Linker DNA Research Articles

DNA tetrahedron-based CRISPR bioassay for treble-self-amplified and multiplex HPV-DNA detection with elemental tagging.

Sensitive quantification of multiple analytes of interest is of great significance for clinical diagnosis. CRISPR Cas platforms offer a strategy for improving the specificity, sensitivity, and speed of nucleic acid-based diagnostics, while their multiplex analysis capability is still limited and challenging. Herein, we develop a novel DNA Tetrahedron (DTN)-supported biosensor based on the spatially separated CRISPR Cas self-amplification strategy and multiple-metal-nanoparticle tagging coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection to improve the sensitivity and feasibility of the platform for multiplex detection of HPV-DNA (HPV-16, HPV-18 and HPV-52). Given target DNA induces robust trans-cleavage activity of the Cas12a/crRNA duplex, and the surrounding corresponding single-stranded DNA (ssDNA) linker are cleaved into short fragments that are unable to bond metal-nanoparticle probes (197Au, 107Ag, 195Pt) onto DTN modified magnetic beads probe (MBs-DTN), resulting in obvious ICP-MS signal change. Of note, compared with ssDNA functionalized MBs, a higher Signal-to-Noise Ratio was obtained by using MBs-DTN in our system, further amplifying the signal by regulating probes on the surface of MBs. As expected, the HPV-DNA could be detected with detection limits as low as 218fM and be multiplexed assayed at one test with high accuracy and specificity by this proposed strategy. Furthermore, we demonstrated that the HPV-DNA in cervical swab samples could be detected, showing high consistency with DNA sequencing results. We believe that this work provides a promising option in designing CRISPR based multiplex detection system for high sensitivity, good specificity, and clinical molecular diagnostics.

Specific Detection of Proteins by a Nanobody-Functionalized Nanopore Sensor

Nanopores are label-free single-molecule analytical tools that show great potential for stochastic sensing of proteins. Here, we described a ClyA nanopore functionalized with different nanobodies through a 5-6 nm DNA linker at its periphery. Ty1, 2Rs15d, 2Rb17c, and nb22 nanobodies were employed to specifically recognize the large protein SARS-CoV-2 Spike, a medium-sized HER2 receptor, and the small protein murine urokinase-type plasminogen activator (muPA), respectively. The pores modified with Ty1, 2Rs15d, and 2Rb17c were capable of stochastic sensing of Spike protein and HER2 receptor, respectively, following a model where unbound nanobodies, facilitated by a DNA linker, move inside the nanopore and provoke reversible blockade events, whereas engagement with the large- and medium-sized proteins outside of the pore leads to a reduced dynamic movement of the nanobodies and an increased current through the open pore. Exploiting the multivalent interaction between trimeric Spike protein and multimerized Ty1 nanobodies enabled the detection of picomolar concentrations of Spike protein. In comparison, detection of the smaller muPA proteins follows a different model where muPA, complexing with the nb22, moves into the pore, generating larger blockage signals. Importantly, the components in blood did not affect the sensing performance of the nanobody-functionalized nanopore, which endows the pore with great potential for clinical detection of protein biomarkers.

Self‐Assembled L–DNA Linkers for Rapid Construction of Multi‐Specific Antibody‐Drug Conjugates Library

AbstractOne of the key challenges of improving clinical outcomes of antibody drug conjugates (ADCs) is overcoming cancer resistance to the antibody and/or drug components of ADCs, and hence the need for ADC platforms with high combinatory flexibility. Here, we introduce the use of self‐assembled left‐handed DNA (L–DNA) oligonucleotides to link combinatory single‐domain antibodies and toxin payloads for tunable and adaptive delivery of ADCs. We demonstrate that the method allows convenient construction of a library of ADCs with multi‐specific targeting, multi‐specific payloads, and exact drug‐antibody ratio. The newly constructed ADCs with L–DNA scaffold showed favorable properties of in vitro cell cytotoxicity and in vivo suppression and eradication of solid tumors. Collectively, our data suggest that the L–DNA based modular ADC (MADC) platform is a viable option for generating therapeutic ADCs and for potentially expanding ADC therapeutic window via multi‐specificity.

