-lysR and tet (A)-tetR (A) were located in the MDR of pTA7. Both pTA2 and pTA7 were highly stable in E. coli DH5α cells with no fitness cost to the host or disadvantage in growth competition. These results indicate that transposons carrying tet (M) continuously integrate via mediation with an insertion sequence, which accelerates the transmission of tet (M) in E. coli isolates through integration of other drug-resistant genes, thereby posing a potential serious threat to the efficacy of clinical treatment.

Entropy-Driven Catalytic G-Quadruple Cycle Amplification Integrated with Ligases for Label-Free Detection of Single Nucleotide Polymorphisms.

G-Quadruplex/thioflavin (G4/THT) has become a very promising label-free fluorescent luminescent element for nucleic acid detection due to its good programmability and compatibility. However, the weak fluorescence efficiency of single-molecule G4/THT limits its potential applications. Here, we developed an entropy-driven catalytic (EDC) G4 (EDC-G4) cycle amplification technology as a universal label-free signal amplification and output system by properly programming classical EDC and G4 backbone sequences, preintegrated ligase chain reaction (LCR) for label-free sensitive detection of single nucleotide polymorphisms (SNPs). First, the positive strand LCR enabled specific transduction and preliminary signal amplification from single-base mutation information to single-strand information. Subsequently, the EDC-G4 cycle amplification reaction was activated, accompanied by the production of a large number of G4/THT luminophores to output fluorescent signals. The EDC-G4 system was proposed to address the weak fluorescence of G4/THT and obtain a label-free fluorescence signal amplification. The dual-signal amplification effect enabled the LCR-EDC-G4 detection system to accurately detect mutant target (MT) at concentrations as low as 22.39 fM and specifically identify 0.01% MT in a mixed detection pool. Moreover, the LCR-EDC-G4 system was further demonstrated for its potential application in real biological samples. Therefore, this study not only contributes ideas for the development of label-free fluorescent biosensing strategies but also provides a high-performance and practical SNP detection tool in parallel.

The DUF579 proteins GhIRX15s regulate cotton fiber development by interacting with proteins involved in xylan synthesis

Cotton provides the most abundant natural fiber for the textile industry. The mature cotton fiber largely consists of secondary cell walls with the highest proportion of cellulose and a small amount of hemicellulose and lignin. To dissect the roles of hemicellulosic polysaccharides during fiber development, four IRREGULAR XYLEM 15 (IRX15) genes, GhIRX15-1/-2/-3/-4, were functionally characterized in cotton. These genes encode DUF579 domain-containing proteins, which are homologs of AtIRX15 involved in xylan biosynthesis. The four GhIRX15 genes were predominantly expressed during fiber secondary wall thickening, and the encoded proteins were localized to the Golgi apparatus. Each GhIRX15 gene could restore the xylan deficient phenotype in the Arabidopsis irx15irx15l double mutant. Silencing of GhIRX15s in cotton resulted in shorter mature fibers with a thinner cell wall and reduced cellulose content as compared to the wild type. Intriguingly, GhIRX15-2 and GhIRX15-4 formed homodimers and heterodimers. In addition, the GhIRX15s showed physical interaction with glycosyltransferases GhGT43C, GhGT47A and GhGT47B, which are responsible for synthesis of the xylan backbone and reducing end sequence. Moreover, the GhIRX15s can form heterocomplexes with enzymes involved in xylan modification and side chain synthesis, such as GhGUX1/2, GhGXM1/2 and GhTBL1. These findings suggest that GhIRX15s participate in fiber xylan biosynthesis and modulate fiber development via forming large multiprotein complexes.

Recombinational exchange of M-fibril and T-pilus genes generates extensive cell surface diversity in the global group A Streptococcus population.

Among genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as "pilin types." Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools (pubmlst.org) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies.IMPORTANCEPrecision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.

