CRISPR-Cas12a biosensing technology advances and applications in precision diagnostics and cancer research.

First report of CRISPR/Cas13a-based rapid detection of groundnut bud necrosis virus without amplification.

Neutral sphingomyelinase 2 knockdown attenuates disease severity and modulates immune responses in enterovirus A71-infected mice.

Crustacean cardioactive peptide activates pupal ecdysial behavior in the 28-spotted larger potato ladybird.

Combined toxicity prediction of deoxynivalenol and fumonisin B1 by physiologically based toxicokinetic modelling and in vitro toxicity mechanism study on astrocyte-like C6 cells.

We report a series of uracil-triazole-pyrene peptidomimetics (2-4) designed to achieve modular geometric control through peptide linkers of varying length and flexibility. All three compounds exhibited submicromolar affinity for ds-DNA, while compound 4 also showed strong binding to ds-RNA, demonstrating the advantage of combining a uracil recognition unit with an extended pyrene aromatic surface compared to the Pyr-Trp reference ligand and the phenanthridine analog 4'. Structural variations strongly affected Cu(II) coordination. Although Pyr-Trp bound Cu(II) approximately 1000-fold more strongly than the short, rigid compound 2, elongation and increased linker flexibility restored high affinity in 4. Notably, replacing phenanthridine (4') with pyrene (4) enhanced Cu(II) binding by nearly 100-fold, highlighting the superior coordination properties of the pyrene scaffold. Cu(II) complexation significantly enhanced nucleic acid binding exclusively for compound 2, increasing its DNA/RNA affinity 15-fold. The resulting 2-Cu(II) complex displayed exceptional selectivity for poly rA-poly rU, exceeding that of the Pyr-Trp-Cu(II) analog by more than 10-fold, consistent with uracil-mediated Hoogsteen-type recognition. Membrane interactions with POPC MLVs were linker-dependent, producing fluorescence enhancements of up to 1400% (2), 650% (3), and 100% (4). Together with negligible cytotoxicity, these findings indicate that compounds 2-4 represent promising multifunctional platforms for Cu2+ sensing and photoinduced therapeutic applications.

RNA binding proteins (RBPs) are multi-faceted proteins that interact with transcripts in various RNA driven processes and functions. However, in situ covalent capture techniques for screening authentic RNA substrates of RBPs remain challenging in terms of reproducibility, specificity, and sensitivity. Here, we developed CuCLIP-seq (CuAAC-Crosslinking and Immunoprecipitation Sequencing), an alternative in situ covalent capture sequencing method which utilizes CuAAC reaction to crosslink RBP-RNA between azido groups of RBPs and ethynyl groups of RNA molecules, followed by streptavidin-mediated enrichment of RNA substrates and high-throughput sequencing. We demonstrate the reliability of CuCLIP-seq by identifying substrate RNAs of several RBPs, including PTBP1, ADAR2, SRSF2, HNRNPA1, and PINX1, especially for capturing low-abundance targets. Additionally, this approach is authenticated by specifically resolving the alternative splicing transcripts targeted by PTBP1 and RNA targets by PINX1. Thus, this technique offers a sensitive and specific approach for detecting RBP substrates with high reproducibility, potential scalability, and wide applicability.

Hexanucleotide repeat expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. These expansions give rise to pathogenic sense and antisense repeat RNAs that form nuclear foci and undergo repeat-associated non-AUG translation, producing dipeptide repeat proteins with cellular toxicity. Directly targeting the causative repeat RNAs with antisense oligonucleotides represents a promising therapeutic strategy. One barrier to further development is the propensity of this G-rich repeat-containing RNA target to form stable secondary structures, which may hinder efficient hybridization. In this study, we designed a panel of fluorine-modified ASOs that target the sense repeat expansions. We identified C-rich F-ASO gapmers that reduced translation from sense repeat RNAs in a cell-based reporter assay and lowered the RNA foci burden in patient-derived cells. Structural analyses in vitro revealed that the 2'F-RNA gapmer formed a stable hairpin structure. Our results demonstrate that structural properties of fluorine modifications can be leveraged for effective binding of repeat RNA and highlight the potential for F-ASOs to serve as therapeutic tools when targeting toxic repeat RNAs in C9ORF72-mediated FTD/ALS and other repeat expansion diseases.