Kinetically limited valence of colloidal particles with surface mobile DNA linkers

We characterize the self-assembly of colloidal particles with surface mobile DNA linkers under kinetically limited valence conditions. For this, we put forward a computer simulation model that captures the interplay between the main dynamic processes governing these systems and allows the simulation of the long time scales reached in experiments, predicting the final structures from a set of physical parameters mapped to experimental conditions. The model is validated by direct comparison with available experimental results, showing an overall good agreement that includes measurements of the average effective valence and its probability distribution as a function of the density of DNA linkers on the particles surface. We show that our simulation model is capable to not only reproduce the experimental structures but that it also provides dynamic information on the self-assembly process and shows how particle concentration and characteristic hybridization time influence the effective valence of the particles.

Abstract 4728: Biochemical and genomic approaches for high throughput drug discovery in chromatin remodeling research

Abstract Chromatin remodeling is mediated by ATP-dependent enzymes that play key roles regulating gene expression and genome replication/repair. Aberrant nucleosome organization from dysregulated chromatin remodeling can severely alter chromatin accessibility and disrupt these important processes, thereby driving various cancers. Remarkably, nearly 20% of all human cancers contain mutations in subunits from the SWI/SNF family of chromatin remodeling complexes, making them of great interest to basic research and therapeutic intervention. In vitro studies on the remodeling enzymes (and their multi-subunit complexes) are challenging, partially due to the strong preference for nucleosome-based substrates (the physiological target of these enzymes). We have created the EpiDyne® nucleosome portfolio to examine chromatin remodeler activity in biochemical assays, and here present the development of novel readouts (-PicoGreenTM and -TR-FRET). These nonradioactive plate-based assays are automation adaptable, ready for high-throughput inhibitor screening, and can be customized for various remodeling enzymes that exhibit preferences in nucleosome composition (e.g. histone type or DNA linker length).For parallel in vivo studies we note that genome-wide remodeler localization and open chromatin mapping are fundamental for understanding the function/activity of these enzymes in cancer development and inhibitor response. However, traditional genomic approaches have significant issues: e.g. ChIP-seq is unable to effectively map ATPases without heavily modified high-noise protocols; while ATAC-seq to map open regions cannot deal with cross-linking that could stabilize transient states on interest. To this end, we have optimized the CUTANA™ CUT&RUN approach to efficiently capture the localization of all major classes of chromatin remodelers with high signal to background. We have also adopted NicE-seq for chromatin accessibility profiling in cross-linked material. As complementary tools to the EpiDyne platform, CUT&RUN and NicE-seq facilitate epigenomic research on chromatin remodelers in cancer therapeutic intervention. Keywords: Chromatin Remodeling, EpiDyne, Nucleosome repositioning, high-throughput screening (HTS), CUTANA™ CUT&RUN, NicE-seq Citation Format: Lu Sun, Hannah Willis, Matthew R. Marunde, Vishnu U. Kumary, Matthew J. Meiners, Saarang Gopinath, Jonathan M. Burg, Bryan J. Venters, Allison Hickman, Zu-Wen Sun, Martis W. Cowles, Pierre Esteve, Hang Gyeong Chin, Chaithanya Ponnaluri, Sriharsa Pradhan, Michael-Christopher Keogh. Biochemical and genomic approaches for high throughput drug discovery in chromatin remodeling research. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4728.

