RNA molecules adopt complex structures that perform essential biological functions across all forms of life, making them promising candidates for therapeutic applications. However, our ability to design new RNA structures remains limited by an incomplete understanding of their folding principles. While global metrics such as the minimum free energy are widely used, they are at odds with naturally occurring structures and incompatible with established design rules. Here, we introduce local stability compensation (LSC), a principle that RNA folding is governed by the local balance between destabilizing loops and their stabilizing adjacent stems, challenging the focus on global energetic optimization. Analysis of over 100 000 RNA structures revealed that LSC signatures are particularly pronounced in bulges and their adjacent stems, with distinct patterns across different RNA families that align with their biological functions. To validate LSC experimentally, we systematically analyzed thousands of RNA variants using DMS chemical mapping. Our results demonstrate that stem folding, as measured by reactivity, correlates with LSC (R² = 0.458 for hairpin loops) and that instabilities show no significant effect on folding for distal stems. These findings demonstrate that LSC can be a guiding principle for understanding RNA function and for the rational design of custom RNAs.

Xenobiotic nucleic acids (XNAs) significantly expand the range of genetic polymers and serve as promising alternatives to DNA and RNA for numerous biological applications. However, the extensive exploration and application of XNAs are limited by low sustainability and yields in solid-phase oligonucleotide synthesis, as well as by the unavailability of efficient XNA polymerases for polymerase-mediated XNA production. To address the limitations in XNA production, we developed a solid-phase enzymatic XNA oligonucleotide synthesis platform using a laboratory-evolved XNA polymerase, SFM5-7, which exhibits excellent activity for synthesizing DNA, RNA, 2′-fluoroarabinonucleic acid (FANA), and other 2′-modified XNA oligonucleotides. This platform employs ribonucleotide insertion and alkaline cleavage of the oligonucleotide product before and after SFM5-7-mediated XNA synthesis, enabling recycled XNA synthesis through the reuse of a bead-immobilized self-priming hairpin DNA template. The platform’s potential and versatility are demonstrated by the production of FANA, 2′-modified RNAs, chimeric XNAs, 5′-end-labeled FANA, and an active FANAzyme. This platform should facilitate the customized production of functional XNAs with programmable modifications, accelerating their applications in biotechnology and biomedicine.

The growing demand for sustainable solutions in agriculture, which are critical for crop productivity and food quality in the face of climate change and the need to reduce agrochemical usage, has brought biostimulants into the spotlight as valuable tools for regenerative agriculture. With their diverse biological activities, biostimulants can contribute to crop growth, nutrient use efficiency, and abiotic stress resilience, as well as to the restoration of soil health. Biomolecules include humic substances, protein lysates, phenolics, and carbohydrates have undergone thorough investigation because of their demonstrated biostimulant activities. Here, we review the process of the discovery and development of extract-based biostimulants, and propose a practical step-by-step pipeline that starts with initial identification of biomolecules, followed by extraction and isolation, determination of bioactivity, identification of active compound(s), elucidation of mechanisms, formulation, and assessment of effectiveness. The different steps generate a roadmap that aims to expedite the transfer of interdisciplinary knowledge from laboratory-scale studies to pilot-scale production in practical scenarios that are aligned with the prevailing regulatory frameworks.

CRISPR–Cas12a has been demonstrated to be activated for its trans-cleavage activity by single- and double-stranded DNA containing a protospacer adjacent motif (PAM), but other types of activators have remained undiscovered. In this work, we found that a hairpin-structured substrate can activate the trans-cleavage activity of Cas12a without a PAM, and the parameters of the hairpin loop obviously affect the activity. Cas12a exhibits sequence preference for proximal loops, preferring to recognize polyadenine hairpin loop activators. Molecular docking and dynamic calculations provide a theoretical basis for the activation of Cas12a by hairpin activators. Leveraging the efficient activation capability of the hairpin activator, we constructed an allosteric detection platform for non-nucleic acid targets, capable of sensitively and specifically detecting hypochlorous acid and calcium ions. This novel activator of Cas12a holds enormous potential for the development of multi-functional biological platforms.

