Nucleic Acids Research

Escherichia coli couples the initiation of DNA replication with cell size by modulating the activity of the replication initiator protein DnaA. The activity of DnaA is regulated by both its interconversion between an active and inactive form and its titration on binding sites on the chromosome. Whereas its interconversion has been thoroughly studied, the extent to which DnaA titration can control replication initiation is poorly understood. Here, we describe the control of E. coli DNA replication via titration by modulating the expression of an always-active DnaA variant in four growth conditions. While we obtained stable cell cycles during slow growth, faster growth associated with overlapping replication forks led to replicative instability and DNA damage. Overall, our results provide insights into the limits of titration-based systems in the control of genome replication and their potential role in the evolutionary trajectory of E. coli. Finally, this study provides design principles for a simplified, titration-only regulatory mechanism for DNA replication in synthetic cells.

Ribozyme-based permuted intron-exon (PIE) systems offer a protein-independent route to circRNA production, but existing platforms require elevated temperatures that promote RNA degradation. Here we report the first application of the Candida albicans mitochondrial large subunit (C.a.mtLSU) group I intron as a PIE platform for circRNA synthesis, which we term PCanPIE (Pyle lab Candida PIE). We evaluated three peripheral stems, P5, P6b, and P8, as permutation sites and demonstrated that all three support circularization under near-physiological conditions (25{degrees}C, 6 mM MgCl2), without the 55{degrees}C heating step required by existing PIE systems. Kinetic analysis revealed that permutation site does not affect the observed splicing rate constant but does influence PCanPIE folding and therefore influences circularization efficiency. The P6b permutation yielded the highest circularization efficiency, with 95 % of the precursor splicing to produce circRNA. Optimization of spacer sequences flanking the circRNA payload eliminated interference from structured native exon sequences and enabled efficient circularization of RNAs up to 1,657 nt, including structured, repetitive, and naturally occurring sequences. Together, these results establish PCanPIE as a versatile and near-physiologically active addition to the group I intron PIE toolkit.

The eukaryotic ribosomal genes are multi-copy genes, transcribed from the rDNA, and approximately one third of them is actively transcribed in differentiated cells. A number of lncRNAs have been identified from the intergenic spacer between the rRNA genes, among those the spacer RNA and PAPAS that are involved silencing of rRNA gene copies by altering the chromatin configuration. Here, we have identified lncRNAs that are transcribed from the human rDNA loci and modulate the loci; IGS38 positively regulates rRNA gene transcription by associating to the 47S rRNA gene promoter and modulating the rRNA promoter accessibility while IGS32as associates with heterochromatin. IGS38 binds to the 47S gene promoter through the RNA pol I factors TAF1C and RRN3 as well as the Williams Syndrome Transcription Factor (WSTF), a component of the B-WICH chromatin remodelling complex. The increased accessibility of the promoter stabilises the architectural protein Upstream Binding Factor (UBF) at the rRNA promoter, thereby facilitating RNA pol I promoter escape. Furthermore, IGS38 knock down displays and increased dsRNA abundance in the cytoplasm with a weak induction of the dsRNA sensor OAS2, typically induced by interferon and viral dsRNA. Overall, the both IGS38 and IGS32as are chromatin associated lncRNAs involved in rDNA chromatin changes, and IGS38 is stimulating, together with WSTF, rRNA gene transcription in human cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/722362v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@14d4159org.highwire.dtl.DTLVardef@fd773forg.highwire.dtl.DTLVardef@a0030dorg.highwire.dtl.DTLVardef@1285301_HPS_FORMAT_FIGEXP M_FIG C_FIG IGS stabilises 47S rRNA transcription, disruption of IGS38 expression leads to the release of dsRNA in the cytoplasm and a weak immune activation of OAS2. Created by biorender (https://biorender.com/shortURL)