Monitoring the compaction of single DNA molecules in Xenopus egg extract in real time

DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed Xenopus egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions. Depletion of the core histone chaperone ASF1, which inhibits nucleosome assembly, decreased the final degree of metaphase fiber compaction by 29%, while depletion of linker histone H1 had a greater effect, reducing total compaction by 40%. Compared to controls, both depletions reduced the rate of compaction, led to more short periods of decompaction, and increased the speed of force-induced fiber disassembly. In contrast, depletion of condensin from metaphase extract strongly inhibited fiber assembly, resulting in transient compaction events that were rapidly reversed under high force. Altogether, these findings support a speculative model in which condensin plays the predominant role in mitotic DNA compaction, while core and linker histones act to reduce slippage during loop extrusion and modulate the degree of DNA compaction.

NIR Photocontrolled Fluorescent Nanosensor under a Six-Branched DNA Nanowheel-Induced Nucleic Acid Confinement Effect for High-Performance Bioimaging.

Although DNA nanotechnology is a promising option for fluorescent biosensors to perform bioimaging, the uncontrollable target identification during biological delivery and the spatially free molecular collision of nucleic acids may cause unsatisfactory imaging precision and sensitivity, respectively. Aiming at solving these challenges, we herein integrate some productive notions. On the one hand, the target recognition component is inserted with a photocleavage bond and a core-shell structured upconversion nanoparticle with a low thermal effect is further employed to act as the ultraviolet light generation source, under which a precise near-infrared photocontrolled sensing is achieved through a simple external 808 nm light irradiation. On the other hand, the collision of all of the hairpin nucleic acid reactants is confined by a DNA linker to form a six-branched DNA nanowheel, after which their local reaction concentrations are vastly enhanced (∼27.48 times) to induce a special nucleic acid confinement effect to guarantee highly sensitive detection. By selecting a lung cancer-associated short noncoding microRNA sequence (miRNA-155) as a model low-abundance analyte, it is demonstrated that the newly established fluorescent nanosensor not only presents good in vitro assay performance but also exhibits a high-performance bioimaging competence in live biosystems including cells and mouse body, propelling the progress of DNA nanotechnology in the biosensing field.

MORF and MOZ acetyltransferases target unmethylated CpG islands through the winged helix domain

Human acetyltransferases MOZ and MORF are implicated in chromosomal translocations associated with aggressive leukemias. Oncogenic translocations involve the far amino terminus of MOZ/MORF, the function of which remains unclear. Here, we identified and characterized two structured winged helix (WH) domains, WH1 and WH2, in MORF and MOZ. WHs bind DNA in a cooperative manner, with WH1 specifically recognizing unmethylated CpG sequences. Structural and genomic analyses show that the DNA binding function of WHs targets MORF/MOZ to gene promoters, stimulating transcription and H3K23 acetylation, and WH1 recruits oncogenic fusions to HOXA genes that trigger leukemogenesis. Cryo-EM, NMR, mass spectrometry and mutagenesis studies provide mechanistic insight into the DNA-binding mechanism, which includes the association of WH1 with the CpG-containing linker DNA and binding of WH2 to the dyad of the nucleosome. The discovery of WHs in MORF and MOZ and their DNA binding functions could open an avenue in developing therapeutics to treat diseases associated with aberrant MOZ/MORF acetyltransferase activities.

DNA Origami Curvature Sensors for Nanoparticle and Vesicle Size Determination with Single-Molecule FRET Readout.

Particle size is an important characteristic of materials with a direct effect on their physicochemical features. Besides nanoparticles, particle size and surface curvature are particularly important in the world of lipids and cellular membranes as the cell membrane undergoes conformational changes in many biological processes which leads to diverging local curvature values. On account of that, it is important to develop cost-effective, rapid and sufficiently precise systems that can measure the surface curvature on the nanoscale that can be translated to size for spherical particles. As an alternative approach for particle characterization, we present flexible DNA nanodevices that can adapt to the curvature of the structure they are bound to. The curvature sensors use Fluorescence Resonance Energy Transfer (FRET) as the transduction mechanism on the single-molecule level. The curvature sensors consist of segmented DNA origami structures connected via flexible DNA linkers incorporating a FRET pair. The activity of the sensors was first demonstrated with defined binding to different DNA origami geometries used as templates. Then the DNA origami curvature sensors were applied to measure spherical silica beads having different size, and subsequently on lipid vesicles. With the designed sensors, we could reliably distinguish different sized nanoparticles within a size range of 50-300 nm as well as the bending angle range of 50-180°. This study helps with the development of more advanced modular-curvature sensing devices that are capable of determining the sizes of nanoparticles and biological complexes.