The global crisis of plastic waste accumulation threatens wildlife and ecosystems. Catalytic processes that convert plastic waste into valuable chemicals and fuels offer promising solutions. Recycling or upcycling of real-life plastic mixtures is challenging owing to their diverse composition and structure. Here we propose a product-oriented strategy leveraging the orthogonality in reactivities of different functional groups in plastic mixtures to yield valuable products. This approach involves identifying functional groups followed by converting a selective component in the mixture to valuable products. We use mixtures of polystyrene, polylactic acid, polyurethane, polycarbonate, polyvinyl chloride, polyethylene terephthalate, polyethylene and polypropylene, as well as real-life plastics, to demonstrate the feasibility and effectiveness of the proposed strategy. The diverse physical and chemical properties of these components, which typically hinder direct recovery, offer opportunities for extraction and transformation with the proposed strategy. From a 20-g mixture of real-life plastics, including polystyrene foam, a polylactic acid straw, a polyurethane tube, a polycarbonate mask, a polyvinyl chloride bag, a polyethylene terephthalate bottle, a polyethylene dropper and a polypropylene bottle, we obtained more than 8 separate chemicals: 1.3 g of benzoic acid, 0.5 g of plasticizer, 0.7 g of alanine, 0.7 g of lactic acid, 1.4 g of aromatic amine salt, 2.1 g of bisphenol A, 2.0 g of terephthalic acid and 3.5 g of C3–C6 alkanes. This study reveals the potential for designing transformation strategies for complex plastic waste based on their chemical nature and opens paths for managing end-of-life plastic mixtures. A product-oriented strategy that leverages the reactivities of different functional groups in real-life plastic mixtures can be used to obtain valuable products, opening a path for managing end-of-life plastic mixtures.

A molecular beacon (MB) is an oligonucleotide probe with a stem-loop structure designed to fluoresce upon hybridization with a specific target nucleic acid. Herein, we introduce a novel i-motif DNA-based molecular beacon (iMB) designed for nucleic acid detection. DNA chemical sequencing showed that the iMB can be selectively opened when its target is present, thanks to the formation of a duplex between this target and the iMB recognition loop. Phase diagrams were introduced to analyze the binding properties between the iMB and its target, and to optimize the detection conditions with different combinations of pH and temperature. Benefiting from the environment-sensitive stability and special structural feature inherent to the i-motif, this iMB exhibits a controllable sensitivity and specificity through choosing appropriate temperature/pH combination and recognition loop position in the i-motif probe. Leveraging the duplex-specific nuclease (DSN) resistance of the i-motif, we significantly enhanced the sensitivity by more than four orders of magnitude through the implementation of a DSN-assisted amplification strategy. Finally, miRNAs in cell lines and patient plasma were detected by the iMBs, as validated via quantitative reverse transcription polymerase chain reaction (RT-qPCR). This research successfully broadens the structural repertoire of MB stems to include a pH- and temperature-responsive i-motif stem.

Mobile genetic elements such as plasmids play a crucial role in the emergence of antimicrobial resistance. Hence, plasmid maintenance proteins such as ParA of the Walker A-type ATPases/ParA superfamily are potential targets for novel antibiotics. Plasmid partitioning by ParA relies on ATP-dependent dimerization and formation of chemophoretic gradients of ParA-ATP on bacterial nucleoids. Though polymerization of ParA has been reported in many instances, the need for polymerization in plasmid maintenance remains unclear. In this study, we provide insights into the polymerization of ParA and the effect of polymerization on plasmid maintenance. We report two mutations, Q351H and W362E, in ParA from the F plasmid (ParAF) that form cytoplasmic filaments independent of the ParBSF partitioning complex. Both variants fail to partition plasmids, do not bind non-specific DNA, and act as super-repressors to suppress transcription from the ParAF promoter. Further, we show that the polymerization of ParAF requires an ATP-dependent conformational switch. We identify two residues, R320 in helix 12 and E375 in helix 14 at the interface of the predicted ParAF filament structure, whose mutations abolish filament assembly of ParAF W362E and affect plasmid partitioning. Our results thus suggest a role for the C-terminal helix of ParAF in plasmid maintenance and assembly into higher order structures.