Defining high-confidence RNA interaction sites for specific proteins is essential to understand RNA biology, but existing methods face trade-offs between specificity, sensitivity, and experimental accessibility. Here, we present fluorescent Cross-linking and analysis of cDNAs (fCRAC), a mammalian-cell optimized update to the CRAC protocol. In fCRAC, a fluorescent adaptor is used, in place of radiolabeling, to visualize RNA-protein complexes during gel-purification. fCRAC retains the tandem affinity purification and stringent, denaturing conditions of classical CRAC, enabling nucleotide-resolution mapping of protein:RNA interactions with high signal to noise ratio. We initially tested fCRAC using RPP25L, a component the RNase MRP and RNase P complexes. RPP25L almost exclusively bound to predicted, single sites in the RNA components (RMRP and RPPH1), showing excellent selectivity with nucleotide resolution. To support analysis of UV cross-linking data for more complex targets, we developed the trxtools package and example pipeline for standardized processing, quality control, and analysis of data from fCRAC and related methods. We include tailored strategies for repetitive RNA classes, such as tRNA and rRNA, which can be challenging to analyze using other approaches. We applied fCRAC and trxtools to define the RNA interactome of human CYCLON/CCDC86, a nuclear protein previously implicated in oncogenesis. This revealed specific interactions with rRNA, tRNA and ncRNAs involved in pre-rRNA and pre-tRNA processing. HighlightsO_LINucleotide-resolution definition of RNAs interacting with specific proteins, including rRNA and tRNA C_LIO_LIStringent denaturing purifications and robust visualization steps, with no requirement for radioactive labelling C_LIO_LITrxtools provides an integrated analysis pipeline with approaches for analyzing both single and multi-copy RNA species C_LI

Variants affecting RNA splicing are a major contributor to human disease, yet the consequences of variants outside of the canonical splice motifs are often difficult to determine. Here, we present a protocol for minigene-based evaluation of candidate splice-altering variants. The methodology described includes locus-specific insert design, commercial gene fragment synthesis, and long-read sequencing. The combined approach enables rapid assay development and nucleotide level resolution of the effect on splice isoforms in vitro, providing a scalable framework for functional validation of predicted cryptic splice variants. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/723105v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1a88cb5org.highwire.dtl.DTLVardef@adda98org.highwire.dtl.DTLVardef@1ea587corg.highwire.dtl.DTLVardef@574a63_HPS_FORMAT_FIGEXP M_FIG C_FIG

We developed a lightweight codon-based DNA Transformer equipped with multi-head self-attention and an adaptive classifier head, which achieves exon intron classification with high accuracy and also has moderate accuracy in CDS classification and splice site recognition. We named this model as ExIT (Exon-Intron Transformer). We have implemented codon tokenization for this model. This has been validated on the human genome with external validation from the chimpanzee genome. Further benchmarking has implied that our model is better than the existing models in the above tasks.

Type III restriction-modification (RM) enzymes are prominent bacterial defense against bacteriophage and invading foreign DNA that also modulate the hosts epigenetic landscape. Genome analysis of the host-adapted Mycoplasma bovis PG45 that has a very small genome revealed a Type III RM locus comprising one res and three mod genes. We characterized Mbo45V, a representative enzyme encoded by this locus. The enzyme forms a heterotrimeric complex consisting of two Mod subunits and one Res subunit. Mbo45V recognizes the asymmetric sequence 5'-YAATC-3' (Y = T/C) and cleaves DNA having at least two head-to-head oriented sites [~]26-28 bp away from the recognition site. Methylation of the second adenine of the target site using cofactor S-adenosylmethionine (SAM) protects DNA from restriction, while the SAM analogue sinefungin enhances DNA binding and cleavage. Kinetic studies reveal that Mbo45V exhibits relatively weak DNA binding affinity and an unusually high Km for SAM, indicating low cofactor affinity compared to prototypical enzymes such as EcoP15I. ATPase activity is strongly stimulated by cognate DNA and is inhibited upon methylation of the substrate, suggesting a regulatory interplay between methylation and restriction functions. Comparative analysis indicates that, although Mbo45V shares core mechanistic features with prototypes from Escherichia coli, its kinetic parameters are distinct. These differences likely reflect adaptation to the stable intracellular environment of M. bovis, in contrast to the fluctuating conditions encountered by the enteric bacteria.