Single molecule evidence for linker histone H1 DNA sliding and nucleosomal bypassing regulated by the C-terminal disordered domain.

Linker histone H1 proteins are amongst the most ubiquitous chromatin-binding proteins in eukaryotic cells. H1 binds to nucleosomes via interactions of the winged helix domain (WHD) with both the nucleosomal DNA at the dyad and the linker DNA that extends out from the nucleosome. The C-terminal domain (CTD) of H1 is a key domain for linker histone function, which includes regulating nucleosomal DNA wrapping, chromatin compaction, and transcription factor occupancy. Currently, dynamics of the DNA binding interactions between different H1 subtypes in the context of chromatin is poorly understood. Using an optical trap single molecule strategy combined with confocal microscopy, we investigated the DNA binding interactions between two different subtypes of human H1, H1.0 and H1.2, and nucleosomal DNA. We determined the impact of mechanical force on H1 binding interactions with linker and nucleosomal DNA, the role of the H1 CTD in regulating DNA binding interactions, and the regulation of linker histone H1 interactions with DNA by the linker histone chaperone protein Nap1. Our findings reveal the importance of the H1 CTD on how linker histones load onto DNA and find their nucleosome targets. These findings can help advance the biophysical and structural understanding of the mechanism behind how linker histone H1 targets nucleosomes to regulate chromatin compaction and transcription.

A model system reveals how different linker histones direct chromatin to liquid-like or fibre-like condensed states.

In all organisms, metre-length DNA is packaged to a micrometre scale through charge neutralisation by basic polymers. In eukaryotes, the main condensing polymers are the histone proteins and the resulting protein/DNA complex is called chromatin. Chromatin organisation is hierarchical: the basic unit is the nucleosome, arrays of which form “beads on a string” that make up a 10 nm fibre. The second stage of condensation is achieved through binding of a different set of histones, the linker histones. This stage is more enigmatic and many proposals have been made for the structure that results, from a regular 30 nm fibre to a more heterogeneous, dynamic and liquid-like assembly with the 10 nm fibre as the basic unit. It is likely that both states are relevant: the liquid-like condensate allowing transcription, replication and repair, processes that would be shut down on adoption of a fibre-like state. The growing appreciation of dynamics in chromatin packaging has paralleled developments by us and others in our “bottom-up” understanding of the linker histone proteins themselves. We have developed an in vitro model system of linker histone and linker DNA, which although very minimal, displays surprisingly complex behaviour, and is sufficient to model the known states of H1-condensed chromatin: disordered complexes (“open” chromatin), dense liquid-like assemblies (dynamic condensates) and higher-order structures (organised 30 nm fibres). A crucial advantage of such a simple model is that it allows the study of the disordered state at high resolution by NMR, CD and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. With these methods we can rationalise the distinct condensing properties of linker histone variants across species that are encoded by their amino acid sequence.

DNA binding dynamics and target location mechanism of pioneer factor GAF.

