Nucleic Acids Research最新文献

Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate messenger RNA (mRNA) would be an advantageous approach to correct gene expression but has not been evaluated in an in vivo disease model. Here, we investigated whether a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identify and engineer human proteins capable of increasing mRNA translation using the CRISPR-Cas-inspired RNA-targeting system (CIRTS) platform to enable programmable, guide RNA-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3) that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold-key phenotypic indicators of Dravet syndrome. This work validates a strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential of targeted translational activation to address neurological haploinsufficiency.

DNA superhelicity and transcription are intimately related because changes to DNA topology can influence gene expression and vice versa. Information is transferred through the modulation of local DNA torsional stress, where the expression of one gene may influence the superhelical level of neighbouring genes, either promoting or repressing their expression. In this work, we introduce a one-dimensional physical model that simulates supercoiling-mediated regulation. This TORCphysics model takes as input a genome architecture represented either by a plasmid or chromosomal DNA sequence with ends constrained under specific biological conditions and computes the molecule's output. Our findings demonstrate that the expression profiles of genes are directly influenced by the gene circuit design, including gene location, the positions of topological barriers, promoter sequences, and topoisomerase activity. The novelty that TORCphysics offers is versatility, where users can define distinct activity models for different types of proteins and protein-binding sites. The aim of this research is to establish a flexible framework for developing physical simulations of gene circuits to deepen our comprehension of the intricate mechanisms involved in gene regulation.

The expanded backbone chemistries of xeno-nucleic acids (XNAs) hold significant promise for emerging areas of synthetic biology and nanomaterials, but metal-mediated XNA interactions remain largely unexplored. Here, we use a combination of circular dichroism spectroscopy and mass spectrometry to show that XNAs can form Ag+-mediated duplex structures resembling their DNA counterparts. XNAs with a range of different backbone compositions are found to stabilize photoluminescent silver nanoclusters with spectral properties that can be tuned based on their respective backbone chemistry. The resistance of silver nanoclusters to nuclease digestion is also compared for DNA and XNAs. These results show that XNA backbone chemistry provides a new tool beyond nucleobase sequence for controlling and expanding the properties of nucleic acid-stabilized silver nanoclusters and metal-mediated DNA duplexes.

Archaea, a domain of microorganisms found in diverse environments, including the human microbiome, represent the closest known prokaryotic relatives of eukaryotes. This phylogenetic proximity positions them as a relevant model for investigating the evolutionary origins of nucleic acid secondary structures such as G-quadruplexes (G4s) which play regulatory roles in transcription and replication. Although G4s have been extensively studied in eukaryotes, their presence and function in archaea remain poorly characterized. In this study, a genome-wide analysis of the halophilic archaeon Haloferax volcanii identified over 5800 potential G4-forming sequences. Biophysical validation confirmed that many of these sequences adopt stable G4 conformations in vitro. Using G4-specific detection tools and super-resolution microscopy, G4 structures were visualized in vivo in both DNA and RNA across multiple growth phases. Comparable findings were observed in the thermophilic archaeon Thermococcus barophilus. Functional analysis using helicase-deficient H. volcanii strains further identified candidate enzymes involved in G4 resolution. These results establish H. volcanii as a tractable archaeal model for G4 biology.

DNA ends are frequently damaged during the formation of DNA double-strand breaks (DSBs). These ends must be repaired to enable ligation during non-homologous end joining (NHEJ). NHEJ uses several end processing factors to repair DNA ends within the short-range synaptic complex (SRC), including Polymerase λ (Pol λ) which performs gap fill-in. Pol λ possesses a Ku Binding Motif (KBM) within its BRCT domain that interacts with Ku and recruits it to the SRC. Here, we show that in addition to its role in recruitment, Ku also stimulates Pol λ polymerase activity at DSBs. Using a structural prediction approach and biochemical assays, we identify and characterize an autoinhibitory intramolecular interaction between the N-terminal BRCT and C-terminal catalytic domains of Pol λ. Furthermore, single-molecule approaches reveal that Ku increases both the binding rate of Pol λ to primer-template DNA and the rate of nucleotide incorporation, demonstrating that Ku releases Pol λ autoinhibition and stimulates its polymerase activity within the SRC during NHEJ. Combined, these data highlight how intricate protein-protein interactions within the SRC complex are critical to regulate end-processing and maximize the fidelity of DSB repair.

