Nucleic Acids Research最新文献

Gene regulatory mechanisms control cell differentiation and homeostasis but are often undetectable, particularly at the single-cell level. We introduce bayesReact, which quantifies regulatory activities from bulk or single-cell omics data. It is based on an unsupervised generative model, exploiting the fact that each regulator typically targets many genes sharing a sequence motif. Using mRNA expression data, we illustrate and evaluate bayesReact on microRNAs (miRNAs). It outperforms existing methods on sparse bulk data and improves activity inference on single-cell data. Inferred miRNA activities correlate with miRNA expression across pan-cancer TCGA and healthy GTEx tissue samples. The activities capture cancer-type-specific miRNA patterns, e.g., for miR-122-5p and miR-124-3p, which also correlate more strongly with their target genes than their measured expression. This includes a strong negative correlation between miR-124-3p and the anti-neuronal REST transcription factor in nervous system cancers. Analyzing single-cell data, bayesReact detects prominent miRNAs during murine stem cell differentiation, including miR-298-5p, miR-92-2-5p, and the Sfmbt2 cluster (miR-297-669). Furthermore, spatio-temporal inference shows increasing miR-124-3p activity in differentiating neurons during embryonic spinal cord development in mice. bayesReact enables large-scale hypothesis-generating screens for novel regulatory factors and the discovery of condition-specific activities. It is implemented as a user-friendly R package (https://github.com/JakobSkouPedersenLab/bayesReact).

Sense codon reassignment enables ribosomal incorporation of nonproteinogenic amino acids (npAAs) at any of the 61 sense codons. Because npAAs replace proteinogenic amino acids (pAAs), the total number of available building blocks usually remains limited to 20. To overcome this, we previously introduced "artificial codon box division", where four-codon boxes (e.g. Val GUN) are split into distinct sets (e.g. GUY and GUG) using in vitro transcribed transfer RNAs (tRNAs) lacking nucleotide modifications. This allows two different amino acids-a pAA and an npAA-to be assigned within the same original box. While we previously demonstrated this by incorporating 23 amino acids, low incorporation efficiency hindered further expansion. Here, we applied our engineered tRNAs, tRNAPro1E2 and tRNAiniP, to the codon box division framework and optimized translation conditions to facilitate multiple npAA incorporations. Consequently, we successfully expanded the genetic code to 32 amino acids, incorporating 11 elongator npAAs and 1 initiator npAA while maintaining all 20 pAAs. Notably, these npAAs include therapeutically significant monomers such as β-amino, d-amino, and N-methyl amino acids, as well as an initiator N-chloroacetyl-d-tyrosine for peptide macrocyclization. This platform offers vast potential for generating diverse macrocyclic peptide libraries with unique chemical entities for drug discovery.

This study describes the identification and characterization of two new extremophilic phage recombinases, UvsXt and UvsXp, discovered through metagenomic analysis within the Virus-X project, and explores their potential applications in biotechnology. DNA recombinases are essential for maintaining genome integrity across all kingdoms of life by facilitating homologous recombination and repairing double-stranded DNA breaks. Their capacity to bind and stabilize single-stranded DNA (ssDNA) has led to wide-ranging applications in molecular biology. UvsXt and UvsXp show homology with known bacterial RecA and viral UvsX recombinases, including conservation of key catalytic residues and DNA-binding motifs. Biochemical assays reveal that both enzymes exhibit superior DNA strand-exchange activity compared to Escherichia coli RecA. High-resolution crystal structures of UvsXt (2.0 Å) and UvsXp (2.6 Å) confirm a conserved RecA-like core fold, with distinct structural variation at the N-terminus responsible for oligomerization. However, in spite of their similarities, we show that neither enzyme is capable to functionally replace RecA in E. coli. Their remarkable thermostability and functionality across diverse chemical environments highlights their robustness for biotechnological use. Notably, UvsXt enhances loop-mediated isothermal amplification of viral RNA by stabilizing ssDNA intermediates. These findings expand the repertoire of thermostable recombinases with potential utility in diagnostic applications.

DNA is most often found in its canonical B-form double-helical structure, but can also adopt alternative conformations, known as non-B DNA structures. Numerous non-B structures have been characterized, including G-quadruplexes, i-motifs, Z-DNA, hairpins, cruciforms, slipped structures, R-loops, and H-DNA. Non-B DNA motifs are enriched in functional regions, including near transcription start and end sites, topologically associated domains, and replication origins, suggesting their importance in gene regulation, genome organization, and replication. However, these structures are intrinsically prone to error-generating processing, leading to genomic instability and hence have been implicated in the development of human diseases. Here, we discuss recent advances in understanding the biological roles of non-B DNA structures and their contribution to genomic instability in somatic and germline contexts. We highlight how they promote replication stress, transcription stalling, and DNA breaks, resulting in the formation of mutational hotspots. Emerging technologies have enabled the detailed mapping of previously challenging repetitive regions that harbor potential non-B DNA-forming sequences, and are poised to unravel additional contributions in human disease and evolution. Furthermore, we explore the dual role of non-B DNA as a driver of genetic variation that facilitates evolutionary adaptation and as a source of mutations that contribute to tissue dysfunction and aging.