Transcription factors (TFs) regulate gene expression by binding to specific DNA sequences. Widely used models of TF-DNA binding, such as position weight matrices (PWMs) and position-specific affinity matrices (PSAMs), assume binding free energy is the sum of independent base contributions. However, there is ample evidence that non-additive effects significantly influence TF binding. Here, we utilize data from a high-throughput in vitro assay (ivtFOODIE) to generate genome-scale TF-DNA dissociation constants (Kd) and systematically evaluate sequence-to-affinity models of increasing complexity. We demonstrate that pairwise additive models exhibit systematic deviations from the measured affinity landscapes. Models incorporating adjacent dinucleotide interactions and deep learning architectures achieve markedly improved agreement with experimental Kd values. The magnitude of this non-pairwise-additivity depends strongly on the TF family. In silico mutation screening reveals widespread, TF-specific long-range interposition dependencies, highlighting the role of energetic coupling across distant positions in target recognition. These results provide a quantitative framework for comparing non-pairwise-additive energetic effects across diverse TFs.

Double-stranded RNA (dsRNA) is recognized by cellular receptors as a sign of viral infection, triggering the innate immune response. Increasing evidence shows that cellular dysregulation, for example in immune disorders and neurodegenerative diseases, can also lead to accumulation of endogenously produced dsRNA that stimulates a viral-like immune response. Additionally, dsRNA contamination in RNA therapeutics can lead to harmful side effects via a similar pathway. Despite the clinical relevance of dsRNA, reliable tools for its detection remain limited. At present, dsRNA detection relies almost exclusively on the monoclonal antibodies J2 and K1, which suffer from sequence bias and low sensitivity, limiting their reliability. To address this challenge, we aimed to repurpose naturally occurring dsRNA-binding domains (dsRBDs) to produce reliable, pan-specific affinity reagents for dsRNA. We first systematically screened the dsRBDs of the three human adenosine deaminases acting on RNA (ADARs). This analysis identified ADAR3 dsRBDs as promising candidates due to their reduced sequence dependence compared to the dsRBDs of ADAR1 and ADAR2. We then engineered ADAR3-derived dsRBD constructs having varying linker lengths and domain combinations, allowing us to specifically vary the length cutoff of dsRNA detected, thus creating dsRNA accumulation detected by ADAR3 RBDs (dsRADAR) affinity reagents. Finally, we demonstrate the superior performance of dsRADAR over currently available dsRNA antibodies in a cell model of viral infection and a tissue model of gastric inflammation. Together, dsRADAR provides a sensitive and reliable approach for imaging and quantifying diverse dsRNA structures in a variety of biological contexts. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/724404v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d89c30org.highwire.dtl.DTLVardef@1f64fc1org.highwire.dtl.DTLVardef@1ee391forg.highwire.dtl.DTLVardef@e834a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

The three-dimensional folding of DNA is essential for genome function, but its organization remains difficult to summarize quantitatively across genomic scales. Here, we study DNA folding from Hi-C contact data using a network-based notion of fractal dimension. In this representation, genomic loci are treated as nodes, and observed Hi-C contacts define weighted edges, so that frequently interacting loci are closer in the resulting network. We then estimate fractal dimension using two complementary graph-based methods: the correlation dimension and the sandbox dimension. Validation on synthetic networks shows that the proposed estimators detect clear scaling behavior in hierarchical fractal-like networks, while distinguishing them from networks with local clustering but no stable multiscale self-similarity. Applied to intrachromosomal Hi-C data from the IMR90 human cell line, the method reveals approximate linear scaling regimes on log-log plots, suggesting fractal-like organization in chromatin contact networks. At the chromosome level, estimated fractal dimension tends to increase with chromosome size: larger chromosomes often have dimensions closer to 3, consistent with more compact and space-filling organization, whereas shorter chromosomes tend to have lower dimensions, closer to 1, consistent with simpler and more open folding patterns. A sliding-window analysis at 5 kb resolution further shows that fractal organization varies substantially along chromosomes rather than remaining uniform across genomic position. These results suggest that graph-based fractal dimension provides an interpretable summary of DNA folding complexity at both global and local scales. More broadly, the proposed framework offers a quantitative way to study multiscale genome organization from Hi-C data using tools from network geometry.

