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The human glycosylase OGG1 extrudes and excises the oxidized DNA base 8-oxoguanine (8-oxoG) to initiate base excision repair and plays important roles in many pathological conditions such as cancer, inflammation, and neurodegenerative diseases. Previous structural studies have used a truncated protein and short linear DNA, so it has been unclear how full-length OGG1 operates on longer DNA or on nucleosomes. Here we report cryo-EM structures of human OGG1 bound to a 35-bp long DNA containing an 8-oxoG within an unmethylated Cp-8-oxoG dinucleotide as well as to a nucleosome with an 8-oxoG at super-helical location (SHL)-5. The 8-oxoG in the linear DNA is flipped out by OGG1, consistent with previous crystallographic findings with a 15-bp DNA. OGG1 preferentially binds near dsDNA ends at the nucleosomal entry/exit sites. Such preference may underlie the enzyme's function in DNA double-strand break repair. Unexpectedly, we find that OGG1 bends the nucleosomal entry DNA, flips an undamaged guanine, and binds to internal nucleosomal DNA sites such as SHL-5 and SHL+6. We suggest that the DNA base search mechanism by OGG1 may be chromatin context-dependent and that OGG1 may partner with chromatin remodelers to excise 8-oxoG at the nucleosomal internal sites., (© 2024. The Author(s).)

Hydroxamic acids represent a group of weak organic acids, both naturally occurring and synthetically derived, characterized by the general formula RC(= O)N(R'OH). In this study, we investigated the binding behavior of N-m-tolyl-4-chlorophenoxyaceto hydroxamic acid with calf thymus DNA (ct-DNA) and torula yeast RNA (t-RNA) through a combination of techniques including UV-visible spectroscopy, fluorescence emission analysis, viscometry, and computational simulations using AutoDock4 software. Our findings reveal that the mode of binding between the compound and the nucleic acids is consistent with intercalation. Competitive binding experiments demonstrated that the complex competes effectively with ethidium bromide (EB) for binding to ct-DNA/t-RNA, displacing EB from its binding sites. Additionally, the introduction of the compound into the DNA-EB system resulted in a quenching of fluorescence emission peaks. Analysis of absorption spectra indicated a red shift and hypochromic shift when the compound interacted with DNA, further supporting the intercalative binding mode. The calculated binding constant (K b ) value for the compound is 6.62 × 10 4  M -1 and 5.40 × 10 3  M -1 indicating a strong interaction with ct-DNA and t-RNA respectively. We determined the Stern-Volmer constants for ct-DNA and t-RNA as 9.96 × 10 4  M -1 and 8.13 × 10 5  M -1 , respectively. The binding free energy values for ct-DNA/t-RNA were calculated to be - 3.741 × 10 7 and - 5.425 × 10 8  kcal/mol, respectively. Viscometric studies corroborated the UV results, showing a continuous increase in relative viscosity of ct-DNA/t-RNA solutions with the addition of the optimal hydroxamic acid concentration. Furthermore, we assessed the antioxidant activity of the compound using DPPH-radical scavenging and β-carotene linoleic acid assays. Gel electrophoresis results demonstrated the compound's remarkable efficacy in preventing DNA damage. Collectively, all experimental evidence supports the conclusion that N-m-tolyl-4-chlorophenoxyaceto hydroxamic acid binds to ct-DNA/t-RNA through an intercalative mechanism, which is consistent with our molecular docking simulations., (© 2024. The Author(s).)

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Biocompatibility and precise control over their size and shape make DNA origami nanostructures (DONs) promising for drug delivery applications. Whilst many investigations have focused on cancer treatment, this might not be the best fit for DONs that get degraded by nucleases in blood. In comparison, an eye is a uniquely isolated target organ, which could benefit from DONs to achieve and maintain therapeutic concentrations in diseases that threaten the eyesight of millions of patients every year. We investigated the loading of doxorubicin (DOX) as a model drug into three distinct DONs and tested their stability upon storage. Further, we chose one structure (24HB) to probe its stability under physiological conditions in cell media and porcine vitreous, before examining the uptake and effect of DOX-loaded 24HB (24HB-DOX) on the cell viability in a retinal cell line (ARPE-19). Similar to previous reports, the tested low μM loading concentrations of DOX resulted in high drug loadings of up to 34% (m/m), and remained mostly intact in water for at least 2 months at 4 °C. In cell media and porcine vitreous at 37 °C, however, 24HB required additional Mg 2+ supplementation to avoid degradation and the loss of the attached fluorophores. With added Mg 2+ , 24HB remained stable in vitreous for 7 days at 37 °C. The treatment with 24HB-DOX was well tolerated by ARPE-19 cells, compared to the observed higher toxicity of free DOX. Uptake studies revealed, however, that in contrast to free DOX, very little 24HB-DOX was taken up by the cells. Instead, the particles were observed to attach around the cells. Hence, our results suggest that since the uptake seems to be the bottleneck for therapies using DONs, further strategies such as adding ocular targeting moieties are necessary to increase the uptake and efficacy of doxorubicin-loaded DONs.

As with all new fields of discovery, work on the biological role of G-quadruplexes (GQs) has produced a number of results that at first glance are quite baffling, sometimes because they do not fit well together, but mostly because they are different from commonly held expectations. Like other classes of flipons, those that form G-quadruplexes have a repeat sequence motif that enables the fold. The canonical DNA motif (G 3 N 1-7 ) 3 G 3 , where N is any nucleotide and G is guanine, is a feature that is under active selection in avian and mammalian genomes. The involvement of G-flipons in genome maintenance traces back to the invertebrate Caenorhabditis elegans and to ancient DNA repair pathways. The role of GQs in transcription is supported by the observation that yeast Rap1 protein binds both B-DNA, in a sequence-specific manner, and GQs, in a structure-specific manner, through the same helix. Other sequence-specific transcription factors (TFs) also engage both conformations to actuate cellular transactions. Noncoding RNAs can also modulate GQ formation in a sequence-specific manner and engage the same cellular machinery as localized by TFs, linking the ancient RNA world with the modern protein world. The coevolution of noncoding RNAs and sequence-specific proteins is supported by studies of early embryonic development, where the transient formation of G-quadruplexes coordinates the epigenetic specification of cell fate.