The chromatin landscape regulates transcription by localizing and regulating the activity of sequence-specific DNA binding proteins, chromatin remodeling enzymes and transcription machinery. Pioneer transcription factors can target DNA motifs on closed chromatin and trigger local chromatin remodeling to increase chromatin accessibility. Despite this knowledge, how pioneer factors search for their cognate motif on chromatin remains poorly understood. Here, we use multicolor single-molecule fluorescence resonance energy transfer (smFRET) to investigate how a prototypical Drosophila pioneer factor GAF locates its cognate motif on chromatin. To investigate how GAF searches for its cognate motif on DNA and nucleosomes, we strategically placed fluorophores on the DNA binding domain of GAF (GAF-DBD) and DNA motifs, allowing distinction of motif-specific binding and nonspecific DNA binding by FRET. We observed GAF-DBD dwells on a DNA molecule containing one GAF binding motif for 3-4 seconds before dissociation. Interestingly, on a DNA sequence with two GAF binding motifs, GAF-DBD can slide between two motifs while staying bound to the DNA. This suggests GAF monomers use one-dimensional (1D) sliding to search for its motif on nucleosome-free DNA. We next investigated the scenario where one of the two motifs is protected by a nucleosome. If the motif is located at the edge of a 601 nucleosome (SHL7 location), GAF-DBD more frequently lands on the other motif on linker DNA before it slides to the nucleosomal motif. Nucleosomes effectively block 1D sliding when the motif is located more internally on the nucleosome, at SHL5 or SHL3. However, on a nucleosome reconstituted on a native Drosophila DNA sequence, GAF-DBD can directly target internal motifs by three-dimensional diffusion, instead of 1D sliding. Our findings reveal different search modes that can be used by pioneer factors to locate their cognate motifs on chromatin.

Understanding the three-dimentional structures of chromatins at nucleosome resolution.

In human cells, the genomic DNA are associated with proteins to form compact chromatin structures in the nucleus. In the past decades, significant efforts have been made to obtain the three-dimensional (3D) structures of chromatin and to understand the mechanism driving the folding process of the genome, with the goal of understanding the relationship between the 3D structures and the gene regulation and expression. Recently, the advance of experimental techniques makes it possible to investigate the structural features in chromatin, such as compartmentalization and loops, at fine resolutions, which also inspired us to develop a physical model for understanding the folding mechanism of chromatin structures at comparable resolutions. Here, we introduce a theoretical model at nucleosome resolution which not only generates chromatin conformations consistent with experimental data, but also facilitates the investigation the folding mechanisms, such as loop formation as well as potential underlying regulations between different loops. In this model, every nucleosome in the chromatin is represented by a bead with the same diameter of the nucleosome while neighboring beads are connected at the distance comparable to the length of linker DNA between nucleosomes, which makes it possible to precisely compare the simulated chromatin conformations with experimentally measurable quantities. As an example, we calculate the chromatin volume concentrations from the simulated conformations, which are comparable to the experimental measures using ChromEMT. This model provides significant insights to chromatin 3D structures at fine resolutions, which also serves as a powerful tool for future investigations such as how structures affect gene regulations and expressions.

Extension and long-range contacts drive chromatin clutches and aggregates.

Chromatin organization plays a vital role in gene expression and DNA replication. Whether the local arrangement in vivo is a 10-nm array or 30-nm fiber remains controversial. Moreover, it remains unclear whether the structure and dynamics are closer to a liquid or solid on a larger scale. Multiple factors, such as DNA linker lengths, chromatin remodelers, histone modifications, and ionic environments, can affect the structure of chromatin. Using a near-atomistic coarse-grained model without additional assumptions, we studied the force-extension behavior of uniform canonical chromatin with 12 nucleosomes and 20-bp linkers. We found that forces below 3 pN result in a spring-like linear extension as nucleosomes undergo shear motions. However, forces beyond 3.5 pN break chromatin into trimer and tetramer clutches, thus enabling a dramatic non-uniform extension. Meanwhile, inter-chain contacts and interdigitation can facilitate chromatin unfolding. Under a crowding environment, chromatin unfolds and exposes more nucleosomal surfaces, allowing favorable inter-chain inter-nucleosomal interactions, such as stackings. These results reconcile observed ordered fibers in vitro and disordered arrays in vivo. The high nucleosome concentration and the tension provided by molecular motors can cause disordered chromatin arrays to prevail in vivo. These factors may be relevant to the irregularity of heterochromatin and mitotic chromosomes. Furthermore, the strong inter-chain contacts can lead to the formation of condensates. By applying such models under different nucleosome and salt concentrations, we will provide more insights into whether chromatin favors liquid- or solid-like states. We can also test whether long-range contacts can drive percolation transitions. Our simulations provide a detailed microscopic description of genome organization and characterize chromatin's local and bulk properties.