150 Backbone Spiking Sequence as a Basis for Preplay, Replay, and Default States in Human Cortex

INTRODUCTION: Sequences of spiking activity are a fundamental feature of many neural systems, and recent evidence suggests that they play an important role in information coding in the human brain. A parallel set of studies also asserts that sequential activity may in part derive from a pre-existing network structure. Sequential spiking activity therefore may arise as a natural property of the neurophysiologic properties of the human cortex. METHODS: We measured single unit spiking activity in the human cortex by implanting Utah micro-electrode arrays into participants who performed an episodic memory task while undergoing invasive monitoring for potential resective epilepsy surgery. RESULTS: We confirm the presence of an average backbone spiking sequence identified during pre-task rest that is stable over time and different cognitive states. These resting backbone sequences served as a scaffold for cognitively relevant sequential spiking activity occurring later in the recording session. We further demonstrate that backbone sequences are composed of both rigid and flexible sequence elements, and that flexible elements serve to promote memory specificity when forming and retrieving new memories. CONCLUSIONS: Here we demonstrate that backbone sequences can be measured in the human cortex and that they persist over time and through different cognitive states. Our data show that backbone sequences are composed of both rigid and flexible elements, and flexibility around the backbone allows for the formation of memory specific spiking sequences. Taken together, our results provide the first evidence for backbone spiking sequences in the human cortex, and more generally suggest a cognitive role of predetermined spiking network dynamics in the human brain.

Direct and Detailed Site-Specific Glycopeptide Characterization by Higher-Energy Electron-Activated Dissociation Tandem Mass Spectrometry.

Glycosylation is widely recognized as the most complex post-translational modification due to the widespread presence of macro- and microheterogeneities, wherein its biological consequence is closely related to both the glycosylation sites and the glycan fine structures. Yet, efficient site-specific detailed glycan characterization remains a significant analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform, higher-energy electron-activated dissociation (heExD) tandem mass spectrometry (MS/MS) revealed extraordinary efficacy for the structural characterization of intact glycopeptides. HeExD produced extensive fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments (with or without the glycan). The intensity of cross-ring cleavage and backbone fragments retaining the intact glycan was highly dependent on the electron energy. Among the four electron energy levels investigated, electronic excitation dissociation (EED) provided the most comprehensive structural information, yielding a complete series of glycosidic fragments for accurate glycan topology determination, a wealth of cross-ring fragments for linkage definition, and the most extensive peptide backbone fragments for accurate peptide sequencing and glycosylation site localization. The glycan fragments observed in the EED spectrum correlated well with the fragmentation patterns observed in EED MS/MS of the released glycans. The advantages of EED over higher-energy collisional dissociation (HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy collisional dissociation (EThcD) were demonstrated for the characterization of a glycopeptide bearing a biantennary disialylated glycan. EED can produce a complete peptide backbone and glycan sequence coverage even for doubly protonated precursors. The exceptional performance of heExD MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization on an Orbitrap-Omnitrap hybrid instrument presents a novel option for in-depth glycosylation analysis.

A skeletal randomization strategy for high-performance quinoidal-aromatic polymers.

Enhancing the solution-processability of conjugated polymers (CPs) without diminishing their thin-film crystallinity is crucial for optimizing charge transport in organic field-effect transistors (OFETs). However, this presents a classic "Goldilocks zone" dilemma, as conventional solubility-tuning methods for CPs typically yield an inverse correlation between solubility and crystallinity. To address this fundamental issue, a straightforward skeletal randomization strategy is implemented to construct a quinoid-donor conjugated polymer, PA4T-Ra, that contains para-azaquinodimethane (p-AQM) and oligothiophenes as repeat units. A systematic study is conducted to contrast its properties against polymer homologues constructed following conventional solubility-tuning strategies. An unusually concurrent improvement of solubility and crystallinity is realized in the random polymer PA4T-Ra, which shows moderate polymer chain aggregation, the highest crystallinity and the least lattice disorder. Consequently, PA4T-Ra-based OFETs, fabricated under ambient air conditions, deliver an excellent hole mobility of 3.11 cm2 V-1 s-1, which is about 30 times higher than that of the other homologues and ranks among the highest for quinoidal CPs. These findings debunk the prevalent assumption that a random polymer backbone sequence results in decreased crystallinity. The considerable advantages of the skeletal randomization strategy illuminate new possibilities for the control of polymer aggregation and future design of high-performance CPs, potentially accelerating the development and commercialization of organic electronics.