Cascade-enzyme reaction systems have emerged as promising tools for treating malignant tumors by efficiently converting nutrients into toxic substances. However, the challenges of poor localized retention capacity and utilization of highly active enzymes often result in extratumoral toxicity and reduced therapeutic efficacy. In this study, we introduced a cell membrane-DNA nanoanchor (DNANA) with a spatially confined cascade enzyme for in vivo tumor therapy. The DNANAs are constructed using a polyvalent cholesterol-labeled DNA triangular prism, ensuring high stability in cell membrane attachment. Glucose oxidase (GOx) and horseradish peroxidase (HRP), both modified with streptavidin, are precisely confined to biotin-labeled DNANAs. Upon intratumoral injection, DNANA enzymes efficiently colonize the tumor site through cellular membrane engineering strategies, significantly reducing off-target enzyme leakage and the associated risks of extratumoral toxicity. Furthermore, DNANA enzymes demonstrated effective cancer therapy in vitro and in vivo by depleting glucose and producing highly cytotoxic hydroxyl radicals in the vicinity of tumor cells. This membrane-engineered cascade-enzyme reaction system presents a conceptual approach to tumor treatment.

Neural networks enable the processing of large, complex data sets with applications in disease diagnosis, cell profiling, and drug discovery. Beyond electronic computers, neural networks have been implemented using programmable biomolecules such as DNA; this confers unique advantages, such as greater portability, electricity-free operation, and direct analysis of patterns of biomolecules in solution. Analogous to bottlenecks in electronic computers, the computing power of DNA-based neural networks is limited by the ability to add more computing units, i.e., neurons. This limitation exists because current architectures require many nucleic acids to model a single neuron. Each additional neuron compounds existing problems such as long assembly times, high background signal, and cross-talk between components. Here, we test three strategies to solve this limitation and improve the scalability of DNA-based neural networks: (i) enzymatic synthesis for high-purity neurons, (ii) spatial patterning of neuron clusters based on their network position, and (iii) encoding neuron connectivity on a universal single-stranded DNA backbone. We show that neurons implemented via these strategies activate quickly, with a high signal-to-background ratio and process-weighted inputs. We rewired our modular neurons to demonstrate basic neural network motifs such as cascading, fan-in, and fan-out circuits. Finally, we designed a prototype two-layer microfluidic device to automate the operation of our circuits. We envision that our proposed design will help scale DNA-based neural networks due to its modularity, simplicity of synthesis, and compatibility with various neural network architectures. This will enable portable computing power for applications in portable diagnostics, compact data storage, and autonomous decision making for lab-on-a-chips.

Studies on the molecular mechanisms of heavy metal toxicity in invertebrate reproduction are limited. Given that PARP-catalysed ADP-ribosylation is also involved in counteracting heavy metal toxicity and maintaining genomic integrity, and that PARylation is implicated in chromatin remodelling but its role in sperm chromatin remains to be elucidated, we investigated the effects of chromium(VI) at 1, 10 and 100 nM on the reproductive health of Mytilus galloprovincialis. The damage to the gonads was assessed by morphological analyses and the damage indices PARP and ɣH2A.X were measured. Changes in the binding of protamine-like (PL) to DNA and the possibility of poly(ADP-ribosyl)ation of PL proteins were also investigated. Gonadal chromium accumulation and morphological damage were found, especially when the mussels were exposed to the highest dose of chromium(VI). In addition, the maximum expression of gonadal ɣH2A.X and PARP were obtained at 100 and 10 nM Cr(VI), respectively. Interestingly, for the first time in all exposed conditions, poly(ADP)-ribosylation was detected on PL-II, which, together with PL-III and PL-IV, are the major nuclear basic proteins of Mytilus galloprovincialis sperm chromatin. Since PL-II is involved in the final high level of sperm chromatin compaction, this post-translational modification altered the binding of the PL protein to DNA, favouring the action of micrococcal nuclease on sperm chromatin. This study provides new insights into the effects of chromium(VI) on Mytilus galloprovincialis reproductive system and proposes a molecular mechanism hypothesis describing the toxic effects of this metal on PL-DNA binding, sperm chromatin and gonads., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The 2024 Albert Lasker Basic Medical Research Award was attributed to Zhijian (James) Chen for "the discovery of the cGAS enzyme that senses foreign and self DNA, solving the mystery of how DNA stimulates immune and inflammatory responses." Bringing to bear an ingenious in vitro complementation system, an astute insight, and superlative biochemistry, Chen and colleagues identified cGAS (cGAMP synthase) as both the molecule that perceives cytosolic DNA in infected, stressed, or dying cells and the enzyme that catalyzes the synthesis of cGAMP (cyclic GMP-AMP), a critical second messenger along the route to inflammatory cytokine production. These findings cleared up the reigning confusion surrounding a major mechanism of inciting the innate immune system, with therapeutic implications for fighting infections, containing tumors, and extinguishing autoimmune and inflammatory diseases., Competing Interests: Competing interests statement:The author declares no competing interest.

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRβ H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient., Competing Interests: Competing interests statement:The authors declare no competing interest.