Rfam is a comprehensive database of non-coding RNA (ncRNA) families providing curated sequence alignments, consensus secondary structures, and covariance models for thousands of RNA families. The database is essential for identifying structured non-coding RNAs in newly sequenced genomes and understanding RNA structure-function relationships. Here we present computational protocols for automated ncRNA annotation of viral genomes, and for programmatic interaction with Rfam through its RESTful API. We showcase genome-wide RNA structure visualization from a genome sequence and from a multiple sequence alignment by generating comprehensive 2D structure diagrams using newly developed features in R2DT. We also present practical examples for retrieving family metadata, downloading alignments, accessing secondary structures, and searching user sequences from the Rfam API. These methods enable researchers in virology and RNA biology to integrate Rfam data into custom bioinformatics pipelines, comparative analyses, and machine learning workflows.

Downstream of Gene (DoG) transcription occurs when RNA polymerase II fails to terminate normally at the transcription end site, resulting in extended transcription downstream of the gene. This is a widespread phenomenon linked to cellular stress, cancer and neurodegeneration. Existing tools for DoG detection from short-read RNA-seq rely on absolute coverage thresholds and sliding window approaches that are sensitive to sequencing depth and expression level. Here we present Dogcatcher2, which applies improved statistical detection methods to gene body-normalized coverage profiles. Using long-read ground truth across multiple datasets, we show that Dogcatcher2 outperforms existing methods in both detection sensitivity and boundary accuracy while maintaining high precision even at low sequencing depths. Dogcatcher2 further improves detection on pseudobulk scRNA-seq and snRNA-seq data. Analysis of DoG regions in human reveals specific enrichment for Alu elements including inverted Alu pairs capable of forming double-stranded RNA, with transposable elements within DoG regions showing elevated expression, connecting readthrough transcription to dsRNA generation and innate immune signaling.

The spatiotemporal control of transcription and the maintenance of germline genome integrity depend on dynamic chromatin architecture. In Drosophila, the actin-related protein Arp6--a core subunit of the SWR1-like Domino chromatin remodeling complex--mediates the deposition of the histone variant H2Av. Previous studies have established H2Av as a key transcriptional regulator that modulates the +1 nucleosome barrier to promote RNA Polymerase II (Pol II) pause release and productive elongation. Conversely, H2Av is also integral to heterochromatin assembly and gene silencing. Here we demonstrate that Arp6 and H2Av are essential for female fertility and the global repression of transposable elements (TEs) in the Drosophila ovary. Rather than repressing TEs directly, we show that Arp6 and H2Av maintain genomic stability indirectly by driving the transcription of core PIWI-interacting RNA (piRNA) pathway genes. Depletion of either chromatin factor leads to a significant loss of piRNAs and reduced non-canonical transcription of dual-strand piRNA clusters. This defect stems from a failure to express the Rhino-Deadlock-Cutoff (RDC) complex, alongside the downregulation of multiple other piRNA biogenesis factors. Genomic profiling confirms that H2Av acts predominantly as an activating signal at host gene promoters. Upon H2Av or Arp6 depletion, genes that rely on H2Av for their expression exhibit a distinct upstream shift and more precise spatial localization of the Pol II peak at the TSS, indicating an impaired transition from transcription initiation into productive elongation. Together, our findings build upon the known transcriptional activation functions of the Arp6-H2Av axis, revealing that this established chromatin mechanism is critical for licensing piRNA-mediated genome defense and ensuring germline maintenance.

The long non-coding RNA MALAT1 is a conserved oncogenic driver whose function relies on a 3 triple-helix motif. While its biochemistry is well-characterized in vitro, the endogenous requirement for this motif in regulating the stability of the transcript and other genes residing in its locus remains unclear. In this study, we employed a dual-sgRNA CRISPR-Cas9 approach to systematically excise triple-helix-forming sequences from the native MALAT1 locus in gastric (AGS) and breast (MCF7) cancer cells. Our findings demonstrate that the 3 end functions as a binary structural switch. Any perturbation (ranging from genomic deletions to a single-base insertion) triggers total transcript collapse and rapid exonucleolytic decay. This instability leads to locus-wide transcriptomic failure, characterized by the precipitous loss of the antisense transcript TALAM1, while the biogenesis of the small RNA mascRNA (a byproduct of MALAT1, also involved in cancer) remains decoupled and unaffected. In cellulo, DMS probing reveals that edited transcripts retain structural complexity. Phenotypically, structural disruption of the 3 end significantly impairs the proliferative capacity of both cancer cellular models. These results identify the 3 triple-helix as an indispensable determinant of MALAT1 stability and provide endogenous validation for its role in cancer cells.