Chemiluminescence Sensor for miRNA-21 Detection Based on CRISPR-Cas12a and Cation Exchange Reaction

Herein, a chemiluminescence (CL) biosensor based on CRISPR-Cas12a and cation exchange reaction was constructed to detect the biomarker microRNA-21 (miRNA-21). The rolling circle amplification (RCA) reaction was introduced to convert each target RNA strand into a long single-stranded DNA with repeated sequences, which acted as triggers to initiate the transcleavage activity of CRISPR-Cas12a. The activated Cas12a could cleave the biotinylated linker DNA of CuS nanoparticles (NPs) to inhibit the binding of CuS NPs to streptavidin immobilized on the surface of the microplate, which strongly reduced the generation of Cu2+ from a cation exchange between CuS NPs and AgNO3, and thus efficiently suppressed the CL of Cu2+-luminol-H2O2 system, giving a "signal off" biosensor. With the multiple amplification, the detection limit of the developed sensor for miRNA-21 reached 16 aM. In addition, this biosensor is not only suitable for a professional chemiluminescence instrument but also for a smartphone used as a detection tool for the purpose of portable and low-cost assay. This method could be used to specifically detect quite a low level of miRNA-21 in human serum samples and various cancer cells, indicating its potential in ultrasensitive molecular diagnostics.

Role of Histone Tails and Single Strand DNA Breaks in Nucleosomal Arrest of RNA Polymerase.

Transcription through nucleosomes by RNA polymerases (RNAP) is accompanied by formation of small intranucleosomal DNA loops (i-loops). The i-loops form more efficiently in the presence of single-strand breaks or gaps in a non-template DNA strand (NT-SSBs) and induce arrest of transcribing RNAP, thus allowing detection of NT-SSBs by the enzyme. Here we examined the role of histone tails and extranucleosomal NT-SSBs in i-loop formation and arrest of RNAP during transcription of promoter-proximal region of nucleosomal DNA. NT-SSBs present in linker DNA induce arrest of RNAP +1 to +15 bp in the nucleosome, suggesting formation of the i-loops; the arrest is more efficient in the presence of the histone tails. Consistently, DNA footprinting reveals formation of an i-loop after stalling RNAP at the position +2 and backtracking to position +1. The data suggest that histone tails and NT-SSBs present in linker DNA strongly facilitate formation of the i-loops during transcription through the promoter-proximal region of nucleosomal DNA.

Nucleosome dyad determines the H1 C-terminus collapse on distinct DNA arms

Nucleosomes are symmetric structures. However, binding of linker histones generates an inherently asymmetric H1-nucleosome complex, and whether this asymmetry is transmitted to the overall nucleosome structure, and therefore also to chromatin, is unclear. Efforts to investigate potential asymmetry due to H1s have been hampered by the DNA sequence, which naturally differs in each gyre. To overcome this issue, we designed and analyzed by cryo-EM a nucleosome reconstituted with a palindromic (601L) 197-bp DNA. As in the non-palindromic 601 sequence, H1 restricts linker DNA flexibility but reveals partial asymmetrical unwrapping. However, in contrast to the non-palindromic nucleosome, in the palindromic nucleosome H1 CTD collapses to the proximal linker. Molecular dynamics simulations show that this could be dictated by a slightly tilted orientation of the globular domain (GD) of H1, which could be linked to the DNA sequence of the nucleosome dyad.

Histone H3 core domain in chromatin with different DNA linker lengths studied by 1H-Detected solid-state NMR spectroscopy.