FOXA1 serves as a crucial pioneer transcription factor during developmental processes and plays a pivotal role as a mitotic bookmarking factor to perpetuate gene expression profiles and maintain cellular identity. During mitosis, the majority of FOXA1 dissociates from specific DNA binding sites and redistributes to non-specific binding sites; however, the regulatory mechanisms governing molecular dynamics and activity of FOXA1 remain elusive. Here, we show that mitotic kinase Aurora B specifies the different DNA binding modes of FOXA1 and guides FOXA1 biomolecular condensation in mitosis. Mechanistically, Aurora B kinase phosphorylates FOXA1 at Serine 221 (S221) to liberate the specific, but not the non-specific, DNA binding. Interestingly, the phosphorylation of S221 attenuates the FOXA1 condensation that requires specific DNA binding. Importantly, perturbation of the dynamic phosphorylation impairs accurate gene reactivation and cell proliferation, suggesting that reversible mitotic protein phosphorylation emerges as a fundamental mechanism for the spatiotemporal control of mitotic bookmarking., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Transcriptional factors (TFs) in bacteria play a crucial role in gene regulation by binding to specific DNA sequences, thereby assisting in the activation or repression of genes. Despite their central role, deciphering shape recognition of bacterial TFs-DNA interactions remains an intricate challenge. A deeper understanding of DNA secondary structures could greatly enhance our knowledge of how TFs recognize and interact with DNA, thereby elucidating their biological function. In this study, we employed machine learning algorithms to predict transcription factor binding sites (TFBS) and classify them as directed-repeat (DR) or inverted-repeat (IR). To accomplish this, we divided the set of TFBS nucleotide sequences by size, ranging from 8 to 20 base pairs, and converted them into thermodynamic data known as DNA duplex stability (DDS). Our results demonstrate that the Random Forest algorithm accurately predicts TFBS with an average accuracy of over 82% and effectively distinguishes between IR and DR with an accuracy of 89%. Interestingly, upon converting the base pairs of several TFBS-IR into DDS values, we observed a symmetric profile typical of the palindromic structure associated with these architectures. This study presents a novel TFBS prediction model based on a DDS characteristic that may indicate how respective proteins interact with base pairs, thus providing insights into molecular mechanisms underlying bacterial TFs-DNA interaction., (© The Author(s) 2024. Published by Oxford University Press.)

Accurate prediction of transcription factor binding sites (TFBSs) is essential for understanding gene regulation mechanisms and the etiology of diseases. Despite numerous advances in deep learning for predicting TFBSs, their performance can still be enhanced. In this study, we propose MLSNet, a novel deep learning architecture designed specifically to predict TFBSs. MLSNet innovatively integrates multisize convolutional fusion with long short-term memory (LSTM) networks to effectively capture DNA-sparse higher-order sequence features. Further, MLSNet incorporates super token attention and Bi-LSTM to systematically extract and integrate higher-order DNA shape features. Experimental results on 165 ChIP-seq (chromatin immunoprecipitation followed by sequencing) datasets indicate that MLSNet consistently outperforms several state-of-the-art algorithms in the prediction of TFBSs. Specifically, MLSNet reports average metrics: 0.8306 for ACC, 0.8992 for AUROC, and 0.9035 for AUPRC, surpassing the second-best methods by 1.82%, 1.68%, and 1.54%, respectively. This research delineates the effectiveness of combining multi-size convolutional layers with LSTM and DNA shape-based features in enhancing predictive accuracy. Moreover, this study comprehensively assesses the variability in model performance across different cell lines and transcription factors. The source code of MLSNet is available at https://github.com/minghaidea/MLSNet., (© The Author(s) 2024. Published by Oxford University Press.)

Tandem repeat proteins (TRPs) are widely distributed and bind to a wide variety of ligands. DNA-binding TRPs such as zinc finger (ZNF) and transcription activator-like effector (TALE) play important roles in biology and biotechnology. In this study, we first conducted an extensive analysis of TRPs in public databases, and found that the enormous diversity of TRPs is largely unexplored. We then focused our efforts on identifying novel TRPs possessing DNA-binding capabilities. We established a protein language model for DNA-binding protein prediction (PLM-DBPPred), and predicted a large number of DNA-binding TRPs. A subset was then selected for experimental screening, leading to the identification of 11 novel DNA-binding TRPs, with six showing sequence specificity. Notably, members of the STAR (Short TALE-like Repeat proteins) family can be programmed to target specific 9 bp DNA sequences with high affinity. Leveraging this property, we generated artificial transcription factors using reprogrammed STAR proteins and achieved targeted activation of endogenous gene sets. Furthermore, the members of novel families such as MOON (Marine Organism-Originated DNA binding protein) and pTERF (prokaryotic mTERF-like protein) exhibit unique features and distinct DNA-binding characteristics, revealing interesting biological clues. Our study expands the diversity of DNA-binding TRPs, and demonstrates that a systematic approach greatly enhances the discovery of new biological insights and tools., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