Programmed ribosomal frameshifting (PRF) is an essential strategy used by many RNA viruses to expand their coding capacity within compact genomes. This process is governed by frameshifting elements (FSEs), specialized RNA structures that regulate translation through dynamic secondary and tertiary conformations. While the structural adaptability of the SARS-CoV-2 FSE has been extensively characterized, the conformational landscapes of FSEs across other pathogenic viruses remain poorly understood. Here, we present a comparative structural analysis of FSEs from Japanese Encephalitis Virus (JEV), West Nile Virus (WNV), Hepatitis C Virus (HCV), and Human Immunodeficiency Virus (HIV) using integrative computational modeling and molecular simulations. Our analysis reveals previously uncharacterized, virus-specific conformational ensembles, alongside a conserved core architecture that exhibits pronounced length-dependent structural plasticity. Extension of flanking sequences induces substantial conformational rearrangements, highlighting the role of sequence context in shaping FSE topology and potentially modulating PRF efficiency. Importantly, we demonstrate that antisense oligonucleotide (ASO) binding can reprogram FSE architectures, disrupting native structural motifs and stabilizing alternative conformations with altered thermodynamic stability. Collectively, these findings establish viral FSEs as dynamic RNA ensembles governed by sequence context and external interactions, and position ASO-mediated structural perturbation as a promising strategy for modulating frameshifting and viral gene expression.

DNA topology is a key regulator of chromatin structure and transcription, yet its direct role in transcription factor recognition remains unclear. Here, we investigate how distinct DNA topological states modulate binding of the Saccharomyces cerevisiae bZIP transcription factor GCN4 using topologically defined plasmids. By combining, complementary biochemical approaches, including Bio-Layer Interferometry applied here for the first time to topology-dependent protein-DNA interactions, we show that DNA supercoiling directly reshapes GCN4-DNA recognition. Positively supercoiled DNA forms more stable and persistent complexes, whereas negatively supercoiled DNA retains greater conformational heterogeneity. To interpret these effects, we performed multiscale molecular simulations. Coarse-grained simulations of plasmids recapitulate the global topology-dependent trends observed experimentally, while matched minicircle models reproduce the same behaviour at the local scale. In strong agreement with experimental data, simulations reveal that DNA topology modulates the conformational ensemble of the GCN4 basic region. Overall, positively supercoiled DNA promotes a more ordered binding mode and localized protein distribution, whereas negatively supercoiled DNA supports increased structural plasticity. These findings identify DNA topology as an active determinant of transcription factor recognition and provide a multiscale framework linking global DNA mechanics to local protein-DNA interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/722604v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@18f8ba9org.highwire.dtl.DTLVardef@11a395dorg.highwire.dtl.DTLVardef@ac093borg.highwire.dtl.DTLVardef@923212_HPS_FORMAT_FIGEXP M_FIG C_FIG

DNA helicases are ATP-dependent motor proteins that catalyze duplex DNA unwinding and are involved in DNA repair, recombination and replication restart. Prominent members of the non-hexameric SF1A UvrD-family helicases are E. coli UvrD, Rep, B. stearothermophilus PcrA and M. tuberculosis UvrD1. SF1A monomers are processive 3 to 5 single stranded DNA translocases, but need to be activated to become DNA helicases. One mechanism of activation is dimerization. Whereas Rep, UvrD and PcrA form non-covalent dimers, the Mtb UvrD1 helicase forms a redox-dependent covalent dimer. Dimerization of Mtb UvrD1 occurs between the same regulatory domain (2B) within each subunit stabilized by a disulfide bond formed between the same cysteine (Cys451) within each subunit. Dimerization relieves an inhibitory interaction between the 2B domain and duplex DNA within the monomer-DNA complex. We show here that Rep, UvrD and PcrA dimerize using the same 2B-2B interface. By placing a Cys residue within the 2B domains of Rep, UvrD and PcrA in the structurally equivalent position occupied by Cys451 of Mtb UvrD1, all three enzymes form redox-dependent covalent dimers that are constitutively active helicases with increased processivity compared to the non-covalent dimers. Hence, the 2B domain is a general dimerization domain for UvrD-family SF1A helicases.