Chromatin, a dynamic protein-DNA complex that regulates eukaryotic genome accessibility and essential functions, is composed of nucleosomes connected by linker DNA with each nucleosome consisting of DNA wrapped around an octamer of histones H2A, H2B, H3 and H4. Magic angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy can yield unique insights into histone structure and dynamics in condensed nucleosomes and nucleosome arrays representative of chromatin at physiological concentrations. Recently we used J-coupling-based solid-state NMR methods to investigate with residue-specific resolution the conformational dynamics of histone H3 N-terminal tails in 16-mer nucleosome arrays containing 15, 30 or 60 bp DNA linkers. Here, we probe the H3 core domain in the 16-mer arrays as a function of DNA linker length via dipolar coupling-based 1H-detected solid-state NMR techniques. Specifically, we established nearly complete assignments of backbone chemical shifts for H3 core residues in arrays with 15-60 bp DNA linkers reconstituted with 2H,13C,15N-labeled H3. Overall, these chemical shifts were similar irrespective of the DNA linker length indicating no major changes in H3 core conformation. Notably, however, multiple residues at the H3-nucleosomal DNA interface in arrays with 15 bp DNA linkers exhibited relatively pronounced differences in chemical shifts and line broadening compared to arrays with 30 and 60 bp linkers. These findings are consistent with increased heterogeneity in nucleosome packing and structural strain within arrays containing short DNA linkers that likely leads to side-chains of these interfacial residues experiencing alternate conformations or shifts in their rotamer populations relative to arrays with the longer DNA linkers.

H2A Ubiquitination Alters H3-tail Dynamics on Linker-DNA to Enhance H3K27 Methylation

Polycomb repressive complex 1 (PRC1) and PRC2 are responsible for epigenetic gene regulation. PRC1 ubiquitinates histone H2A (H2Aub), which subsequently promotes PRC2 to introduce the H3 lysine 27 tri-methyl (H3K27me3) repressive chromatin mark. Although this mechanism provides a link between the two key transcriptional repressors, PRC1 and PRC2, it is unknown how histone-tail dynamics contribute to this process. Here, we have examined the effect of H2A ubiquitination and linker-DNA on H3-tail dynamics and H3K27 methylation by PRC2. In naïve nucleosomes, the H3-tail dynamically contacts linker DNA in addition to core DNA, and the linker-DNA is as important for H3K27 methylation as H2A ubiquitination. H2A ubiquitination alters contacts between the H3-tail and DNA to improve the methyltransferase activity of the PRC2–AEBP2–JARID2 complex. Collectively, our data support a model in which H2A ubiquitination by PRC1 synergizes with linker-DNA to hold H3 histone tails poised for their methylation by PRC2–AEBP2–JARID2.

Programming rigidity into size-defined wireframe DNA nanotubes.

Nanotubes built from DNA hold promise for several biological and materials applications, due to their high aspect ratio and encapsulation potential. A particularly appealing goal is to control the size, shape, and dynamic behaviour of DNA nanotubes with minimal design alteration, as nanostructures of varying morphologies and lengths have been shown to exhibit distinct cellular uptake, encapsulation behaviour, and in vivo biodistribution. Herein, we report a systematic investigation, combining experimental and computational design, to modulate the length, flexibility, and longitudinal patterns of wireframe DNA nanotubes. Subtle design changes govern the structure and properties of our nanotubes, which are built from a custom-made, long, and size-defined template strand to which DNA rungs and linkers are attached. Unlike DNA origami, these custom-made strands possess regions with repeating sequences at strategic locations, thereby reducing the number of strands necessary for assembly. Through strand displacement, the nanotubes can be reversibly altered between extended and collapsed morphologies. These design concepts enable fine-tuning of the nanotube stiffness and may pave the way for the development of designer nanotubes for a variety of applications, including the study of cellular internalization, biodistribution, and uptake mechanisms for structures of varied shapes and sizes.