Does human homology reduce the potential immunogenicity of non-antibody scaffolds?

Biologics developers are moving beyond antibodies for delivery of a wide range of therapeutic interventions. These non-antibody modalities are often based on 'natural' protein scaffolds that are modified to deliver bioactive sequences. Both human-derived and non-human-sourced scaffold proteins have been developed. New types of "non-antibody" scaffolds are still being discovered, as they offer attractive alternatives to monoclonals due to their smaller size, improved stability, and ease of synthesis. They are believed to have low immunogenic potential. However, while several human-sourced protein scaffolds have not been immunogenic in clinical studies, this may not predict their overall performance in other therapeutic applications. A preliminary evaluation of their potential for immunogenicity is warranted. Immunogenicity risk potential has been clearly linked to the presence of T "helper" epitopes in the sequence of biologic therapeutics. In addition, tolerogenic epitopes are present in some human proteins and may decrease their immunogenic potential. While the detailed sequences of many non-antibody scaffold therapeutic candidates remain unpublished, their backbone sequences are available for review and analysis. We assessed 12 example non-antibody scaffold backbone sequences using our epitope-mapping tools (EpiMatrix) for this perspective. Based on EpiMatrix scoring, their HLA DRB1-restricted T cell epitope content appears to be lower than the average protein, and sequences that may act as tolerogenic epitopes are present in selected human-derived scaffolds. Assessing the potential immunogenicity of scaffold proteins regarding self and non-self T cell epitopes may be of use for drug developers and clinicians, as these exciting new non-antibody molecules begin to emerge from the preclinical pipeline into clinical use.

A geometrical framework for thinking about proteins.

We present a model, based on symmetry and geometry, for proteins. Using elementary ideas from mathematics and physics, we derive the geometries of discrete helices and sheets. We postulate a compatible solvent-mediated emergent pairwise attraction that assembles these building blocks, while respecting their individual symmetries. Instead of seeking to mimic the complexity of proteins, we look for a simple abstraction of reality that yet captures the essence of proteins. We employ analytic calculations and detailed Monte Carlo simulations to explore some consequences of our theory. The predictions of our approach are in accord with experimental data. Our framework provides a rationalization for understanding the common characteristics of proteins. Our results show that the free energy landscape of a globular protein is pre-sculpted at the backbone level, sequences and functionalities evolve in the fixed backdrop of the folds determined by geometry and symmetry, and that protein structures are unique in being simultaneously characterized by stability, diversity, and sensitivity.

Backbone spiking sequence as a basis for preplay, replay, and default states in human cortex

Sequences of spiking activity have been heavily implicated as potential substrates of memory formation and retrieval across many species. A parallel line of recent evidence also asserts that sequential activity may arise from and be constrained by pre-existing network structure. Here we reconcile these two lines of research in the human brain by measuring single unit spiking sequences in the temporal lobe cortex as participants perform an episodic memory task. We find the presence of an average backbone spiking sequence identified during pre-task rest that is stable over time and different cognitive states. We further demonstrate that these backbone sequences are composed of both rigid and flexible sequence elements, and that flexible elements within these sequences serve to promote memory specificity when forming and retrieving new memories. These results support the hypothesis that pre-existing network dynamics serve as a scaffold for ongoing neural activity in the human cortex.

Minimized antibiotic-free plasmid vector for gene therapy utilizing a new toxin-antitoxin system