CRISPR-based methods enable genome modifications for diverse applications but often face challenges, such as inconsistent efficiencies, reduced performance in iterative modifications, and difficulties generating high-quality datasets for high-throughput genome engineering. Here, we present SELECT (SOS Enhanced programmabLE CRISPR-Cas ediTing), a novel strategy integrating the CRISPR–Cas system with the DNA damage response. By employing designed and optimized double-strand break induced promoters that are activated upon genome editing, SELECT enables a counter-selection process to eliminate unedited cells, ensuring high-fidelity editing. This approach achieves up to 100% efficiency for point mutations, iterative knockouts, and insertions. In high-throughput library editing, SELECT achieved up to 94.2% efficiency and preserved higher library diversity compared with conventional methods. Application of SELECT in flaviolin biosynthesis resulted in a 3.97-fold increase in production. Furthermore, integration with machine learning tools allowed rapid mapping of genotype–phenotype relationships. SELECT provides a versatile platform for precision genome engineering in Escherichia coli and Saccharomyces cerevisiae.

Metabolomics, a critical tool for analyzing small-molecule metabolites, integrates with genomics, transcriptomics, and proteomics to provide a systems-level understanding of fungal biology. By mapping metabolic networks, it elucidates regulatory mechanisms driving physiological and ecological adaptations. In fungal pathogenesis, metabolomics reveals host-pathogen dynamics, identifying virulence factors like gliotoxin in Aspergillus fumigatus and metabolic shifts, such as glyoxylate cycle upregulation in Candida albicans. Ecologically, it highlights fungal responses to abiotic stressors, including osmolyte production like trehalose, enhancing survival in extreme environments. These insights highlight metabolomics’ role in decoding fungal persistence and niche colonization. In drug discovery, it aids target identification by profiling biosynthetic pathways, supporting novel antifungal and nanostructured therapy development. Combined with multi-omics, metabolomics advances insights into fungal pathogenesis, ecological interactions, and therapeutic innovation, offering translational potential for addressing antifungal resistance and improving treatment outcomes for fungal infections. Its progress shed light on complex fungal molecular profiles, advancing discovery and innovation in fungal biology.

Global agriculture stands at a critical juncture, facing the dual challenge of sustaining food production for a rapidly growing population while mitigating the environmental consequences of intensive farming. The overuse of chemical fertilizers and pesticides has accelerated soil degradation, biodiversity loss, and ecological imbalances, threatening long-term viability. Synthetic microbial communities (SynCom) have emerged as a promising approach to reshape plant-microbe interactions, offering a precise, scalable, and ecologically sustainable alternative to conventional agrochemicals. Unlike native microbial communities, which form naturally and vary with environmental conditions, SynComs are deliberately assembled consortium of multiple microbial strains selected for their complementary functions, ecological compatibility, and ability to perform targeted roles within a host or environment. By engineering microbes with targeted functional traits, SynComs enhance nutrient assimilation, bolster plant defence, and fortify resilience against biotic and abiotic stresses. The understanding of SynCom design, exploring their composition, functional dynamics, and mechanisms for optimizing plant health is crucial for effective synthesis and application, alongside cutting-edge computational tools and genomic databases that enable precision engineering of microbial communities. Despite their transformative potential, large-scale application of SynComs remains constrained by challenges related to field efficacy, regulatory frameworks, and long-term microbial persistence. Addressing these barriers through interdisciplinary research and policy innovation is imperative. As environmental microbiome moves towards sustainability-driven solutions, SynComs hold the key to revolutionizing farming practices, reducing chemical dependence, and ensuring global food security in an era of mounting environmental stressors.