The union of two germ cells to form a zygote, and subsequent early embryo development, are marked by radical remodeling of virtually every major class of biomolecules as the specialized germline states give way to the rapid and active growth that marks early development. In recent years, advances in ultra-low input genome-wide methods have enabled systematic analyses of mRNA abundance, and of chromatin organization, throughout early development in a variety of model systems. Here, we extend these efforts to the study of RNA binding protein (RBP) function in early mouse embryos, adapting REMORA 1 - based on fusing an RNA-editing enzyme to an RBP of interest - for use in early embryos. We benchmark our approach for several well-studied RBPs, successfully recovering expected features of their RNA cargos, and assayed the RNA cargos for 17 RBPs of interest for early gene regulation. Analysis of changes in mRNA metabolism following knockdowns of the RBPs surveyed here allowed us to identify direct regulatory functions for a subset of RBPs in the early mammalian embryo, including an unanticipated role for the RNA export adaptor Alyref in control of 3 polyadenylation sites. Together, our data provide a proof of concept resource for systematically exploring RBP functions in mammalian embryogenesis.

Abstract/SummaryGross chromosomal rearrangements are a hallmark of many diseases and cancers. The study of their biogenesis and the mechanisms underlying their formation is greatly facilitated by the availability of genetic reporter assays in model organisms. We present here a novel GCR assay developed in fission yeast, a highly relevant model for understanding genome instability related to human biology. The reporter employs canavanine counter-selection to detect GCRs within a chromosomal context. Using this assay, we identified natural hotspots for GCRs, including inverted long terminal repeats (IR-LTRs). Structural analysis of GCR events showed that IR-LTR-induced GCRs mainly result in either terminal deletions with adjacent inverted duplications or repair via long-range break-induced replication (BIR). Deleting IR-LTRs reduces the GCR rate and reveals another hotspot driven by BIR between homeologous aldo/keto reductase genes on opposite arms of chromosome I. This is the first evidence that BIR can occur in S. pombe on long tracks reaching up to 600 kb. Besides highlighting genome rearrangement hotspots, the assay also identifies regulators of genome instability in fission yeast. Loss of Nup132, a component of the nuclear pore complex, increases IR-LTRs-induced GCRs, while the budding yeast homolog Nup133 has no effect on the stability of a structurally similar IR. In contrast, disrupting djc9, which encodes a conserved histone H3-H4 binding protein, decreases GCR rates. Overall, this sensitive GCR assay enables the identification of factors that control spontaneous and fragile motif-induced chromosomal instability, including those conserved in humans but lost through evolution in other organisms.

Chromosome conformation capture (3C)-derived methods have become an indispensable tool in the study of gene regulation. The three-dimensional contacts probed by 3C methods depend strongly on the properties of the enzyme used to fragment chromatin prior to proximity-driven ligation. Micrococcal nuclease (MNase), used in Micro-C, increases resolution at the expense of low ligation efficiency and the need for extensive enzyme titration. To overcome these limitations, we engineered a highly active, TEV protease-activatable caspase-activated DNase (CAD) to enable an efficient, low-sequence-bias, and high-resolution proximity ligation assay we call CAD-C. CAD-C was successful on the first attempt for each human cell line tested and the resulting datasets capture loops, TADs, compartments, and stripes similarly to Micro-C. However, compared to Micro-C and Hi-C, CAD-C shows enhanced sensitivity for promoter-enhancer loops. Leveraging the ligation-competent DNA ends produced by CAD cleavage, we show that CAD-C is compatible with a highly streamlined, repair-free protocol and produces multi-step CADwalks, consecutive ligations between nucleosomal or sub-nucleosomal fragments. With these walks, we probe local chromatin fiber folding contacts, nucleosomal and sub-nucleosomal footprints, and long-range nuclear organization regimes in human cell lines. CAD-C is an efficient, robust chromatin structure assay that can span sub-nucleosomal to chromosomal length scales in a single experiment.