The ribosomal RNA gene cluster (rDNA) in Saccharomyces cerevisiae consists of about 150 tandem copies, making it a fragile site prone to copy number changes through recombination among the repeat. While extensive research has been conducted to understand the mechanisms for rDNA stability maintenance, the relationship between the stability maintenance of rDNA and other genomic regions remains unclear. In this study, we identified a mutant, sic1, that exhibited instability in both rDNA and chromosome IV (chr.IV). We revealed that Ty element-mediated ectopic recombination leads to partial duplication and elongation of chr.IV. Furthermore, we found that rDNA instability is caused by an increased SIR4 gene dosage resulting from this partial duplication. These findings suggest a link between the stability of rDNA and other genomic regions.

Chronic hepatitis B virus (HBV) persistence relies on the chromatin plasticity of covalently closed circular DNA (cccDNA), a viral minichromosome resistant to current therapies. Using proximity labeling (TurboID-dCas9), ChIP-seq and DNA pull-down assays, we identified SMARCC2-a BAF scaffolding subunit-bound to cccDNA enhancer-promoter regions (EnhⅠ/XP, CP/EnhII), where it sustains nucleosome-depleted regions (NDRs) and recruits RNA polymerase II. Genetic or pharmacological BAF inhibition compacted cccDNA chromatin, reduced histone acetylation (AcH3/AcH4), and enhanced SMC5/6-mediated silencing to suppress transcription, with the BAF ATPase inhibitor FHT-2344 reducing serum HBV DNA by 50% (P <.05) and intrahepatic HBV RNA by 70% (P <.01) without cccDNA loss, indicating epigenetic silencing. Mechanistically, BAF maintains NDRs by counteracting nucleosome retention and recruiting host transcription factors such as HNF4α. This work concludes that BAF safeguards cccDNA chromatin plasticity to enable viral persistence, and targeting BAF (e.g. FHT-2344) epigenetically silences cccDNA, offering a novel strategy for functional cure.

Temperature stress is a fundamental challenge for all organisms. While two-component systems (TCSs) are known to transduce environmental signals in microbes, their role in thermal sensing remains underexplored. Here, we unveil a novel thermosensitive TCS, DhqSR, in the thermophile Thermus thermophilus HB27. We demonstrate that the histidine kinase DhqS perceives thermal cues and autophosphorylates at His327. Its cognate response regulator, DhqR, is activated through a unique tyrosine-phosphorylation mechanism: phosphorylation at a unique Tyr84 residue, rather than the canonical aspartate. This atypical DhqSR system orchestrates cellular thermoadaptation by directly regulating a key enzyme in the shikimate pathway, type II 3-dehydroquinate dehydratase (DHQase). Our findings reveal a novel molecular mechanism of temperature sensing and adaptation, providing a new paradigm for microbial environmental adaptation and offering a unique toolbox for engineering thermotolerance.

DNA origami has become a ubiquitous platform because it enables straightforward design of nanostructures that self-assemble with high yield. The interactions between the cooperative effects involved in its assembly are currently not well understood. Fortunately, the nearly infinite number of choices available to the origami designer provides a rich environment in which to explore cooperativity. The DNA domains comprising origami have predictable energetics, and the sources of cooperativity are conceptually straightforward, and the difficulty in predicting assembly comes from their large number of cooperative interactions. We are able to probe cooperativity by using design variations and measuring their effect on assembly yield. We employ an accelerated assembly protocol that increases the sensitivity of structural perfection, or lack thereof, to design variation, and apply this approach to survey a broad set of design features. Using the resulting dataset, we develop metrics to correlate thermal stability, beneficial cooperativity from short folds, and detrimental cooperativity from long folds, with defectivity. Surprisingly, these metrics can be combined to create a single parameter with a clear correlation to yield, which serves as a useful starting place for a predictive understanding of the interplay between cooperativity and design. In doing so, we also identify qualitative trends that provide useful insight into design best practice.