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The main structural difference between the mutation-susceptible retinal isoforms of inosine 5´-monophosphate dehydrogenase-1 (IMPDH-1) with the canonical form resides in the C- and N-terminal peptide extensions with unknown structural/functional impacts. In this report, we aimed to experimentally evaluate the functional impact of these extensions on the specific/non-specific single-stranded DNA (ssDNA)-binding activities relative to those of the canonical form. Our in silico findings indicated the possible contribution of the C-terminal segment to the reduced flexibility of the Bateman domain of the enzyme. In addition, the in silico data indicated that the N-terminal tail acts by altering the distance between the tetramers in the concave octamer complex (the native form) of the enzyme. The overall impact of these predicted structural variations became evident, first, through higher Km values with respect to either of the substrates relative to the canonical isoform, as reported previously (Andashti et al. in Mol Cell Biochem 465(1):155-164, 2020). Secondary, the binding of the recombinant mouse retinal isoform IMPDH1 (603) to its specific Rhodopsin target gene was significantly augmented while its binding to non-specific ssDNA was lower than that of the canonical isoform. The DNA-binding activity of the other mouse retinal isoform, IMPDH1(546), to specific and non-specific ssDNA was lower than that of the canonical form most probably due to the in silico predicted rigidity created in the Bateman domain by the C-terminal peptide extension. Furthermore, the DNA binding to the Rhodopsin target gene by each of the IMPDH isoforms influenced in the presence of GTP (Guanosine triphosphate) and ATP (Adenosine triphosphate). [ABSTRACT FROM AUTHOR]

Toward the construction of robotic and cybernetic systems with molecular reactions, development of a reaction that can rapidly generate single-stranded DNA (ssDNA) molecules in response to an input signal is an essential demand. This study explored the cascading of DNA generation reactions employing DNA polymerase and nicking endonuclease to achieve significant amplification of ssDNA molecules serving as signals to direct and fuel the operation of DNA-based systems. The modular architecture allows for interconnection with other reactions through primer-binding sequence or template design, making it adaptable to various molecular robotic components. The research aims to contribute to the development of efficient and reliable amplification circuits for molecular robotics and cybernetics. The cascading reactions, implemented up to three layers, exhibit enhanced amplification rates and sensitivities at physiological temperatures, enabling stable hybridization with complementary sequences. The investigation reveals the potential of the proposed approach to bridge the molecular quantity gap and restore signals in molecular systems including molecular robots and related applications. The experimental validation demonstrates the feasibility of achieving up to 100,000-fold amplification in response to low concentrations of primers within two hours, driving the structural transformation of DNA probes and nanomotors, while suppressing non-specific leak amplification, thereby showcasing practical applicability. The study's findings address fundamental challenges in ssDNA amplification and opens avenues for creating intelligent systems composed of molecular components with increased sensitivity and responsiveness. [ABSTRACT FROM AUTHOR]

Aptamers, which are single-stranded DNA or RNA molecules, are increasingly recognized as important tools in diagnostics and therapeutics across various medical disciplines such as oncology, respiratory diseases, and neurological disorders. This review provides a comprehensive evaluation of the recent progress and obstacles encountered in the field of aptamer-based applications. Aptamers have shown promise in oncology for early cancer detection and targeted drug delivery, effectively reducing off-target effects. They also hold potential for significantly impacting the management of respiratory conditions such as asthma and Chronic Obstructive Pulmonary Disease (COPD) by selectively targeting cytokines and regulating the inflammatory response. In the realm of neurological disorders, aptamers offer novel methods by influencing the gut-brain axis and proposing potential approaches for early detection and specific therapy. Despite these notable benefits, persistent challenges remain in areas such as molecular stability, delivery mechanisms, and economic viability. This review offers a comprehensive overview of aptamer-based diagnostics and therapeutics while exploring potential avenues for future research. [ABSTRACT FROM AUTHOR]

Despite remarkable advances in the diagnosis of invasive fungal infections (IFIs), rapid, specific, sensitive, and cost-effective detection methods remain elusive. Due to their stability, ease of production, and specificity to signature molecules of fungal pathogens, short single-stranded sequences of DNA, RNA, and XNA, collectively called aptamers, have emerged as promising diagnostic markers. In this perspective, we summarize recent progress in aptamer-based diagnostic tools for IFIs and discuss how these tools could potentially meet the needs and provide economical and simple solutions for point-of-care for better management of IFIs. [ABSTRACT FROM AUTHOR]

[Display omitted] Peptide nucleic acid (PNA) is a DNA mimic that shows good stability against nucleases and proteases, forming strongly recognized complementary strands of DNA and RNA. However, due to its feeble ability to cross the cellular membrane, PNA activity and its targeting gene action is limited. Halloysite nanotubes (HNTs) are a natural and low-cost aluminosilicate clay. Because of their peculiar ability to cross cellular membrane, HNTs represent a valuable candidate for delivering genetic materials into cells. Herein, two differently charged 12-mer PNAs capable of recognizing as molecular target a 12-mer DNA molecule mimicking a purine-rich tract of neuroglobin were synthetized and loaded onto HNTs by electrostatic attraction interactions. After characterization, the kinetic release was also assessed in media mimicking physiological conditions. Resonance light scattering measurements assessed their ability to bind complementary single-stranded DNA. Furthermore, their intracellular delivery was assessed by confocal laser scanning microscopy on living MCF-7 cells incubated with fluorescence isothiocyanate (FITC)-PNA and HNTs labeled with a probe. The nanomaterials were found to cross cellular membrane and cell nuclei efficiently. Finally, it is worth mentioning that the HNTs/PNA can reduce the level of neuroglobin gene expression, as shown by reverse transcription-quantitative polymerase chain reaction and western blotting analysis. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus (V. parahaemolyticus) is one of the important seafood-borne pathogens that cause a serious gastrointestinal disorder in humans. Recently, biosensors have attracted serious attention for precisely detecting and tracking risk factors in foods. However, a major consideration when fabricating biosensors is to match the low cost of portable devices to broaden its application. In this study, a 3D-printed integrated handheld biosensor (IHB) that combines RPA-CRISPR/Cas12a, a lateral flow strip (LFS), and a handheld device was developed for the ultrasensitive detection of V. parahaemolyticus. Using the preamplification of RPA on tlh gene of V. parahaemolyticus, a specific duplex DNA product was obtained to activate the trans-cleavage activity of CRISPR/Cas12a, which was then utilized to cleave the ssDNA probe. The ssDNA probe was then detected by the LFS, which was negatively correlated with the content of amplified RPA products of the tlh gene. The IHB showed high selectivity and excellent sensitivity for V. parahaemolyticus detection, and the limit of detection was 4.9 CFU/mL. The IHB also demonstrated great promise for the screening of V. parahaemolyticus in samples and had the potential to be applied to the rapid screening of other pathogen risks for seafood and marine environmental safety. [ABSTRACT FROM AUTHOR]