Approaches to improve plasmid-mediated transgene expression are needed for gene therapy and genetic immunization applications. The backbone sequences needed for the production of plasmids in bacterial hosts and the use of antibiotic resistance genes as selection markers represent biological safety risks. Here, we report the development of an antibiotic-free expression plasmid vector with a minimized backbone utilizing a new toxin-antitoxin (TA) system. The Rs_0636/Rs_0637 TA pair was derived from the coral-associated bacterium Roseivirga sp. The toxin gene is integrated into the chromosome of Escherichia coli host cells, and a recombinant mammalian expression plasmid is constructed by replacing the antibiotic resistance gene with the antitoxin gene Rs_0637 (here named Tiniplasmid). The Tiniplasmid system affords high selection efficiency (∼80%) for target gene insertion into the plasmid and has high plasmid stability in E. coli (at least 9 days) in antibiotic-free conditions. Furthermore, with the aim of reducing the size of the backbone sequence, we found that the antitoxin gene can be reduced to 153 bp without a significant reduction in selection efficiency. To develop its applications in gene therapy and DNA vaccines, the biosafety and efficiency of the Tiniplasmid-based eukaryotic gene delivery and expression were further evaluated in CHO–K1 cells. The results showed that Rs_0636/Rs_0637 has no cell toxicity and that the Tiniplasmid vector has a higher gene expression efficiency than the commercial vectors pCpGfree and pSTD in the eukaryotic cells. Altogether, the results demonstrate the potential of the Rs_0636/Rs_0637-based antibiotic-free plasmid vector for the development and production of safe and efficacious DNA vaccines.

De novo design of protein structure and function with RFdiffusion

There has been considerable recent progress in designing new proteins using deep-learning methods1–9. Despite this progress, a general deep-learning framework for protein design that enables solution of a wide range of design challenges, including de novo binder design and design of higher-order symmetric architectures, has yet to be described. Diffusion models10,11 have had considerable success in image and language generative modelling but limited success when applied to protein modelling, probably due to the complexity of protein backbone geometry and sequence–structure relationships. Here we show that by fine-tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topology-constrained protein monomer design, protein binder design, symmetric oligomer design, enzyme active site scaffolding and symmetric motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold diffusion (RFdiffusion), by experimentally characterizing the structures and functions of hundreds of designed symmetric assemblies, metal-binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryogenic electron microscopy structure of a designed binder in complex with influenza haemagglutinin that is nearly identical to the design model. In a manner analogous to networks that produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple molecular specifications.

The cytosolic N-terminal domain of V-ATPase a-subunits is a regulatory hub targeted by multiple signals.

Vacuolar H+-ATPases (V-ATPases) acidify several organelles in all eukaryotic cells and export protons across the plasma membrane in a subset of cell types. V-ATPases are multisubunit enzymes consisting of a peripheral subcomplex, V1, that is exposed to the cytosol and an integral membrane subcomplex, Vo, that contains the proton pore. The Vo a-subunit is the largest membrane subunit and consists of two domains. The N-terminal domain of the a-subunit (aNT) interacts with several V1 and Vo subunits and serves to bridge the V1 and Vo subcomplexes, while the C-terminal domain contains eight transmembrane helices, two of which are directly involved in proton transport. Although there can be multiple isoforms of several V-ATPase subunits, the a-subunit is encoded by the largest number of isoforms in most organisms. For example, the human genome encodes four a-subunit isoforms that exhibit a tissue- and organelle-specific distribution. In the yeast S. cerevisiae, the two a-subunit isoforms, Golgi-enriched Stv1 and vacuolar Vph1, are the only V-ATPase subunit isoforms. Current structural information indicates that a-subunit isoforms adopt a similar backbone structure but sequence variations allow for specific interactions during trafficking and in response to cellular signals. V-ATPases are subject to several types of environmental regulation that serve to tune their activity to their cellular location and environmental demands. The position of the aNT domain in the complex makes it an ideal target for modulating V1-Vo interactions and regulating enzyme activity. The yeast a-subunit isoforms have served as a paradigm for dissecting interactions of regulatory inputs with subunit isoforms. Importantly, structures of yeast V-ATPases containing each a-subunit isoform are available. Chimeric a-subunits combining elements of Stv1NT and Vph1NT have provided insights into how regulatory inputs can be integrated to allow V-ATPases to support cell growth under different stress conditions. Although the function and distribution of the four mammalian a-subunit isoforms present additional complexity, it is clear that the aNT domains of these isoforms are also subject to multiple regulatory interactions. Regulatory mechanisms that target mammalian a-subunit isoforms, and specifically the aNT domains, will be described. Altered V-ATPase function is associated with multiple diseases in humans. The possibility of regulating V-ATPase subpopulations via their isoform-specific regulatory interactions are discussed.