CRISPR-Cas systems function as adaptive immune mechanisms in bacteria and archaea and offer protection against phages and other mobile genetic elements. Among many types of CRISPR-Cas systems, Type I CRISPR-Cas systems are most abundant, with target interference depending on a multi-subunit, RNA-guided complex known as Cascade that recruits a transacting helicase nuclease, Cas3, to degrade the target. While structural studies on several other types of Cas3 have been conducted long ago, it was only recently that the structural study of Type I-C Cas3 in complex with Cascade was revealed, shedding light on how Cas3 achieve its activity in the Cascade complex. In the present study, we elucidated the first structure of standalone Type I-C Cas3 from Neisseria lactamica (NlaCas3). Structural analysis revealed that the histidine-aspartate (HD) nuclease active site of NlaCas3 was bound to two Fe2+ ions that inhibited its activity. Moreover, NlaCas3 could cleave both single-stranded and double-stranded DNA in the presence of Ni2+ or Co2+, showing the highest activity in the presence of both Ni2+ and Mg2+ ions. By comparing the structural studies of various Cas3 proteins, we determined that our NlaCas3 stays in an inactive conformation, allowing us to understand the structural changes associated with its activation and their implication., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Telomeric repeat containing RNA (TERRA) is a noncoding RNA that is transcribed from telomeres. Previous study showed that TERRA trans anneals by invading into the telomeric duplex to form an R-loop in mammalian cells. Here, we elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin proteins, RAD51 and RNase H using single molecule (sm) assays. We demonstrate that TERRA trans annealing into telomeric DNA exhibits dynamic movement that is stabilized by TRF2. TERRA annealing to the telomeric duplex results in the formation of a stable triplex structure which differs from a conventional R-loop. We identified that the presence of a sub-telomeric DNA and a telomeric overhang in the form of a G-quadruplex significantly enhances TERRA annealing to telomeric duplex. We also demonstrate that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Additionally, TRF2 increases TERRA affinity to telomeric duplex and protects it from RNase H digestion. In contrast, TRF1 represses TERRA annealing to telomeric duplex and fails to provide protection against RNase H digestion. Our findings provide an in-depth molecular mechanism underpinning TERRA recruitment and annealing to the telomere., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Xenobiotic nucleic acids (XNAs) are artificial genetic polymers with altered structural moieties and useful features, such as enhanced biological and chemical stability. Enzymatic synthesis and efficient labelling of XNAs are crucial for their broader application. Terminal deoxynucleotidyl transferases (TdTs) have been exploited for the de novo synthesis and labelling of DNA and demonstrated the capability of recognizing various substrates. However, the activities of TdTs for the synthesis and labelling of commonly used XNAs with 2' modifications have not been systematically explored. In this work, we explored and demonstrated the varied activities of three TdTs (bovine TdT, MTdT-evo and murine TdT) for the template-independent incorporation of 2'-methoxy NTPs, 2'-fluoro NTPs and 2'-fluoroarabino NTPs into the 3' ends of single- and double-stranded DNAs and the extension of 2'-modified XNAs with (d)NTPs containing a natural or unnatural nucleobase. Taking advantages of these activities, we established a strategy for protecting single-stranded DNAs from exonuclease I degradation by TdT-synthesized 2'-modified XNA tails and methods for 3'-end labelling of 2'-modified XNAs by TdT-mediated synthesis of G-quadruplex-containing tails or incorporation of nucleotides with a functionalized nucleobase. A DNA-2'-fluoroarabino nucleic acid (FANA) chimeric hydrogel was also successfully constructed based on the extraordinary activity of MTdT-evo for template-independent FANA synthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Current approaches to RNA synthesis/manufacturing require substantial (and incomplete) purification post-synthesis. We have previously demonstrated the synthesis of RNA from a complex in which T7 RNA polymerase is tethered to promoter DNA. In the current work, we extend this approach to demonstrate an extremely stable system of functional co-tethered complex to a solid support. Using the system attached to magnetic beads, we carry out more than 20 rounds of synthesis using the initial polymerase-DNA construct. We further demonstrate the wide utility of this system in the synthesis of short RNA, a CRISPR guide RNA, and a protein-coding mRNA. In all cases, the generation of self-templated double stranded RNA (dsRNA) impurities are greatly reduced, by both the tethering itself and by the salt-tolerance that local co-tethering provides. Transfection of the mRNA into HEK293T cells shows a correlation between added salt in the transcription reaction (which inhibits RNA rebinding that generates RNA-templated extensions) and significantly increased expression and reduced innate immune stimulation by the mRNA reaction product. These results point in the direction of streamlined processes for synthesis/manufacturing of high-quality RNA of any length, and at greatly reduced costs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The chromatin-remodeling enzyme helicase lymphoid-specific (HELLS) interacts with cell division cycle-associated 7 (CDCA7) on nucleosomes and is involved in the regulation of DNA methylation in higher organisms. Mutations in these genes cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, which also results in DNA hypomethylation of satellite repeat regions. We investigated the functional domains of human CDCA7 in HELLS using several mutant CDCA7 proteins. The central region is critical for binding to HELLS, activation of ATPase, and nucleosome sliding activities of HELLS-CDCA7. The N-terminal region tends to inhibit ATPase activity. The C-terminal 4CXXC-type zinc finger domain contributes to CpG and hemimethylated CpG DNA preference for DNA-dependent HELLS-CDCA7 ATPase activity. Furthermore, CDCA7 showed a binding preference to DNA containing hemimethylated CpG, and replication-dependent pericentromeric heterochromatin foci formation of CDCA7 with HELLS was observed in mouse embryonic stem cells; however, all these phenotypes were lost in the case of an ICF syndrome mutant of CDCA7 mutated in the zinc finger domain. Thus, CDCA7 most likely plays a role in the recruitment of HELLS, activates its chromatin remodeling function, and efficiently induces DNA methylation, especially at hemimethylated replication sites., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

While DNA ligase I (LigI) joins most Okazaki fragments, a backup pathway involving poly(ADP-ribose) synthesis, XRCC1 and DNA ligase IIIα (LigIIIα) functions along with the LigI-dependent pathway and is also capable of supporting DNA replication in the absence of LigI. Here we have addressed for the first time the roles of PARP1 and PARP2 in this pathway using isogenic null derivatives of mouse CH12F3 cells. While single and double null mutants of the parental cell line and single mutants of LIG1 null cells were viable, loss of both PARP1 and PARP2 was synthetically lethal with LigI deficiency. Thus, PARP1 and PARP2 have a redundant essential role in LigI-deficient cells. Interestingly, higher levels of PARP2 but not PARP1 associated with newly synthesized DNA in the LIG1 null cells and there was a much higher increase in PARP2 chromatin retention in LIG1 null cells incubated with the PARP inhibitor olaparib with this effect occurring independently of PARP1. Together our results suggest that PARP2 plays a major role in specific cell types that are more dependent upon the backup pathway to complete DNA replication and that PARP2 retention at unligated Okazaki fragments likely contributes to the side effects of current clinical PARP inhibitors., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Development of the malaria parasite, Plasmodium falciparum, is regulated by a limited number of sequence-specific transcription factors (TFs). However, the mechanisms by which these TFs recognize genome-wide binding sites is largely unknown. To address TF specificity, we investigated the binding of two TF subsets that either bind CACACA or GTGCAC DNA sequence motifs and further characterized two additional ApiAP2 TFs, PfAP2-G and PfAP2-EXP, which bind unique DNA motifs (GTAC and TGCATGCA). We also interrogated the impact of DNA sequence and chromatin context on P. falciparum TF binding by integrating high-throughput in vitro and in vivo binding assays, DNA shape predictions, epigenetic post-translational modifications, and chromatin accessibility. We found that DNA sequence context minimally impacts binding site selection for paralogous CACACA-binding TFs, while chromatin accessibility, epigenetic patterns, co-factor recruitment, and dimerization correlate with differential binding. In contrast, GTGCAC-binding TFs prefer different DNA sequence context in addition to chromatin dynamics. Finally, we determined that TFs that preferentially bind divergent DNA motifs may bind overlapping genomic regions due to low-affinity binding to other sequence motifs. Our results demonstrate that TF binding site selection relies on a combination of DNA sequence and chromatin features, thereby contributing to the complexity of P. falciparum gene regulatory mechanisms., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Saccharomyces cerevisiae HMO1 is an architectural nuclear DNA-binding protein that stimulates the activity of some remodelers and regulates the transcription of ribosomal protein genes, often binding to a DNA motif called IFHL. However, the molecular mechanism dictating this sequence specificity is unclear. Our circular dichroism spectroscopy studies show that the HMO1:DNA complex forms without noticeable changes in the structure of DNA and HMO1. Molecular modeling/molecular dynamics studies of the DNA complex with HMO1 Box B reveal two extended sites at the N-termini of helices I and II of Box B that are involved in the formation of the complex and stabilize the DNA bend induced by intercalation of the F114 side chain between base pairs. A comparison of the affinities of HMO1 for 24 bp DNA fragments containing either randomized or IFHL sequences reveals a twofold increase in the stability of the complex in the latter case, which may explain the selectivity in the recognition of the IFHL-containing promoter regions.