Nucleic acid aptamers, often termed "chemical antibodies," are short, single‐stranded DNA or RNA molecules, which are selected by SELEX. In addition to their high specificity and affinity comparable to traditional antibodies, aptamers have numerous unique advantages such as wider identification of targets, none or low batch‐to‐batch variations, versatile chemical modifications, rapid mass production, and lack of immunogenicity. These characteristics make aptamers a promising recognition probe for scientific research or even clinical application. Aptamer‐functionalized nanomaterials are now emerged as a promising drug delivery system for various diseases with decreased side‐effects and improved efficacy. In this review, the technological strategies for generating high‐affinity and biostable aptamers are introduced. Moreover, the development of aptamers for their application in biomedicine including aptamer‐based biosensors, aptamer–drug conjugates and aptamer functionalized nanomaterials is comprehensively summarized. [ABSTRACT FROM AUTHOR]

Nucleic acids are major structures detected by the innate immune system. Although intracellular single-stranded DNA (ssDNA) accumulates during pathogen infection or disease, it remains unclear whether and how intracellular ssDNA stimulates the innate immune system. Here, we report that intracellular ssDNA triggers cytokine expression and cell death in a CGT motif–dependent manner. We identified Schlafen 11 (SLFN11) as an ssDNA-activated RNase, which is essential for the innate immune responses induced by intracellular ssDNA and adeno-associated virus infection. We found that SLFN11 directly binds ssDNA containing CGT motifs through its carboxyl-terminal domain, translocates to the cytoplasm upon ssDNA recognition, and triggers innate immune responses through its amino-terminal ribonuclease activity that cleaves transfer RNA (tRNA). Mice deficient in Slfn9, a mouse homolog of SLFN11, exhibited resistance to CGT ssDNA–induced inflammation, acute hepatitis, and septic shock. This study identifies CGT ssDNA and SLFN11/9 as a class of immunostimulatory nucleic acids and pattern recognition receptors, respectively, and conceptually couples DNA immune sensing to controlled RNase activation and tRNA cleavage. Editor's summary: Single-stranded DNA (ssDNA) generated during microbial infection and in response to DNA damage activates innate immune signaling. Toll-like receptor 9 senses endosomal ssDNA, but whether a designated pattern recognition receptor (PRR) detects ssDNA in other intracellular compartments remains unclear. By screening a library of ssDNA mimics, Zhang et al. identified CGT motifs as immunostimulatory sequences in intracellular ssDNA that lead to cell death and cytokine transcription. After a genome-wide CRISPR-Cas9 screen, they found that SLFN11 (encoding Schlafen 11) and its murine homolog Slfn9 bind CGT ssDNA and activate innate immune responses via the amino-terminal ribonuclease domain. Together, these results demonstrate that Schlafen 11 can function as a PRR for intracellular ssDNA. —Claire Olingy [ABSTRACT FROM AUTHOR]

Cell-free protein expression (CFE) systems have emerged as a critical platform for synthetic biology research. The vectors for protein expression in CFE systems mainly rely on double-stranded DNA and single-stranded RNA for transcription and translation processing. Here, we introduce a programmable vector - circular single-stranded DNA (CssDNA), which is shown to be processed by DNA and RNA polymerases for gene expression in a yeast-based CFE system. CssDNA is already widely employed in DNA nanotechnology due to its addressability and programmability. To apply above methods in the context of synthetic biology, CssDNA can not only be engineered for gene regulation via the different pathways of sense CssDNA and antisense CssDNA, but also be constructed into several gene regulatory logic gates in CFE systems. Our findings advance the understanding of how CssDNA can be utilized in gene expression and gene regulation, and thus enrich the synthetic biology toolbox. Protein expression vectors in cell-free systems mainly rely on double-stranded DNA and single-stranded RNA. Here, authors use circular single-stranded DNA as a programmable vector for gene regulation in a cell-free expression system by identifying its expression pathways. [ABSTRACT FROM AUTHOR]

The degradation of low-density lipoprotein receptor (LDLR) is induced by proprotein convertase subtilisin/kexin type 9 (PCSK9), resulting in elevated plasma concentrations of LDL cholesterol. Therefore, inhibiting the interactions between PCSK9 and LDLR is a desirable therapeutic goal for managing hypercholesterolemia. Aptamers, which are RNA or single-stranded DNA sequences, can recognize their targets based on their secondary structure. Aptamers exhibit high selectivity and affinity for binding to target molecules. The systematic evolution of ligands by exponential enrichment (SELEX), a combination of biological approaches, is used to screen most aptamers in vitro. Due to their unique advantages, aptamers have garnered significant interest since their discovery and have found extensive applications in various fields. Aptamers have been increasingly utilized in the development of biosensors for sensitive detection of pathogens, analytes, toxins, drug residues, and malignant cells. Furthermore, similar to monoclonal antibodies, aptamers can serve as therapeutic tools. Unlike certain protein therapeutics, aptamers do not elicit antibody responses, and their modified sugars at the 2'-positions generally prevent toll-like receptor-mediated innate immune responses. The focus of this review is on aptamer-based targeting of PCSK9 and the application of aptamers both as biosensors and therapeutic agents. [ABSTRACT FROM AUTHOR]