The seed region (positions from g2 to g8) in the guide strand of the small interfering RNA (siRNA) plays a pivotal role in the selective recognition and binding of target mRNA during the human Argonaute 2 (hAGO2)-facilitated gene silencing. Strategic incorporation of specific chemical modifications in the seed region is crucial for enhancing specificity toward the target mRNA and minimizing off-target effects. Using microsecond-scale molecular dynamics (MD) simulations across three different states of hAGO2-siRNA complexes (guide-only, seed-paired guide-target, and fully paired guide-target), we show that 2'-OMe and 2'-F modifications stabilize the C3'-endo sugar conformation in the seed region. The conformational changes enhance Watson-Crick base pairing with the target RNA while maintaining the hAGO2 binding interactions, explaining the improved RNA interference (RNAi) activity. (S)-GNA modification at the g7 position stabilizes the intrinsic kink between the g6 and g7 nucleotides. Additionally, it reduces the base pairing occupancies at the end of the seed region, which rationalizes the mitigation of off-target effects and improved RNAi activity. At the g2 position, (S)-GNA induces large deviations in the backbone torsion angles at the g1-g2 clover-leaf geometry, which could affect the guide strand loading into the RNA-induced silencing complex (RISC). ANA modification at the g7 position maintains the A-type geometry of RNA, Watson-Crick H-bond occupancy, and binding interactions with hAGO2. However, when inserted at the g2 position, ANA induces steric clashes with MID domain residues, which likely reduce the loading of the guide strand into the RISC. Overall, the results demonstrate position-dependent structural and functional effects of seed-region modifications, including 2'-OMe, 2'-F, (S)-GNA, and ANA, providing critical insights for the design and development of next-generation siRNA-based therapeutics.

Abstract Spatiotemporal control of RNA therapeutics remains a fundamental challenge limiting clinical translation. Here, we develop a photoactivatable CRISPR/Cas13d (paCas13d) system that enables non-invasive, light-controlled RNA manipulation in deep tissues. Through structure-guided engineering, we identify optimal split sites within RfxCas13d and create light-switchable fragments using CRY2PHR/CIBN optogenetic dimerization. To overcome the limited tissue penetration of blue light, we engineer polyethylenimine-functionalized upconversion nanoparticles (UCNPs-PEI) that serve dual roles as gene carriers and photon transducers, converting tissue-penetrating near-infrared (NIR) to blue light. The UCNPs-PEI@paCas13d system achieves precise spatiotemporal control of RNA targeting within bone tissue in vivo. In a murine steroid-associated osteonecrosis model, NIR-activated paCas13d achieves robust TET3 knockdown, disrupting the TET3-5hmC-PTEN axis that drives glucocorticoid-induced osteocyte apoptosis. This targeted intervention prevents bone deterioration, with treated mice showing preserved trabecular architecture, enhanced bone volume, and favorable shifts in bone turnover markers, while maintaining systemic glucocorticoid efficacy. Our platform combines the programmability of CRISPR/Cas13d with non-invasive optical control, offering a versatile approach for treating diseases requiring localized RNA modulation while minimizing systemic effects.

Non-canonical proteolytic activation of RNase L by SARS-CoV-2 3CLpro offsets inactivation of OAS1 p46 antiviral signaling.