DNA cleavage by nanomaterials has the potential to be utilized as an innovative tool for gene editing. Numerous nanomaterials exhibiting DNA cleavage properties have been identified and cataloged. Yet, the exploitation of property data through data-driven machine-learning approaches remains unexplored. A database was developed, compiling thirty distinctive characteristics, encompassing physical and chemical properties, as well as experimental conditions of nanomaterials that have demonstrated DNA cleavage capability such as in articles published over the past two decades. The DNA cleavage effect and efficiency of nanomaterials were predicted using machine learning algorithms such as support vector machines, deep neural networks, and random forest, and a classification accuracy of 0.93 for the cleavage effect was achieved. Moreover, the potential of utilizing larger datasets to enhance the predictive capacity of models was discussed. The findings indicate the feasibility of predicting nanomaterial properties based on experimental data. Evaluating the performance and effectiveness of the machine learning models trained using the existing data can furnish valuable insights for future materials research endeavors, especially for the design of DNA cleavage with specific sites., (© 2024. The Author(s).)

RNA-binding proteins (RBPs) are involved in many biological processes. The direct interaction between protein and RNA can be studied using cross-linking immunoprecipitation (CLIP) techniques in living cells. Here, we present a protocol to characterize the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using CLIP. We describe steps for RNA-protein UV-C cross-linking in living cells, isolating RNA-protein complexes, RNA labeling, and extracting nucleic acid. We then detail procedures for nuclease treatment and nucleic acid migration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We previously identified M.ApeKI from Aeropyum pernix K1 as a highly thermostable DNA (cytosine-5)-methyltransferase. M.ApeKI uses the type II restriction-modification system (R-M system), among the best-studied R-M systems. Although endonucleases generally utilize Mg (II) as a cofactor, several reports have shown that MTases exhibit different reactions in the presence of metal ions. This study aim was to evaluate the enzymatic properties of DNA (cytosine-5)-methyltransferase M.ApeKI from archaea in the presence of metal ions. We evaluated the influence of metal ions on the catalytic activity and DNA binding of M.ApeKI. The catalytic activity was inhibited by Cu (II), Mg (II), Mn (II), and Zn (II), each at 5 m m. DNA binding was more strongly inhibited by 5 m m Cu (II) and 10 m m Zn (II). To our knowledge, this is the first report showing that DNA binding of type II MTase is inhibited by metal ions., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Fanzors are recently characterized RNA-guided DNA endonucleases found in eukaryotic organisms. In this issue of Cell, Xu, Saito et al. reveal the structural diversity of Fanzors and identify key features shared with TnpB and Cas12 proteins, providing a comprehensive perspective on their molecular function and evolution., Competing Interests: Declaration of interests O.N. is a co-founder, board member, and scientific advisor of Curreio., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Genetic medicines show promise for treating various diseases, yet clinical success has been limited by tolerability, scalability, and immunogenicity issues of current delivery platforms. To overcome these, we developed a proteolipid vehicle (PLV) by combining features from viral and non-viral approaches. PLVs incorporate fusion-associated small transmembrane (FAST) proteins isolated from fusogenic orthoreoviruses into a well-tolerated lipid formulation, using scalable microfluidic mixing. Screening a FAST protein library, we identified a chimeric FAST protein with enhanced membrane fusion activity that improved gene expression from an optimized lipid formulation. Systemically administered FAST-PLVs showed broad biodistribution and effective mRNA and DNA delivery in mouse and non-human primate models. FAST-PLVs show low immunogenicity and maintain activity upon repeat dosing. Systemic administration of follistatin DNA gene therapy with FAST-PLVs raised circulating follistatin levels and significantly increased muscle mass and grip strength. These results demonstrate the promising potential of FAST-PLVs for redosable gene therapies and genetic medicines., Competing Interests: Declaration of interests D.W.B., P.W., P.B., A.B., L.G., M.H., H.V., D.S., J.A., J.K., M.P.S.A., R.Q., B.R., F.E., N.R., L.M., N.G., B.T., A.D., M.P., J.G., C.M., P.H.B., A.R., R.D., and J.D.L. are employees and/or shareholders of Entos Pharmaceuticals. H.G., M.S., and J.D.L. are employees and/or shareholders of Oisin Biotechnologies. J.A., M.S., and J.D.L. are employees and/or shareholders of OncoSenX. E.K.C., D.P., M.S., A.R., R.D., and J.D.L. are authors on a patent related to this work., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts., Competing Interests: Declaration of interests P.X., M.S., G.F., and F.Z. are coinventors on a patent application (PCT/US2022/081593) related to this work filed by the Broad Institute and MIT. F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, Aera Therapeutics, and Moonwalk Biosciences. F.Z. is a scientific advisor for Octant., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Biomolecular condensates assemble in living cells through phase separation and related phase transitions. An underappreciated feature of these dynamic molecular assemblies is that they form interfaces with other cellular structures, including membranes, cytoskeleton, DNA and RNA, and other membraneless compartments. These interfaces are expected to give rise to capillary forces, but there are few ways of quantifying and harnessing these forces in living cells. Here, we introduce viscoelastic chromatin tethering and organization (VECTOR), which uses light-inducible biomolecular condensates to generate capillary forces at targeted DNA loci. VECTOR can be utilized to programmably reposition genomic loci on a timescale of seconds to minutes, quantitatively revealing local heterogeneity in the viscoelastic material properties of chromatin. These synthetic condensates are built from components that naturally form liquid-like structures in living cells, highlighting the potential role for native condensates to generate forces and do work to reorganize the genome and impact chromatin architecture., Competing Interests: Declaration of interests C.P.B. is a scientific founder, Scientific Advisory Board member, shareholder, and consultant for Nereid Therapeutics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgos) and the DNA defense module DdmDE system. Through biochemical analysis, structural determination, and in vivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Synthesis and maturation of Okazaki Fragments is an incessant and highly efficient metabolic process completing the synthesis of the lagging strands at replication forks during S phase. Accurate Okazaki fragment maturation (OFM) is crucial to maintain genome integrity and, therefore, cell survival in all living organisms. In eukaryotes, OFM involves the consecutive action of DNA polymerase Pol ∂, 5' Flap endonuclease Fen1 and DNA ligase I, and constitutes the best example of a sequential process coordinated by the sliding clamp PCNA. For OFM to occur efficiently, cooperation of these enzymes with PCNA must be highly regulated. Here, we present evidence of a role for the K164-PCNA-deubiquitylase Ubp10 in the maturation of Okazaki fragments in the budding yeast Saccharomyces cerevisiae. We show that Ubp10 associates with lagging-strand DNA synthesis machineries on replicating chromatin to ensure timely ligation of Okazaki fragments by promoting PCNA dissociation from chromatin requiring lysine 164 deubiquitylation., (© 2024. The Author(s).)