Transmembrane proteins transmit chemical signals as well as mechanical cues. The latter is often achieved by coupling to the cytoskeleton. The incorporation of fully engineerable membrane‐spanning structures for the transduction of chemical and, in particular, mechanical signals is therefore a critical aim for bottom‐up synthetic biology. Here, a membrane‐spanning DNA origami signaling units (DOSUs) is designed and mechanically coupled to DNA cytoskeletons encapsulated within giant unilamellar vesicles (GUVs). The incorporation of the DOSUs into the GUV membranes is verified and clustering upon external stimulation is achieved. Dye‐influx assays reveal that clustering increases the insertion efficiency. The transmembrane‐spanning DOSUs act as pores to allow for the transport of single‐stranded DNA into the GUVs. This is employed to trigger the reconfiguration of DNA cytoskeletons within GUVs. In addition to chemical signaling, mechanical coupling of the DOSUs to the internal DNA cytoskeletons is induced. With chemical cues from the environment, clustering of the DOSUs is induced, which triggers a symmetry break in the organization of the DNA cytoskeleton inside of the GUV. DNA‐based transmembrane structures are engineered that transduce signals without transporting the signaling molecule itself—providing a route toward signal processing and adaptive synthetic cells. [ABSTRACT FROM AUTHOR]

Cardiac troponin I (cTnI) is a cardiac biomarker for diagnosing ischemic heart disease and acute myocardial infarction. Current biochemical assays use antibodies (Abs) due to their high specificity and sensitivity. However, there are some limitations, such as the high-cost production of Abs due to complex instruments, reagents, and steps; the variability of Abs quality from batch to batch; the low stability at high temperatures; and the difficulty of chemical modification. Aptamer overcomes the limitations of antibodies, such as relatively lower cost, high reproducibility, high stability, and ease of being chemically modified. Aptamers are three-dimensional architectures of single-stranded RNA or DNA that bind to targets such as proteins. Six aptamers (Tro1-Tro6) with higher binding affinity than an antibody have been identified, but the molecular interaction has not been studied. In this study, six DNA aptamers were modeled and docked to cTnI protein. Molecular docking revealed that the interaction between all aptamer and cTnI happened in the similar cTnI region. The interaction between aptamer and cTnI involved hydrophobic interaction, hydrogen bonds, π-cation interactions, π-stack interactions, and salt-bridge formation. The calculated binding energy of all complexes was negative, which means that the complex formation was thermodynamically favorable. The electrostatic energy term was the main driving force of the interaction between all aptamer and cTnI. This study could be used to predict the behavior of further modified aptamer to improve aptamer performance. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) arises from expanded CAG repeats in exon 1 of the Huntingtin (HTT) gene. The resultant misfolded HTT protein accumulates within neuronal cells, negatively impacting their function and survival. Ultimately, HTT accumulation results in cell death, causing the development of HD. A nonhuman primate (NHP) HD model would provide important insight into disease development and the generation of novel therapies due to their genetic and physiological similarity to humans. For this purpose, we tested CRISPR/Cas9 and a single-stranded DNA (ssDNA) containing expanded CAG repeats in introducing an expanded CAG repeat into the HTT gene in rhesus macaque embryos. Analyses were conducted on arrested embryos and trophectoderm (TE) cells biopsied from blastocysts to assess the insertion of the ssDNA into the HTT gene. Genotyping results demonstrated that 15% of the embryos carried an expanded CAG repeat. The integration of an expanded CAG repeat region was successfully identified in five blastocysts, which were cryopreserved for NHP HD animal production. Some off-target events were observed in biopsies from the cryopreserved blastocysts. NHP embryos were successfully produced, which will help to establish an NHP HD model and, ultimately, may serve as a vital tool for better understanding HD's pathology and developing novel treatments. [ABSTRACT FROM AUTHOR]

The CRISPR‐Cas12a system has emerged as a powerful tool for next‐generation nucleic acid‐based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA‐enabled trans‐cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full‐size RNA targets, although LbCas12a exhibits weaker trans‐cleavage activity than AsCas12a on both single‐stranded DNA and RNA substrates. Remarkably, we find that the RNA‐activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the "Universal Nuclease for Identification of Virus Empowered by RNA‐Sensing" (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM‐less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double‐stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a‐based nucleic acid detection and further enhance the understanding of CRISPR‐Cas biochemistry. [ABSTRACT FROM AUTHOR]

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation. [ABSTRACT FROM AUTHOR]

Nipah virus (NiV), a bat-borne zoonotic viral pathogen with high infectivity and lethality to humans, has caused severe outbreaks in several countries of Asia during the past two decades. Because of the worldwide distribution of the NiV natural reservoir, fruit bats, and lack of effective treatments or vaccines for NiV, routine surveillance and early detection are the key measures for containing NiV outbreaks and reducing its influence. In this study, we developed two rapid, sensitive and easy-to-conduct methods, RAA-CRISPR/Cas12a-FQ and RAA-CRISPR/Cas12a-FB, for NiV detection based on a recombinase-aided amplification (RAA) assay and a CRISPR/Cas12a system by utilizing dual-labeled fluorophore–quencher or fluorophore-biotin ssDNA probes. These two methods can be completed in 45 min and 55 min and achieve a limit of detection of 10 copies per μL and 100 copies per μL of NiV N DNA, respectively. In addition, they do not cross-react with nontarget nucleic acids extracted from the pathogens causing similar symptoms to NiV, showing high specificity for NiV N DNA detection. Meanwhile, they show satisfactory performance in the detection of spiked samples from pigs and humans. Collectively, the RAA-CRISPR/Cas12a-FQ and RAA-CRISPR/Cas12a-FB methods developed by us would be promising candidates for the early detection and routine surveillance of NiV in resource-poor areas and outdoors. [ABSTRACT FROM AUTHOR]