CRISPR-Cas9 knock-in efficiency is often limited by geometric misalignment between donor DNA and the endogenous strand-invasion path. In Aspergillus nidulans, we found that integration drops sharply when the insertion site is offset from the invasion entry point, producing premature annealing or unsupported 3' ends that stall DNA synthesis. Chromatin immunoprecipitation-based profiling shows directional loading of the RAD51 homolog UvsC around Cas9-induced double-strand breaks, thereby defining the spatial origin of strand invasion. Guided by this insight, we introduce a dual-single-guide RNA design that places two cuts flanking the insertion site to create a geometry-matched strand-invasion window. This alignment consistently and markedly increases homology-directed-repair-mediated integration across insert sizes and editing tasks-including C-terminal tagging, bidirectional promoter rewiring, and long-distance dual-site mutagenesis-and generalizes across multiple fungal species. We propose a structural-docking model in which pairing fidelity between the resected chromosomal strand and donor homology arms governs knock-in outcomes, providing a practical design principle for efficient and precise genome engineering at structurally constrained loci.

The spatial organization of RNA-scaffolded condensates is fundamental for understanding of basic cellular functions, but may also provide pivotal insights into diseases. One of the major challenges to understanding the role of condensates is the lack of technologies to map condensate-scale protein architecture at subcompartmental resolution. To address this, we introduce HCR-Proxy, a proximity labelling technique that couples hybridization chain reaction (HCR)-based signal amplification with in situ proximity biotinylation (Proxy), enabling proteomic profiling of RNA-proximal proteomes at subcompartmental resolution. We applied HCR-Proxy to nascent pre-rRNA targets to investigate the distinct proteomic signatures of the nucleolar subcompartments and to uncover a spatial logic of protein partitioning shaped by RNA sequence. Our results demonstrate the ability of HCR-Proxy to provide spatially resolved maps of RNA interactomes within the nucleolus, offering new insights into the molecular organization and compartmentalization of condensates. This subcompartment-specific nucleolar proteome profiling enabled integration with deep learning frameworks, which effectively confirmed a sequence-encoded basis for protein partitioning across nested condensate subcompartments, characterized by antagonistic gradients in charge, molecular weight, and RNA-binding domains. HCR-Proxy thus provides a scalable platform for spatially resolved RNA interactome discovery, bridging transcript localization with proteomic context in native cellular environments.

CRISPR interference (CRISPRi) screens have emerged as powerful tools for dissecting gene function, yet their application to genes with multiple promoters, which comprise over 60% of human genes, remains poorly understood. Here, we demonstrate that CRISPR-dCas9-based screens exhibit widespread promoter specificity, with untargeted promoters often showing compensatory upregulation to maintain gene expression. Leveraging this selective targeting of individual promoters within the same gene, we developed Isoform-Specific single-cell Perturb-Seq to systematically analyse alternative promoter function. Our analysis revealed that alternative promoters in 51.6% of targeted genes drive distinct transcriptional programs. This suggests that promoter selection represents a fundamental mechanism for generating cellular diversity rather than mere transcriptional redundancy. In breast cancer models, this promoter-specific targeting revealed differential effects on drug sensitivity, where distinct estrogen receptor (ESR1) promoters showed opposing influences on tamoxifen response and patient survival. These findings demonstrate the necessity of promoter-level analysis in functional genomics and suggest new strategies for therapeutic intervention through promoter-specific targeting.

Heterochromatin is characterized by an inaccessibility to the transcriptional machinery and is associated with the histone mark H3K9me3. However, studying the functional consequences of heterochromatin loss in human cells has been challenging. Here, we used CRISPRi-mediated silencing of the histone methyltransferase SETDB1 to remove H3K9me3 heterochromatin in human neural progenitor cells. Despite a major loss of H3K9me3 peaks resulting in genome-wide reorganization of heterochromatin domains, silencing of SETDB1 had a limited effect on cell viability. Cells remained proliferative and expressed appropriate marker genes. We found that a key event following the loss of SETDB1-mediated H3K9me3 was the expression of evolutionarily young L1 retrotransposons. Derepression of L1s was associated with a loss of CpG DNA methylation at their promoters, suggesting that deposition of H3K9me3 at the L1 promoter is required to maintain DNA methylation. In conclusion, these results demonstrate that loss of H3K9me3 in human neural somatic cells transcriptionally activates evolutionary young L1 retrotransposons.

Z-DNA is known to be a left-handed alternative form of DNA and has important biological roles in cancer and other genetic diseases. In a recent study, we discovered CBL0137, a curaxin ligand, to enhance cancer immunotherapy by inducing Z-DNA formation and activating the Z-DNA-binding protein ZBP1. However, the structural information on binding complexes between Z-DNA and CBL0137 ligand has not reported to date. Here we present the first high-resolution structure of the complex between a Z-DNA and a curaxin ligand CBL0137. This compound is observed to interact with the Z-DNA through π-stacking and zig-zag localization. Furthermore, we directly observe the complex in living human cells using in-cell 19F NMR for the first time. This structural information provides a platform for the design of topology-specific Z-DNA-targeting compounds and is valuable for the development of new potent anticancer drugs.