Lysosome-targeting chimera (LYTAC) shows great promise for protein-based therapeutics by targeted degradation of disease-associated membrane or extracellular proteins, yet its efficiency is constrained by the limited binding affinity between LYTAC reagents and designated proteins. Here, we established a programmable and multivalent LYTAC system by tandem assembly of DNA into a high-affinity protein degrader, a heterodimer aptamer nanostructure targeting both pathogenic membrane protein and lysosome-targeting receptor (insulin-like growth factor 2 receptor, IGF2R) with adjustable spatial distribution or organization pattern. The DNA-based multivalent LYTACs showed enhanced efficacy in removing immune-checkpoint protein programmable death-ligand 1 (PD-L1) and vascular endothelial growth factor receptor 2 (VEGFR2) in tumor cell membrane that respectively motivated a significant increase in T cell activity and a potent effect on cancer cell growth inhibition. With high programmability and versatility, this multivalent LYTAC system holds considerable promise for realizing protein therapeutics with enhanced activity.

Fourteen cobalt(II) complexes with the non-steroidal anti-inflammatory drugs sodium meclofenamate, tolfenamic acid, mefenamic acid, naproxen, sodium diclofenac, and diflunisal were prepared in the presence or absence of a series of nitrogen-donors (namely imidazole, pyridine, 3-aminopyridine, neocuproine, 2,2'-bipyridine, 1,10-phenanthroline and 2,2'-bipyridylamine) as co-ligands and were characterised by spectroscopic and physicochemical techniques. Single-crystal X-ray crystallography was employed to determine the crystal structure of eight complexes. The biological profile of the complexes was investigated regarding their interaction with serum albumins and DNA, and their antioxidant potency. The interaction of the compounds with calf-thymus DNA takes place via intercalation. The ability of the complexes to cleave pBR322 plasmid DNA at the concentration of 500 μM is rather low. The complexes demonstrated tight and reversible binding to human and bovine serum albumins and the binding site of bovine serum albumin was also examined. In order to assess the antioxidant activity of the compounds, the in vitro scavenging activity towards free radicals, namely 1,1-diphenyl-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), and their ability to reduce H 2 O 2 were studied.

The three-dimensional structure of DNA is a biophysical determinant of transcription. The density of chromatin condensation is one determinant of transcriptional output. Chromatin condensation is generally viewed as enforcing transcriptional suppression, and therefore, transcriptional output should be inversely proportional to DNA compaction. We coupled stable isotope tracers with multi-isotope imaging mass spectrometry to quantify and image nanovolumetric relationships between DNA density and newly made RNA within individual nuclei. Proliferative cell lines and cycling cells in the murine small intestine unexpectedly demonstrated no consistent relationship between DNA density and newly made RNA, even though localized examples of this phenomenon were detected at nuclear-cytoplasmic transitions. In contrast, non-dividing hepatocytes demonstrated global reduction in newly made RNA and an inverse relationship between DNA density and transcription, driven by DNA condensates at the nuclear periphery devoid of newly made RNA. Collectively, these data support an evolving model of transcriptional plasticity that extends at least to a subset of chromatin at the extreme of condensation as expected of heterochromatin., (© 2024 Guillermier et al.)

The transport of biopolymers across nanopores is an important biological process currently under investigation for the rapid analysis of DNA and proteins. While the transport of DNA is generally understood, methods to induce unfolded protein translocation have only recently been discovered (Yu et al., 2023, Sauciuc et al., 2023). Here, we found that during electroosmotically driven translocation of polypeptides, blob-like structures typically form inside nanopores, often obstructing their transport and preventing addressing individual amino acids. This is in contrast with the electrophoretic transport of DNA, where the formation of such structures has not been reported. Comparisons between different nanopore sizes and shapes and modifications by different surface chemistries allowed formulating a mechanism for blob formation. We also show that single-file transport can be achieved by using 1) nanopores that have an entry and an internal diameter smaller than the persistence length of the polymer, 2) nanopores with a nonsticky (i.e ., nonaromatic) inner surface, and 3) moderate translocation velocities. These experiments provide a basis for understanding polypeptide transport under confinement and for improving the design and engineering of nanopores for protein analysis., Competing Interests: Competing interests statement:Giovanni Maglia has equity in and is a founder, director, and shareholder of Portal Biotech Limited, a company engaged in the development of nanopore technologies. This work was not supported by Portal Biotech Limited.