Routinely used metagenomic next-generation sequencing (mNGS) techniques often fail to detect low-level viremia (<104 copies/mL) and appear biased towards viruses with linear genomes. These limitations hinder the capacity to comprehensively characterize viral infections, such as those attributed to the Anelloviridae family. These near ubiquitous non-pathogenic components of the human virome have circular single-stranded DNA genomes that vary in size from 2.0 to 3.9 kb and exhibit high genetic diversity. Hence, species identification using short reads can be challenging. Here, we introduce a rolling circle amplification (RCA)-based metagenomic sequencing protocol tailored for circular single-stranded DNA genomes, utilizing the long-read Oxford Nanopore platform. The approach was assessed by sequencing anelloviruses in plasma drawn from people who inject drugs (PWID) in two geographically distinct cohorts. We detail the methodological adjustments implemented to overcome difficulties inherent in sequencing circular genomes and describe a computational pipeline focused on anellovirus detection. We assessed our protocol across various sample dilutions and successfully differentiated anellovirus sequences in conditions simulating mixed infections. This method provides a robust framework for the comprehensive characterization of circular viruses within the human virome using the Oxford Nanopore. [ABSTRACT FROM AUTHOR]

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a promising nucleic acid-based gene targeting approach for gene expression knock-down and modulation. The RNase P-EGS strategy is unique as an EGS can be designed to basepair any mRNA sequence and recruit intracellular RNase P for hydrolysis of the target mRNA. In this study, we provide the first direct evidence that the RNase P-based approach effectively blocks the gene expression and replication of herpes simplex virus 2 (HSV-2), the causative agent of genital herpes. We constructed EGSs to target the mRNA encoding HSV-2 single-stranded DNA binding protein ICP8, which is essential for viral DNA genome replication and growth. In HSV-2 infected cells expressing a functional EGS, ICP8 levels were reduced by 85%, and viral growth decreased by 3000 folds. On the contrary, ICP8 expression and viral growth exhibited no substantial differences between cells expressing no EGS and those expressing a disabled EGS with mutations precluding RNase P recognition. The anti-ICP8 EGS is specific in targeting ICP8 because it only affects ICP8 expression but does not affect the expression of the other viral immediate-early and early genes examined. This study shows the effective and specific anti-HSV-2 activity of the RNase P-EGS approach and demonstrates the potential of EGS RNAs for anti-HSV-2 applications. [ABSTRACT FROM AUTHOR]

Tethering of a key mutagenic enzyme (activation-induced deaminase, AID) to RNA emanating from RNA polymerase provides insights into its physiological and pathological action. We propose a model where tethering of AID to nascent RNA is important for its action at translocation fragile zones ranging from 20 to 600 bp in size. Tethering likely supports the targeting of AID during its normal function in immunoglobulin class switch recombination and somatic hypermutation. Broader roles of tethering by the enzymes of the APOBEC family may be relevant to APOBEC functions in antiviral defense and other responses to cell stress. One of the two chromosomal breakage events in recurring translocations in B cell neoplasms is often due to the recombination-activating gene complex (RAG complex) releasing DNA ends before end joining. The other break occurs in a fragile zone of 20–600 bp in a non-antigen receptor gene locus, with a more complex and intriguing set of mechanistic factors underlying such narrow fragile zones. These factors include activation-induced deaminase (AID), which acts only at regions of single-stranded DNA (ssDNA). Recent work leads to a model involving the tethering of AID to the nascent RNA as it emerges from the RNA polymerase. This mechanism may have relevance in class switch recombination (CSR) and somatic hypermutation (SHM), as well as broader relevance for other DNA enzymes. [ABSTRACT FROM AUTHOR]

Through controlling the ssDNA product length of rolling circle amplification with AcyNTP, here we develop a nanopore signal enhancement strategy (STSS), which can successfully transfer the short oligonucleotide targets into long ssDNAs with appropriate lengths that can generate significant translocation currents. By labelling the RCA product with tags such as tetrahedral structures and isothermal amplicons, the resolution, signal specificity, and target range of the STSS can be further extended. [ABSTRACT FROM AUTHOR]

DNA replication in Escherichia coli starts with loading of the replicative helicase, DnaB, onto DNA. This reaction requires the DnaC loader protein, which forms a 6:6 complex with DnaB and opens a channel in the DnaB hexamer through which single-stranded DNA is thought to pass. During replication, replisomes frequently encounter DNA damage and nucleoprotein complexes that can lead to replication fork collapse. Such events require DnaB re-loading onto DNA to allow replication to continue. Replication restart proteins mediate this process by recruiting DnaB6/DnaC6 to abandoned DNA replication forks. Several dnaC mutations that bypass the requirement for replication restart proteins or that block replication restart have been identified in E. coli. To better understand how these DnaC variants function, we have purified and characterized the protein products of several such alleles. Unlike wild-type DnaC, three of the variants (DnaC 809, DnaC 809,820, and DnaC 811) can load DnaB onto replication forks bound by single-stranded DNA-binding protein. DnaC 809 can also load DnaB onto double-stranded DNA. These results suggest that structural changes in the variant DnaB6/DnaC6 complexes expand the range of DNA substrates that can be used for DnaB loading, obviating the need for the existing replication restart pathways. The protein product of dnaC1331, which phenocopies deletion of the priB replication restart gene, blocks loading through the major restart pathway in vitro. Overall, the results of our study highlight the utility of bacterial DnaC variants as tools for probing the regulatory mechanisms that govern replicative helicase loading. [ABSTRACT FROM AUTHOR]

Molecular strain can be introduced to influence the outcome of chemical reactions. Once a thermodynamic product is formed, however, reversing the course of a strain-promoted reaction is challenging. Here, a reversible, strain-promoted polymerization in cyclic DNA is reported. The use of nonhybridizing, single-stranded spacers as short as a single nucleotide in length can promote DNA cyclization. Molecular strain is generated by duplexing the spacers, leading to ring opening and subsequent polymerization. Then, removal of the strain-generating duplexers triggers depolymerization and cyclic dimer recovery via enthalpy-driven cyclization and entropy-mediated ring contraction. This reversibility is retained even when a protein is conjugated to the DNA strands, and the architecture of the protein assemblies can be modulated between bivalent and polyvalent states. This work underscores the utility of using DNA not only as a programmable ligand for assembly but also as a route to access restorable bonds, thus providing a molecular basis for DNA-based materials with shape-memory, self-healing, and stimuli-responsive properties. [ABSTRACT FROM AUTHOR]