Mono-material product design with bio-based, circular, and biodegradable polymers

Mono-material product design with bio-based, circular, and biodegradable polymers

Modified Linear Peptides Effectively Silence STAT-3 in Breast Cancer and Ovarian Cancer Cell Lines

RNA interference (RNAi) has drawn enormous attention as a powerful tool because of its capability to interfere with mRNA and protein production. However, designing a safe and efficient delivery system in RNAi therapeutics remains challenging. Herein, we have designed and synthesized several linear peptides containing tryptophan (W) and arginine (R) residues separated by the β-alanine (βA) spacer and attached to a lipophilic fatty acyl chain, cholesterol, or PEG. The peptide backbone sequences were: Ac-C-βA-βA-W4-βA-βA-R4-CO-NH2 and Ac-K-βA-βA-W4-βA-βA-R4-CO-NH2, with only a difference in N-terminal amino acid. The cysteine side chain in the first sequence was used for the conjugation with PEG2000 and PEG550. Alternatively, the side chain of lysine in the second sequence was used for conjugation with cholesterol or oleic acid. We hypothesized that amphiphilic peptides and optimum fatty acyl chain or PEG could function as an effective siRNA carrier by complementing each structural component's self-assembly and membrane internalization properties. None of the designed peptides showed cytotoxicity up to 10 µM. Serum stability studies suggested that the newly designed peptides efficiently protected siRNA against early degradation by nucleases. Flow cytometry analysis indicated 50-90% cellular uptake of siRNA using the newly developed modified linear peptides (MLPs). Western blot results revealed more than 90% protein downregulation after targeting STAT3 in MDA-MB-231 and SKOV-3 cell lines. In summary, a new peptide class was developed to safely and efficiently deliver siRNA.

The influence of chirality on the behavior of oligonucleotides inside cells: revealing the potent cytotoxicity of G-rich l-RNA.

Due to their intrinsic nuclease resistance, mirror image l-oligonucleotides are being increasingly employed in the development of biomedical research tools and therapeutics. Yet, the influence of chirality on the behavior of oligonucleotides in living systems, and specifically, the extent to which l-oligonucleotides interact with endogenous biomacromolecules and the resulting consequences remain unknown. In this study, we characterized the intracellular behavior of l-oligonucleotides for the first time, revealing important chirality-dependent effects on oligonucleotide cytotoxicity. We show that exogenously delivered l-oligonucleotides have the potential to be highly cytotoxic, which is dependent on backbone chemistry, sequence, and structure. Notably, for the sequences tested, we found that single-stranded G-rich l-RNAs are more cytotoxic than their d-DNA/RNA counterparts, exhibiting low nanomolar EC50 values. Importantly, RNA-seq analysis of differentially expressed genes suggests that G-rich l-RNAs stimulate an innate immune response and pro-inflammatory cytokine production. These data not only challenge the general perception that mirror image l-oligonucleotides are nontoxic and nonimmunogenic, but also reveal previously unrecognized therapeutic opportunities. Moreover, by establishing sequence/structure toxicity relationships, this work will guide how future l-oligonucleotide-based biotechnologies are designed and applied.

The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome

TheBacteroides thetaiotaomicronhas developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RG-II depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of the module at the C-terminal domain, which we designated BT0996-C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical β-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.

High-level periodic conjugated terpolymers through AA/BB monomer pair-type terpolymerization improve performance of polymer solar cells

With a growing interest in developing high-performance π-conjugated terpolymers via random terpolymerization, new synthetic approaches resulting in higher-quality terpolymers with improved backbone regularity are needed to help make organic optoelectronic and photovoltaic applications more prosperous. Herein, we report an AA/BB monomer pair-type terpolymerization protocol that renders semi-sequence-controlled (SSC) terpolymers by polymerizing the aperiodically repeating units into the preferentially formed periodic, alternating polymeric block sequences, unlike the conventional random terpolymerization for producing terpolymers with irregular repeating sequences. A close comparison of SSC- and conventional terpolymerization-derived terpolymers indicate detectable differences in the absorption, energetic properties, carrier transport, energy loss, and morphological characteristics. The optimal SSC terpolymer-based polymer solar cells (PSCs) outperform the conventional terpolymerization-derived counterparts in power conversion efficiency (PCE). Moreover, the optimal SSC terpolymer-based PSCs processed by an eco-friendly solvent/additive system also exhibit outstanding PCEs of 16.52 % (small area of 0.041 cm2) and 15.50 % (large area of 1 cm2). To the best our knowledge, it is the highest value among tetrahydrofuran-processed PSCs. This SSC terpolymerization shows considerable promise in synthesizing high-quality in-chain terpolymers with high backbone sequence control without sacrificing their inherent beneficial features, providing high potential for further improving their PSC performances.