The concerted action of regulatory RNA and RNA binding proteins (RBPs) provides cells with highly versatile and transient tools to fine-tune gene expression in a broad variety of cellular systems (Unfried and Ulitsky, Nat Cell Biol 24: 608-615 [2022]; Hentze et al., Nat Rev Mol Cell Biol 19: 327-341 [2018]; Suzuki et al., Nat Genet 50: 657-661 [2018]). In this work, we explore the function of a specific interaction between PTBP1 and the cytoplasmic long noncoding RNA (lncRNA) CyCoNP, highly expressed in neural progenitors (Desideri et al., NAR 52: 9936-9952 [2024]), in which the RBP regulates the abundance of the lncRNA by a miRNA-mediated mechanism. PTBP1 is a well-known splicing regulator, although limited and peculiar examples of its involvement in other cellular processes, such as IRES-dependent translation and miRNA recognition of target RNAs, have been described (Dorn et al., Cell Death Dis 14: 6429 [2023]; Kim et al., Nat Commun 12: 5057 [2021]). We have recently characterized CyCoNP lncRNA as a regulator of NCAM1, which acts through a mechanism that involves direct RNA-RNA interaction with NCAM1 mRNA, balancing the availability and the localization of miR-4492 in its vicinity (Desideri et al., NAR 52: 9936-9952 [2024]). Here we expand the repertoire of molecular players acting in this circuitry by describing a direct interaction between PTBP1 and CyCoNP lncRNA. Through endogenous RNA purification, protein immunoprecipitation, and exploiting CyCoNP mutant constructs, we found that PTBP1, when interacting with CyCoNP, hampers miR-4492 binding to the lncRNA and in turn impedes its regulation on NCAM1 mRNA. This work aims to expand the biochemical characterization of regulatory networks relying on RBPs and their cognate target RNAs, highlighting the relevance of the analysis of the subcellular environment for each case of study.

RNA remains a largely untapped target for covalent small-molecule intervention due to the lack of electrophiles with predictable reactivity and stability in biological settings. Here, a mechanistically defined and tunable class of epoxide- and aziridine-2-carboxamide electrophiles that enable structure-guided covalent targeting of RNA is described. These warheads arise from an unexpected hydrolytic rearrangement of 3-chloropivalamide precursors under physiological conditions and selectively react with guanine N7, with reactivity and stability controlled by substitution pattern, linkage chemistry, and stereochemistry. Application to two distinct RNA targets demonstrates generality: epoxide- and aziridine-based ligands covalently modify pathogenic r(CUG)exp repeat RNA and disrupt RNA-protein interactions in vitro and in cells, while structure-guided placement on a flavin scaffold yields stereoselective covalent modulators of the flavin mononucleotide riboswitch with validated reaction site and cellular activity. Together, this work demonstrates epoxide- and aziridine-2-carboxamides as a versatile platform for covalent RNA targeting and provides a general framework for the rational design of stereochemically controlled RNA-reactive small molecules.

ConspectusBiomolecular condensates are membrane-less organelles formed via liquid-liquid phase separation (LLPS) in cells, which play crucial roles in organizing biochemical reactions, regulating gene expression, and responding to environmental stimuli. These dynamic membrane-less organelles, such as stress granules and nucleoli, could concentrate specific proteins and nucleic acids for spatiotemporally controlling cellular processes. The engineering of synthetic condensates is beneficial for understanding condensates formation, simulating cellular behavior, and exploration of biological pathologies.Nucleic acid, as an important component of biomolecular condensates in cells, offers a unique platform to engineer synthetic condensates due to its programmability and precise and predictable Watson-Crick base pairing. The nucleic acid-based condensates were assembled through multivalent forces among nucleic acids or nucleic acid-peptide complexes. By designing and modifying nucleic acid sequences, the interaction forces could be regulated with external stimuli to control the formation and decomposition of nucleic acid-based condensates for various fields application. Our group has constructed various nucleic acid-based biomolecular condensates and applied them in biosensing and cellular regulation. We designed CUG repeats-based condensates for improving fluorescent RNA aptamer properties (enzymatic degradation resistance, thermal stability, photostability, and binding affinity to fluorophores) and detecting in vitro and intracellular biomolecules (adenosylmethionine and tetracycline), as well as target cells with overexpressed epithelial cell adhesion molecules. In addition, we leveraged the strong Watson-Crick base pairing ability to recruit the intracellular target RNA into condensates for cellular regulation.In this Account, we give an overview of nucleic acid-based biomolecular condensates. We first discuss the intermolecular interactions and forces involved in the formation of nucleic acid-based biomolecular condensates. Subsequently, we summarize recent research about nucleic acid-based condensates and their applications in the fields of biological imaging and biosensing, cell simulation, cellular regulation, and drug delivery. Finally, we outline the current challenges and future opportunities of nucleic acid-based biomolecular condensates. We hope that this Account will afford significant inspiration in the design of nucleic acid-based condensates and the applications in cell biology and biomedicines.