Introduction: Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. Methods: This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays. Results: The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/mL, and both assays were completed in 30 min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays. Discussion: The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication. [ABSTRACT FROM AUTHOR]

Encapsulating individual mammalian cells with biomimetic materials holds potential in ex vivo cell culture and engineering. However, current methodologies often present tradeoffs between homogeneity, stability, and cell compatibility. Here, inspired by bacteria that use proteins stably anchored on their outer membranes to nucleate biofilm growth, we develop a single‐cell encapsulation strategy by using a DNA framework structure as a nucleator (DFN) to initiate the growth of DNA hydrogels under cell‐friendly conditions. We find that among the tested structures, the tetrahedral DFN can evenly and stably reside on cell membranes, effectively initiating hybridization chain reactions which generate homogeneously dense yet flexible single‐cell encapsulation for diverse cell lines. The encapsulation persists for up to 72 hours in a serum‐containing cell culture environment, representing a ~70‐fold improvement compared to encapsulations mediated by single‐stranded DNA nucleators. The metabolism and proliferation of the encapsulated cells are suppressed, but can be restored to the original efficiencies upon release, suggesting the superior cell compatibility of the encapsulation. We also find that compared to naked cells, the encapsulated cells exhibit a lower autophagy level after undergoing mechanical stress, suggesting the protective effect of the DNA encapsulation. This method may provide a new tool for ex vivo cell engineering. [ABSTRACT FROM AUTHOR]

Nitrofuran (NF) contamination in food products is a global problem resulting in the banned utilization and importation of nitrofuran contaminated products. A novel chromogenic detection method using a specific DNA aptamer with high affinity and specificity to nitrofurans was developed. Single-stranded DNA aptamers specific to nitrofuran metabolites, including 3-amino-2-oxazolidinone (AOZ), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), were isolated using magnetic bead-SELEX. The colorimetric detection of nitrofurans using gold nanoparticles (AuNPs) exhibited an AOZ detection range of 0.01–0.06 ppb with a limit of detection (LOD) of 0.03 ppb. At the same time, this system could detect AMOZ and AHD at a range of 0.06 ppb and 10 ppb, respectively. The fast nitrofuran extraction method was optimized for food, such as fish tissues and honey, adjusted to be completed within 3–6 h. This novel apta-chromogenic detection method could detect NF metabolites with a sensitivity below the minimum required performance limit (MPRL). This analysis will be valuable for screening, with a shortened time of detection for aquaculture products such as shrimp and fish muscle tissues. [ABSTRACT FROM AUTHOR]

Polyomavirus (PyV) Large T-antigen (LT) is the major viral regulatory protein that targets numerous cellular pathways for cellular transformation and viral replication. LT directly recruits the cellular replication factors involved in initiation of viral DNA replication through mutual interactions between LT, DNA polymerase alpha-primase (Polprim), and single-stranded DNA binding complex, (RPA). Activities and interactions of these complexes are known to be modulated by post-translational modifications; however, high-sensitivity proteomic analyses of the PTMs and proteins associated have been lacking. High-resolution liquid chromatography tandem mass spectrometry (LC–MS/MS) of the immunoprecipitated factors (IPMS) identified 479 novel phosphorylated amino acid residues (PAARs) on the three factors; the function of one has been validated. IPMS revealed 374, 453, and 183 novel proteins associated with the three, respectively. A significant transcription-related process network identified by Gene Ontology (GO) enrichment analysis was unique to LT. Although unidentified by IPMS, the ETS protooncogene 1, transcription factor (ETS1) was significantly overconnected to our dataset indicating its involvement in PyV processes. This result was validated by demonstrating that ETS1 coimmunoprecipitates with LT. Identification of a novel PAAR that regulates PyV replication and LT's association with the protooncogenic Ets1 transcription factor demonstrates the value of these results for studies in PyV biology. [ABSTRACT FROM AUTHOR]

Single‐stranded DNA oligonucleotides wrapping on the surface of single‐walled carbon nanotubes (SWCNTs), described as DNA corona, are often used as a dispersing agent for SWCNTs. The uneven distribution of DNA corona along SWCNTs is related to the photoelectric properties and the surface activity of SWCNTs. An ionic strength‐mediated "DNA corona defects" (DCDs) strategy is proposed to acquire an exposed surface of SWCNTs (accessible surface) as large as possible while maintaining good dispersibility via modulating the conformation of DNA corona. By adjusting the solution ionic strength, the DNA corona phase transitioned from an even‐distributed and loose conformation to a locally compact conformation. The resulting enlarged exposed surface of SWCNTs is called DCDs, which provide active sites for molecular adsorption. This strategy is applied for the arrangement of SWCNTs on DNA origami. SWCNTs with ≈11 nm DCD, providing enough space for the adsorption of "capture ssDNA" (≈7 nm width required for 24‐nt) extended from DNA origami structures are fabricated. The DCD strategy has potential applications in SWCNT‐based optoelectronic devices. [ABSTRACT FROM AUTHOR]

DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules, enabling the transport of one duplex through the transient double-stranded break of the other, remains elusive owing to structures derived solely from single linear duplex DNAs lacking topological constraints. Using cryo-electron microscopy, we solved the structure of Escherichia coli DNA gyrase bound to a negatively supercoiled minicircle DNA. We show how DNA gyrase captures a DNA crossover, revealing both conserved molecular grooves that accommodate the DNA helices. Together with molecular tweezer experiments, the structure shows that the DNA crossover is of positive chirality, reconciling the binding step of gyrase-mediated DNA relaxation and supercoiling in a single structure. [ABSTRACT FROM AUTHOR]