Gene knockdown by structure defined single-stem loop small non-coding RNAs with programmable regulatory activities

Gene regulation by trans-acting small RNAs (sRNAs) has considerable advantages over other gene regulation strategies. However, synthetic sRNAs mainly take natural sRNAs (MicC or SgrS) as backbones and comprise three functional elements folding into two or more stem-loop structures: an mRNA base pairing region, an Hfq-binding structure, and a rho-independent terminator. Due to limited numbers of natural sRNAs and complicated backbone structures, synthetic sRNAs suffer from low activity programmability and poor structural modularity. Moreover, natural sRNA backbone sequences may increase the possibility of unwanted recombination. Here, we present a bottom-up approach for creating structure defined single-stem loop small non-coding RNAs (ssl-sRNAs), which contain a standardized scaffold of a 7 bp-stem-4 nt-loop-polyU-tail and a 24 nt basing pairing region covering the first eight codons. Particularly, ssl-sRNA requires no independent Hfq-binding structure, as the polyU tail fulfills the roles of binding Hfq. A thermodynamic-based scoring model and a web server sslRNAD (http://www.kangzlab.cn/) were developed for automated design of ssl-sRNAs with well-defined structures and programmable activities. ssl-sRNAs displayed weak polar effects when regulating polycistronic mRNAs. The ssl-sRNA designed by sslRNAD showed regulatory activities in both Escherichia coli and Bacillus subtilis. A streamlined workflow was developed for the construction of customized ssl-sRNA and ssl-sRNA libraries. As examples, the E. coli cell morphology was easily modified and new target genes of ergothioneine biosynthesis were quickly identified with ssl-sRNAs. ssl-sRNA and its designer sslRNAD enable researchers to rapidly design sRNAs for knocking down target genes.

Alternating Isotactic Polyhydroxyalkanoates via Site- and Stereoselective Polymerization of Unsymmetrical Diolides.

Naturally produced, biodegradable polyhydroxyalkanoates (PHAs) promise more sustainable alternatives to nonrenewable/degradable plastics, but biological PHA's stereomicrostructures are strictly confined to isotactic (R)-polymers or copolymers of random sequences. Chemical synthesis via catalyzed ring-opening polymerization (ROP) of cyclic (di)esters offers expedient access to diverse PHA microstructures, including those with defined comonomer sequences and tacticities. However, the synthesis of alternating isotactic PHAs has not been achieved by the existing methodologies. Here, we report the design of unsymmetrically disubstituted eight-membered diolides (rac-8DLR1-R2) and their site- and stereoselective ROP with discrete chiral catalysts, enabling the synthesis of alternating isotactic PHAs, poly(3-hydroxybutyrate-alt-3-hydroxyvalerate) (alt-P3HBV) and poly(3-hydroxybutyrate-alt-3-hydroxyheptanoate) (alt-P3HBHp), with high to quantitative (>99%) alternation and isotacticity and Mn up to 113 kDa and Đ = 1.01. Physical properties of such PHAs are substantially determined by the degree of backbone sequence alternation and tacticity, ranging from amorphous to semi-crystalline materials. The alt-P3HBV shows significantly improved mechanical performance relative to the constituent homopolymers. Intriguingly, enantiomeric (R)-alt-P3HBV and (S)-alt-P3HBV, synthesized by kinetically resolved ROP of rac-8DLMe-Et, form a stereocomplex with a significantly enhanced Tm (by 53 °C), while the enantiomeric homopolymers do not form a stereocomplex.