CRISPR/Cas13a is a powerful RNA-targeting platform for molecular diagnostics, but conventional single-effector systems typically require contiguous RNA targets longer than ∼20-28nt, limiting sensitivity and target flexibility. CRISPR/Cas13a-CTAM is presented as a compensatory target activation mechanism that facilitates synergistic Cas13a activation through two independently programmable short RNA effectors. By functionally decoupling allosteric activation and binding stabilization, CRISPR/Cas13a-CTAM supports robust activation by ultra-short RNA targets as short as 13nt, substantially expanding the detectable target range. Compared with traditional single-effector Cas13a assays, CRISPR/Cas13a-CTAM achieves a detection limit of 1 fM for a 13-nt RNA target, representing an approximately tenfold sensitivity improvement. Notably, a single-nucleotide mismatch within the 13-nt target induces up to a 35-fold reduction in apparent cleavage rate, corresponding to a sevenfold enhancement in mismatch discrimination. The dual-effector architecture further enables simultaneous dual-target detection, demonstrated by dual miRNA profiling related to COVID-19 and combined detection of exosome membrane proteins. Moreover, the weakly activating effector was utilized as an anchoring module to achieve the first functional immobilization of Cas13a on a sensing surface, enabling in situ electrochemical miRNA detection. By overcoming the reliance on long RNA targets, CRISPR/Cas13a-CTAM provides a sensitive, programmable platform for RNA diagnostics and integrated biosensor development.

Achieving stable, multiplexed, and affordable nucleic acid detection in a true one-pot format remains a long-standing challenge for molecular diagnostics. Here, we report PANDA, a photo-activated rolling circle amplification (RCA)-Argonaute (Ago) cascade that encodes a stringent molecular AND logic gate between optical activation and target RNA recognition to enable highly sensitive, programmable RNA detection. In this system, ultraviolet (UV) irradiation triggers photolysis of the precursor probe, simultaneously generating a 5'-phosphorylated DNA guide and circularizable padlock templates, while target RNA selectively initiates ligation and RCA. Signal generation occurs only upon convergence of these two orthogonal inputs, enforcing strict dual-input gating prior to Ago activation. Once engaged, Ago catalyzes continuous guide regeneration and sequence-directed reporter cleavage, producing signal boosting outputs within a single reaction vessel. The modular architecture further enables parallel assembly of multiple AND logic gates synchronized by a common optical trigger yet paired with distinct RNA targets, allowing controllable multichannel detection without physical compartmentalization. By integrating optochemical control with RCA-Ago-mediated catalytic turnover, PANDA establishes a streamlined, stable, and cost-efficient framework for logic-gated nucleic acid diagnostics and multiplexed RNA sensing.

RNA-targeting CRISPR-Cas13 enzymes are robust RNA knockdown tools with both on-target and collateral cleavage activities. However, to date, the in vivo RNA cleavage mechanisms remain poorly understood. Here, we combine in vitro and in vivo methods to elucidate the exact cleavage sites of Cas13. We reveal that some subtypes of Cas13, including Cas13b and Cas13bt, cleave the target RNA at predominant positions, and rational engineering of Cas13 further improves precision. Building on these findings, we develop RNA segment editing (RSE), a targeted RNA cleavage and repair method, to restore dysfunctional RNA in cells. We anticipate that RSE will enable precision RNA engineering for therapeutics and basic research.