The current challenge in using extracellular vesicles (EVs) as drug delivery vehicles is to precisely control their membrane permeability, specifically in the ability to switch between permeable and impermeable states without compromising their integrity and functionality. Here, we introduce a rapid, efficient, and gentle loading method for EVs based on tonicity control (TC) using a lab-on-a-disc platform. In this technique, a hypotonic solution was used for temporarily permeabilizing a membrane ("on" state), allowing the influx of molecules into EVs. The subsequent isotonic washing led to an impermeable membrane ("off" state). This loading cycle enables the loading of different cargos into EVs, such as doxorubicin hydrochloride (Dox), ssDNA, and miRNA. The TC approach was shown to be more effective than traditional methods such as sonication or extrusion, with loading yields that were 4.3-fold and 7.2-fold greater, respectively. Finally, the intracellular assessments of miRNA-497-loaded EVs and doxorubicin-loaded EVs confirmed the superior performance of TC-prepared formulations and demonstrated the impact of encapsulation heterogeneity on the therapeutic outcome, signifying potential opportunities for developing novel exosome-based therapeutic systems for clinical applications. [ABSTRACT FROM AUTHOR]

The power of novel vaccination technologies and their rapid development were elucidated clearly during the COVID‐19 pandemic. At the same time, it also became clear that there is an urgent need to discover and manufacture new antivirals that target emerging viral threats. Toxic species of cyanobacteria produce a range of bioactive compounds that makes them good candidates for drug discovery. Nevertheless, few studies demonstrate the antiviral potential of cyanobacteria. This is partly due to the lack of specific and simple protocols designed for the rapid detection of antiviral activity in cyanobacteria and partly because specialized facilities for work with pathogenic viruses are few and far between. We therefore developed an easy method for the screening of cyanobacterial cultures for antiviral activity and used our private culture collection of non‐pathogenic virus isolates to show that antiviral activity is a prominent feature in the cyanobacterium Microcystis aeruginosa. In this proof‐of‐concept study, we show that M. aeruginosa extracts from three different cyanobacterial strains delay infection of diatom‐infecting single‐stranded DNA and single‐stranded RNA viruses by up to 2 days. Our work shows the ease with which cyanobacteria from culture collections can be screened for antiviral activity and highlights the potential of cyanobacteria as an excellent source for the discovery of novel antiviral compounds, warranting further investigation. [ABSTRACT FROM AUTHOR]

Highly compliant structures such as microbeams can deform substantially in response to interactions between molecules adsorbed on their surface. To understand such systems and improve their detection signals, a mechano-electro-chemical coupling model for mechanical deformations of the microbeams immobilized single-stranded DNA (ssDNA) is established due to flexoelectricity. The governing equations and corresponding boundary conditions of ssDNA microbeams are derived by using the variational principle. The bending deformations of ssDNA microbeams (one for cantilever beam and another for simply supported beam) are derived. The electric potential in the regions inside and outside the ssDNA layer is obtained by linear Poisson-Boltzmann equation for different electrolyte solutions. The analytical expressions to quantify the beam deflection and the potential difference of ssDNA layer are presented. The theoretical predictions are compared with the experimental data to validate the applicability of the present model. Numerical results reveal that the solution types, thickness, and elastic modulus of substrate materials have an obvious influence on the deflections of ssDNA microbeams. Therefore, the present model can help to improve the reading of the bending deformation signal of the microbeam biosensors. [ABSTRACT FROM AUTHOR]

Members of the Geminviridae family are circular single-stranded DNA plant-infecting viruses, some of which impact global food production. Geminiviruses are vectored by sap-feeding insects such as leafhoppers, treehoppers, aphids, and whiteflies. Additionally, geminivirus sequences have also been identified in other insects such as dragonflies, mosquitoes, and stingless bees. As part of a viral metagenomics study on honeybees and solitary bees (Nomia sp.), two geminivirus genomes were identified. These represent a novel citlodavirus (from honeybees collected from Westmoreland, Jamaica) and a mastrevirus-like genome (from a solitary bee collected from Tempe, Arizona, USA). The novel honeybee-derived citlodavirus genome shares ~61 to 69% genome-wide nucleotide pairwise identity with other citlodavirus genome sequences and is most closely related to the passion fruit chlorotic mottle virus identified in Brazil. Whereas the novel solitary bee-derived mastrevirus-like genome shares ~55 to 61% genome-wide nucleotide identity with other mastreviruses and is most closely related to tobacco yellow dwarf virus identified in Australia, based on pairwise identity scores of the full genome, replication-associated protein, and capsid protein sequences. Previously, two geminiviruses in the Begomovirus genus were identified in samples of stingless bee (Trigona spp.) samples. Here, we identify viruses that represent two new species of geminiviruses from a honeybee and a solitary bee, which continues to demonstrate that plant pollinators can be utilized for the identification of plant-infecting DNA viruses in ecosystems. [ABSTRACT FROM AUTHOR]

The ability to direct cell–cell interactions through engineered cell adhesion molecules (CAMs) represents an emerging frontier in studying cell biology and in many therapeutic applications. Engineered CAM methods include chemical methods (e.g., click chemistry and single-stranded DNA) as well as protein-based methods (e.g., coiled coils and nanobody–antigen). Whether an engineered CAM system is compatible with a target application requires consideration of both molecular and application-specific characteristics. The number of currently available engineered CAMs only represent a fraction of the quantity and capabilities that can theoretically be achieved. Existing engineered CAMs can already be directly applied to advance research within immunology, developmental biology, organoid, tissue engineering, and neuroscience fields. Intercellular interactions form the cornerstone of multicellular biology. Despite advances in protein engineering, researchers artificially directing physical cell interactions still rely on endogenous cell adhesion molecules (CAMs) alongside off-target interactions and unintended signaling. Recently, methods for directing cellular interactions have been developed utilizing programmable domains such as coiled coils (CCs), nanobody–antigen, and single-stranded DNA (ssDNA). We first discuss desirable molecular- and systems-level properties in engineered CAMs, using the helixCAM platform as a benchmark. Next, we propose applications for engineered CAMs in immunology, developmental biology, tissue engineering, and neuroscience. Biologists in various fields can readily adapt current engineered CAMs to establish control over cell interactions, and their utilization in basic and translational research will incentivize further expansion in engineered CAM capabilities. [ABSTRACT FROM AUTHOR]