Protein-DNA interactions and protein-mediated DNA compaction play key roles in a range of biological processes. The length scales typically involved in DNA bending, bridging, looping, and compaction (≥1 kbp) are challenging to address experimentally or by all-atom molecular dynamics simulations, making coarse-grained simulations a natural approach. Here, we present a simple and generic coarse-grained model for DNA-protein and protein-protein interactions and investigate the role of the latter in the protein-induced compaction of DNA. Our approach models the DNA as a discrete worm-like chain. The proteins are treated in the grand canonical ensemble, and the protein-DNA binding strength is taken from experimental measurements. Protein-DNA interactions are modeled as an isotropic binding potential with an imposed binding valency without specific assumptions about the binding geometry. To systematically and quantitatively classify DNA-protein complexes, we present an unsupervised machine learning pipeline that receives a large set of structural order parameters as input, reduces the dimensionality via principal-component analysis, and groups the results using a Gaussian mixture model. We apply our method to recent data on the compaction of viral genome-length DNA by HIV integrase and find that protein-protein interactions are critical to the formation of looped intermediate structures seen experimentally. Our methodology is broadly applicable to DNA-binding proteins and protein-induced DNA compaction and provides a systematic and semi-quantitative approach for analyzing their mesoscale complexes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of interests The authors declare no competing interests.

DNA-based nanostructures and nanodevices have recently been employed for a broad range of applications in modulating the assemblies and interaction patterns of different cell membrane receptors. These versatile nanodevices can be rationally designed with modular structures, easily programmed and tweaked such that they may act as smart chemical biology and cell biology tools to reveal insights into complicated cellular signaling processes. Their outstanding in vitro and cellular features have also begun to be further validated for some in vivo applications and demonstrated their great biomedical potential. In this review, we will highlight some key current advances in the molecular engineering and biological applications of DNA-based functional nanodevices, with a focus on how these tools have been used to respond and modulate membrane receptor dimerizations and/or oligomerizations, as a way to control cellular signaling processes. Some current challenges and future directions to further develop and apply these DNA nanodevices will also be discussed.

Binary (also known as split) nucleic acid enzymes have emerged as novel tools in biosensors. We report a new split strategy to split the DNAzyme kinase into two independent and non-functional fragments, denoted Dk1sub and Dk1enz. In the presence of the specific target, their free ends are brought sufficiently close to interact with each other without the formation of Watson-Crick base pairings between Dk1sub and Dk1enz, thus allowing the DNA phosphorylation reaction. We term this approach proximity-dependent activation of split DNAzyme kinase (ProxSDK). The utility of ProxSDK is demonstrated by engineering a biosensing system that is capable of measuring specific DNA-protein interactions. We envision that the approach described herein will find useful applications in biosensing, imaging, and clinical diagnosis., (© 2024 Wiley-VCH GmbH.)

Knowing the conformational ensembles formed by mismatches is crucial for understanding how they are generated and repaired and how they contribute to genomic instability. Here, we review structural and energetic studies of the A-C mismatch in duplex DNA and use the information to identify critical conformational states in its ensemble and their significance in genetic processes. In the 1970s, Topal and Fresco proposed the A-C wobble stabilized by two hydrogen bonds, one requiring protonation of adenine-N1. Subsequent NMR and X-ray crystallography studies showed that the protonated A-C wobble was in dynamic equilibrium with a neutral inverted wobble. The mismatch was shown to destabilize duplex DNA in a sequence- and pH-dependent manner by 2.4-3.8 kcal/mol and to have an apparent pKa ranging between 7.2 and 7.7. The A-C mismatch conformational repertoire expanded as structures were determined for damaged and protein-bound DNA. These structures included Watson-Crick-like conformations forming through tautomerization of the bases that drive replication errors, the reverse wobble forming through rotation of the entire nucleotide proposed to increase the fidelity of DNA replication, and the Hoogsteen base-pair forming through the flipping of the adenine base which explained the unusual specificity of DNA polymerases that bypass DNA damage. Thus, the A-C mismatch ensemble encompasses various conformational states that can be selectively stabilized in response to environmental changes such as pH shifts, intermolecular interactions, and chemical modifications, and these adaptations facilitate critical biological processes. This review also highlights the utility of existing 3D structures to build ensemble models for nucleic acid motifs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Studies have shown that disrupting the formation of the ligand-RET-GFRα complex could be an effective way of treating pain and itch. Compared to traditional high-throughput screens, DNA encoded libraries (DELs) have distinguished themselves as a powerful technology for hit identification in recent years. The present work demonstrates the use of DEL technology identifying compound 16 as the first GFRa2/GFRa3 small molecule inhibitor (0.1/0.2 μM respectively) selective over RET. This molecule represents an opportunity to advance the development of small-molecule inhibitors targeting the GFRα-RET interface for the treatment of pain and itch., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The nucleosome including H2A.B, a mammalian-specific H2A variant, plays pivotal roles in spermatogenesis, embryogenesis, and oncogenesis, indicating unique involvement in transcriptional regulation distinct from canonical H2A nucleosomes. Despite its significance, the exact regulatory mechanism remains elusive. This study utilized solid-state nanopores to investigate DNA unwinding dynamics, applying local force between DNA and histones. Comparative analysis of canonical H2A and H2A.B nucleosomes demonstrated that the H2A.B variant required a lower voltage for complete DNA unwinding. Furthermore, synchronization analysis and molecular dynamics simulations indicate that the H2A.B variant rapidly unwinds DNA, causing the H2A-H2B dimer to dissociate from DNA immediately upon disassembly of the histone octamer. In contrast, canonical H2A nucleosomes unwind DNA at a slower rate, suggesting that the H2A-H2B dimer undergoes a state of stacking at the pore. These findings suggest that nucleosomal DNA in the H2A.B nucleosomes undergoes a DNA unwinding process involving histone octamer disassembly distinct from that of canonical H2A nucleosomes, enabling smoother unwinding. The integrated approach of MD simulations and nanopore measurements is expected to evolve into a versatile tool for studying molecular interactions, not only within nucleosomes but also through the forced dissociation of DNA-protein complexes., (© 2024. The Author(s).)

With recent advances in multi-color super-resolution light microscopy, it is possible to simultaneously visualize multiple subunits within biological structures at nanometer resolution. To optimally evaluate and interpret spatial proximity of stainings on such an image, colocalization analysis tools have to be able to integrate prior knowledge on the local geometry of the recorded biological complex. We present MultiMatch to analyze the abundance and location of chain-like particle arrangements in multi-color microscopy based on multi-marginal optimal unbalanced transport methodology. Our object-based colocalization model statistically addresses the effect of incomplete labeling efficiencies enabling inference on existent, but not fully observable particle chains. We showcase that MultiMatch is able to consistently recover existing chain structures in three-color STED images of DNA origami nanorulers and outperforms geometry-uninformed triplet colocalization methods in this task. MultiMatch generalizes to an arbitrary number of color channels and is provided as a user-friendly Python package comprising colocalization visualizations., (© 2024. The Author(s).)