Similar to proteins, many RNAs fold into three-dimensional (3D) structures to perform biological functions. Here we present the trRosettaRNA server, a web-based platform for automated RNA 3D structure prediction using deep learning. The primary input is the nucleotide sequence of a target RNA, with the option to upload custom multiple sequence alignments and secondary structures. The server uses an end-to-end neural network for automated 3D structure prediction, followed by an energy optimization step to resolve structural violations. As an automated server, trRosettaRNA is distinguished by its state-of-the-art modeling accuracy, flexible input options and comprehensive visualization of prediction results. trRosettaRNA has been successfully applied in various contexts, including predicting structures for Rfam families lacking known 3D structures, where representative cases of high-confidence structure predictions were found to align well with subsequent experimental observations. Utilizing up to 5 central processing unit (CPU) cores in parallel on our computer cluster, the server takes a median time of about 1 h to predict structures for RNA sequences with about 200 nucleotides. The standalone package for trRosettaRNA offers distinct advantages such as enhanced data privacy for sensitive sequences, the ability to bypass server queues and integration into high-throughput automated pipelines. Importantly, the open-source nature of the package empowers researchers to directly modify the codebase for specialized research needs or to develop derivative tools by fine-tuning the underlying neural network. The web server and standalone package of trRosettaRNA are available at https://yanglab.qd.sdu.edu.cn/trRosettaRNA/ and https://github.com/YangLab-SDU/trRosettaRNA2 , respectively.

The global rise in antibiotic resistance necessitates new agents targeting essential bacterial processes like protein synthesis. Structure-based virtual screening enables the rapid identification of drug candidates from large chemical libraries, accelerating drug discovery. Here, we report an integrated computational and experimental pipeline to identify small-molecule translation inhibitors targeting the catalytic cavity of the E. coli ribosome. A consensus docking strategy using Glide and AutoDock Vina, combined with pharmacophore filtering and interaction analysis, was applied to FDA-approved, experimental, and investigational drug libraries to prioritize candidate compounds. The binding free energies of the compounds were estimated using restrained molecular dynamics (MD) simulations coupled with the MM-GBSA method, where the computational efficiency was improved by truncating the ribosome-ligand complexes. Guided by these results and our previous work on the E. coli 30S decoding center, 14 hit compounds were selected for the in vitro antibacterial and translation inhibition assays. Among these, Mitoxantrone (IC50 = 14.10 ± 0.38 µM) was identified as a translation inhibitor with a bacteriostatic effect comparable to the antibiotic Clindamycin. Whereas Plerixafor (IC50 = 62.30 ± 6.47 µM), Olcegepant (IC50 = 144.30 ± 16.41 µM), and Ziritaxestat (IC50 = 224.30 ± 25.02 µM) showed inhibitory effects at higher concentrations. Notably, Mitoxantrone has the potential to be an anticancer agent and a translation inhibitor that may significantly benefit cancer patients by addressing secondary bacterial infections. The pharmacokinetic and toxicological profiles of these compounds are already well-characterized. Overall, this work illustrates a useful drug discovery strategy combining virtual screening, MD simulations, and experimental validation to identify ribosome-targeting inhibitors and can be extended to other challenging RNA targets and protein-RNA complexes.

Targeting RNA G-quadruplexes (rG4s) with photosensitizers offers a mechanistically distinct approach for cancer therapy by integrating molecular targeting with photodynamic activity. Here, we report TO-ISe, a selenium-containing rG4-targeting photosensitizer designed for combined photodynamic therapy and immunomodulation. TO-ISe binds rG4s with high affinity (Kd = 860 nM) and exhibits pronounced Type I photodynamic activity. rG4 complex formation enhances radical generation, resulting in selective phototoxicity toward cancer cells (IC50 = 0.16-0.27 μM) with substantially lower toxicity in nonmalignant cells (IC50 = 5.9-7.8 μM). Under dark conditions, TO-ISe suppresses rG4-regulated oncogenes (c-MYC, NRAS and hTERT), leading to mitochondrial dysfunction, ferroptosis, and apoptosis. Upon white-light irradiation (22.1 mW·cm-2), TO-ISe further potentiates antiproliferative efficacy and induces immunogenic cell death via activation of the cGAS-STING pathway. In an orthotopic 4T1 tumor model, TO-ISe (0.5 mg kg-1) with irradiation achieved up to 72.8% tumor growth inhibition. These results highlight TO-ISe as a dual-function rG4-targeting photodynamic immunotherapeutic agent.