Natural compounds have a high potential for the treatment of various conditions, including infections, inflammatory diseases, and cancer. However, they usually present poor pharmacokinetics, low specificity, and even toxicity, which limits their use. Therefore, targeted drug delivery systems, typically composed of a carrier and a targeting ligand, can enhance natural product selectivity and effectiveness. Notably, aptamers—short RNA or single-stranded DNA molecules—have gained attention as promising ligands in targeted drug delivery since they are simple to synthesize and modify, and they present high tissue permeability, stability, and a wide array of available targets. The combination of natural products, namely plant-based compounds, with a drug delivery system utilizing aptamers as targeting agents represents an emerging strategy that has the potential to broaden its applications. This review discusses the potential of aptamers as targeting agents in the delivery of natural compounds, as well as new trends and developments in their utilization in the field of medicine. [ABSTRACT FROM AUTHOR]

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is activated by binding to DNA. Activated cGAS produces 2'3'-cGAMP, which subsequently binds to the adaptor protein STING (stimulator of interferon genes). This interaction triggers the cGAS/STING signaling pathway, leading to the production of type I interferons. Three types of DNA, namely double-stranded DNA longer than 40 base pairs, a 70-nucleotide single-stranded HIV-1 DNA known as SL2, and Y-form DNA with unpaired guanosine trimers (G3 Y-form DNA), induce interferon production by activating cGAS/STING signaling. However, the extent of cGAS activation by each specific DNA type remains unclear. The comparison of cGAS stimulation by various DNAs is crucial for understanding the mechanisms underlying cGAS-mediated type I interferon production in the innate immune response. Here, we revealed that cGAS produces 2'3'-cGAMP at a significantly lower rate in the presence of single-stranded SL2 DNA than in the presence of double-stranded DNA or G3 Y-form DNA. Furthermore, the guanine-to-cytosine mutations and the deletion of unpaired guanosine trimers significantly reduced the 2'3'-cGAMP production rate and the binding of cGAS to Y-form DNA. These studies will provide new insights into the cGAS-mediated DNA-sensing in immune response. [ABSTRACT FROM AUTHOR]

Linear dichroism spectroscopy is used to investigate the structure of RecA family recombinase filaments (RecA and Rad51 proteins) with DNA for clarifying the molecular mechanism of DNA strand exchange promoted by these proteins and its activation. The measurements show that the recombinases promote the perpendicular base orientation of single‐stranded DNA only in the presence of activators, indicating the importance of base orientation in the reaction. We summarize the results and discuss the role of DNA base orientation. [ABSTRACT FROM AUTHOR]

MiR-451a is a biomolecule tightly related to resistance development during treatment with selective estrogen-receptor modulators for breast cancer. Accurately and sensitively monitoring its level in cells is of great significance for the drug selection and the evaluation of this cancer. However, the commonly obtainable ssDNA probe cannot efficiently enter the cell to sensitively emit reliable signals by avoiding nuclease digestion. Herein, a sensing system is reported to monitor miR-451a in living cells with high specificity and sensitivity. The system consisted of reduced graphene oxide (rGO), duplex-specific nuclease (DSN), and a fluorescence-labeled ssDNA nanoprobe. The fluorescence probe on the rGO surface provided an ultra-low background and resistance to endonuclease digestion. Duplex-specific nuclease sequentially triggered the hybridization cycle and the cleavage of ssDNA nanoprobe. The signal amplification provided a detection limit of 1 fM miR-451a using the optimal conditions with a linear range from 1 fM to 100 nM. Finally, the developed method was used to determine miR-451a in breast cancer tissue. This strategy provides a promising tool for clinical diagnosis and evaluation of miR-451a related cancer. [ABSTRACT FROM AUTHOR]

RAD51 is a highly conserved recombinase involved in the strand invasion/exchange of double-stranded DNA by homologous single-stranded DNA during homologous recombination repair. Although a majority of existing literature associates RAD51 with the pathogenesis of various types of cancer, recent reports indicate a role of RAD51 in maintenance of fertility. The present study reviews the role of RAD51 and its interacting proteins in spermatogenesis/oogenesis and additionally reports the findings from the molecular genetic screening of RAD51 135 G > C polymorphism in infertile cases and controls. Fifty-nine articles from PubMed and Google Scholar related to the reproductive role of RAD51 were reviewed. For case-control study, the PCR-RFLP method was used to screen the RAD51 135 G > C polymorphism in 201 infertile cases (100 males, 101 females) and 201 age- and gender-matched healthy controls (100 males, 101 females) from Punjab, North-West India. The review of literature shows that RAD51 is indispensable for spermatogenesis and oogenesis in animal models. Reports on the role of RAD51 in human fertility are limited, however it is involved in the pathogenesis of infertility in both males and females. Molecular genetic analyses in the infertile cases and healthy controls showed no statistically significant difference in the genotypic and allelic frequencies for RAD51 135 G > C polymorphism, even after segregation of the cases by type of infertility (primary/secondary). Therefore, the present study concluded that the RAD51 135 G > C polymorphism was neither associated with male nor female infertility in North-West Indians. This is the first report on RAD51 135 G > C polymorphism and infertility. [ABSTRACT FROM AUTHOR]

Carvacrol expresses a wide range of biological activities, but the studies of its mechanisms focused on bacteria, mainly involving the destruction of the plasma membrane. In this study, carvacrol exhibited strong activities against several phytopathogenic fungi and demonstrated a novel antifungal mechanism against Lasiodiplodia theobromae. RNA sequencing indicated that many genes of L. theobromae hyphae were predominately induced by carvacrol, particularly those involved in replication and transcription. Hyperchromic, hypsochromic, and bathochromic effects in the UV-visible absorption spectrum were observed following titration of calf thymus DNA (ctDNA) and carvacrol, which indicated the formation of a DNA–carvacrol complex. Circular dichroism (CD) spectroscopy indicated that the response of DNA to carvacrol was similar to that of 4′,6-diamidino-2-phenylindole (DAPI) but different from that of ethidium bromide (EB), implying the ionic bonds between carvacrol and ctDNA. Fluorescence spectrum (FS) analysis indicated that carvacrol quenched the fluorescence of double-stranded DNA (dsDNA) more than single-stranded DNA, indicating that carvacrol mainly bound to dsDNA. A displacement assay showed that carvacrol reduced the fluorescence intensity of the DNA–DAPI complex through competition with DAPI, but this did not occur for DNA–EB. The FS assay revealed that carvacrol bound to the AAA sequence on the minor groove of ds-oligonucleotides. The hydroxyl of carvacrol was verified to bind to ctDNA through a comparative test in which structural analogs of carvacrol, including thymol and 4-ethyl-1,2-dimethyl, were analyzed. The current study indicated carvacrol can destruct plasma membranes and bind to the minor groove of DNA, inhibiting fungal proliferation by disturbing the stability of dsDNA. [ABSTRACT FROM AUTHOR]