Extracellular vesicles (EVs) have emerged as key players in intercellular communication, facilitating the transfer of crucial cargo between cells. Liquid biopsy, particularly through the isolation of EVs, has unveiled a rich source of potential biomarkers for health and disease, encompassing proteins and nucleic acids. A milestone in this exploration occurred a decade ago with the identification of extracellular vesicle-associated DNA (EV-DNA) in the bloodstream of a patient diagnosed with pancreatic cancer. Subsequent years have witnessed substantial advancements, deepening our insights into the molecular intricacies of EV-DNA emission, detection, and analysis. Understanding the complexities surrounding the release of EV-DNA and addressing the challenges inherent in EV-DNA research are pivotal steps toward enhancing liquid biopsy-based strategies. These strategies, crucial for the detection and monitoring of various pathological conditions, particularly cancer, rely on a comprehensive understanding of why and how EV-DNA is released. In our review, we aim to provide a thorough summary of a decade's worth of research on EV-DNA. We will delve into diverse mechanisms of EV-DNA emission, its potential as a biomarker, its functional capabilities, discordant findings in the field, and the hurdles hindering its clinical application. Looking ahead to the next decade, we envision that advancements in EV isolation and detection techniques, coupled with improved standardization and data sharing, will catalyze the development of novel strategies exploiting EV-DNA as both a source of biomarkers and therapeutic targets., (© 2024. The Author(s).)

DNase II, identified in 1947 and named in 1953, is an acidic DNA endonuclease prevalent across organisms and crucial for normal growth. Despite its expression in nearly all human tissues, as well as its biological significance, DNase II's detailed functions and corresponding mechanisms remain unclear. Although many groups are trying to figure this out, progress is very limited. It is very hard to connect its indispensability with its DNA cleavage activity. In this study, we find that DNase II secreted to saliva can digest RNA in mildly acidic conditions, prompting us to hypothesize that salivary DNase II might digest RNA in the stomach. This finding is consistent with the interesting discovery reported in 1964 that RNA could inhibit DNase II's activity, which has been largely overlooked. This RNA digestion activity is further confirmed by using purified DNase II, showing activity to digest both DNA and RNA effectively. Here, we suggest redesignating DNase II as DNase II (RNase). The biological functions of DNase II are suggested to recycle intracellular RNA or digest external nucleic acids (both RNA and DNA) as nutrients. This discovery may untangle the mystery of DNase II and its significant biofunctions.

This study investigates the synthesis and optimization of nanobots (NBs) loaded with pDNA using the layer-by-layer (LBL) method and explores the impact of their collective motion on the transfection efficiency. NBs consist of biocompatible and biodegradable poly(lactic- co -glycolic acid) (PLGA) nanoparticles and are powered by the urease enzyme, enabling autonomous movement and collective swarming behavior. In vitro experiments were conducted to validate the delivery efficiency of fluorescently labeled NBs, using two-dimensional (2D) and three-dimensional (3D) cell models: murine urothelial carcinoma cell line (MB49) and spheroids from human urothelial bladder cancer cells (RT4). Swarms of pDNA-loaded NBs showed enhancements of 2.2- to 2.6-fold in delivery efficiency and 6.8- to 8.1-fold in material delivered compared to inhibited particles (inhibited enzyme) and the absence of fuel in a 2D cell culture. Additionally, efficient intracellular delivery of pDNA was demonstrated in both cell models by quantifying and visualizing the expression of eGFP. Swarms of NBs exhibited a >5-fold enhancement in transfection efficiency compared to the absence of fuel in a 2D culture, even surpassing the Lipofectamine 3000 commercial transfection agent (cationic lipid-mediated transfection). Swarms also demonstrated up to a 3.2-fold enhancement in the amount of material delivered in 3D spheroids compared to the absence of fuel. The successful transfection of 2D and 3D cell cultures using swarms of LBL PLGA NBs holds great potential for nucleic acid delivery in the context of bladder treatments.

Background: Mutations occurring in nucleic acids or proteins may affect the binding affinities of protein-nucleic acid interactions. Although many efforts have been devoted to the impact of protein mutations, few computational studies have addressed the effect of nucleic acid mutations and explored whether the identical methodology could be applied to the prediction of binding affinity changes caused by these two mutation types., Results: Here, we developed a generalized algorithm named PNBACE for both DNA and protein mutations. We first demonstrated that DNA mutations could induce varying degrees of changes in binding affinity from multiple perspectives. We then designed a group of energy-based topological features based on different energy networks, which were combined with our previous partition-based energy features to construct individual prediction models through feature selections. Furthermore, we created an ensemble model by integrating the outputs of individual models using a differential evolution algorithm. In addition to predicting the impact of single-point mutations, PNBACE could predict the influence of multiple-point mutations and identify mutations significantly reducing binding affinities. Extensive comparisons indicated that PNBACE largely performed better than existing methods on both regression and classification tasks., Conclusions: PNBACE is an effective method for estimating the binding affinity changes of protein-nucleic acid complexes induced by DNA or protein mutations, therefore improving our understanding of the interactions between proteins and DNA/RNA., (© 2024. The Author(s).)

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro . We identified the first dsTEL repeat nearest the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats, while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5'-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared with POT1 alone by bridging single- and double-stranded telomeric tracts.

Allostery is a hallmark of cellular function and important in every biological system. Still, we are only starting to mimic it in the laboratory. Here, we introduce an approach to study aspects of allostery in artificial systems. We use a DNA origami domino array structure which-upon binding of trigger DNA strands-undergoes a stepwise allosteric conformational change. Using two FRET probes placed at specific positions in the DNA origami, we zoom in into single steps of this reaction cascade. Most of the steps are strongly coupled temporally and occur simultaneously. Introduction of activation energy barriers between different intermediate states alters this coupling and induces a time delay. We then apply these approaches to release a cargo DNA strand at a predefined step in the reaction cascade to demonstrate the applicability of this concept in tunable cascades of mechanochemical coupling with both spatial and temporal control., (© 2024. The Author(s).)