Upon replication fork stalling, the RPA-coated single-stranded DNA (ssDNA) formed behind the fork activates the ataxia telangiectasia-mutated and Rad3-related (ATR) kinase, concomitantly initiating Rad18-dependent monoubiquitination of PCNA. However, whether crosstalk exists between these two events and the underlying physiological implications of this interplay remain elusive. In this study, we demonstrate that during replication stress, ATR phosphorylates human Rad18 at Ser403, an adjacent residue to a previously unidentified PIP motif (PCNA-interacting peptide) within Rad18. This phosphorylation event disrupts the interaction between Rad18 and PCNA, thereby restricting the extent of Rad18-mediated PCNA monoubiquitination. Consequently, excessive accumulation of the tumor suppressor protein SLX4, now characterized as a novel reader of ubiquitinated PCNA, at stalled forks is prevented, contributing to the prevention of stalled fork collapse. We further establish that ATR preserves telomere stability in alternative lengthening of telomere (ALT) cells by restricting Rad18-mediated PCNA monoubiquitination and excessive SLX4 accumulation at telomeres. These findings shed light on the complex interplay between ATR activation, Rad18-dependent PCNA monoubiquitination, and SLX4-associated stalled fork processing, emphasizing the critical role of ATR in preserving replication fork stability and facilitating telomerase-independent telomere maintenance. Synopsis: The exact molecular interplay between ATR activation and Rad18-dependent PCNA monoubiquitination, along with the broader physiological implications of this interaction, remain unclear. This study reveals that ATR restricts Rad18-mediated PCNA monoubiquitination to maintain replication fork and telomerase-independent telomere stability. ATR phosphorylates human Rad18 at Serine 403 in response to replication stress. ATR restrains excessive SLX4 accumulation at stalled forks by phosphorylating Rad18. SLX4 recognizes monoubiquitinated PCNA to promote fork collapse. ATR preserves telomere stability in ALT cells by restricting Rad18-mediated PCNA monoubiquitination and excessive SLX4 accumulation at telomeres. Replication stress induced phosphorylation of Rad18 restrains excessive SLX4 nuclease accumulation at stalled forks and ALT telomeres. [ABSTRACT FROM AUTHOR]

Over 70,000 DNA lesions occur in the cell every day, and the inability to properly repair them can lead to mutations and destabilize the genome, resulting in carcinogenesis. The base excision repair (BER) pathway is critical for maintaining genomic integrity by repairing small base lesions, abasic sites and single‐stranded breaks. Monofunctional and bifunctional glycosylases initiate the first step of BER by recognizing and excising specific base lesions, followed by DNA end processing, gap filling, and finally nick sealing. The Nei‐like 2 (NEIL2) enzyme is a critical bifunctional DNA glycosylase in BER that preferentially excises cytosine oxidation products and abasic sites from single‐stranded, double‐stranded, and bubble‐structured DNA. NEIL2 has been implicated to have important roles in several cellular functions, including genome maintenance, participation in active demethylation, and modulation of the immune response. Several germline and somatic variants of NEIL2 with altered expression and enzymatic activity have been reported in the literature linking them to cancers. In this review, we provide an overview of NEIL2 cellular functions and summarize current findings on NEIL2 variants and their relationship to cancer. [ABSTRACT FROM AUTHOR]

A sensitive electrochemical genosensor was introduced and developed for a tumor suppressor gene, p53, detection. An Au screen-printed electrode coated with polyaniline film and ceria nanoparticles decorated on reduced graphene oxide was employed. To generate the genosensor, a suitable ssDNA probe sequence was immobilized on the modified surface of a screen-printed electrode without requiring any labeling or tagging moieties. The surface properties of the resulting electrodes were evaluated through scanning electron microscopy (SEM) and atomic force microscopy (AFM). The hybridization phenomena of the probe and its target sequence were followed by a differential pulse voltammetric signal of tris(bipyridine) ruthenium(II) chloride as an electrochemical probe. The detection limit was found 1.3 fM, and the DPV current was proportional to the logarithm of the p53 ssDNA concentration from 10 fM to 0.1 nM. The proposed genosensor showed excellent sensitivity, high selectivity, and reasonable reproducibility, which can be useful in future cancer diagnosis microdevice development. [ABSTRACT FROM AUTHOR]

The effectiveness and scalability of site-directed mutagenesis are constrained by the limited number of mutations and the intricate cloning process required for isolation of the target sequence. Here, we present a method for precise introduction of multiple non-contiguous mutations. The mutant strands are collected through one specially designed magnetic bead separation in alkaline conditions, efficiently removing their complementary partner strands with the original sequences. In a proof-of-concept test, a green fluorescent protein (GFP) was simultaneously mutated in 1–3 specific amino acids, successfully shifting its fluorescence spectrum. The precise mutation rates for single-, double- and triple-site mutations reached 100%, 76% and 70%, respectively. This multiple non-contiguous mutagenesis method may offer a fast and cost-effective approach for customizable construction of gene library.

Targeted protein degradation (TPD) is a powerful technique for the regulation of protein homeostasis. Current TPD mainly focuses on the therapeutical consequences rather than the operation processes of the molecular tools. Herein, a platform for precise manipulation of the protein degradation by activatable lysosome targeting DNA nanodevices is constructed. In the design, a lysosome‐targeting CD63 aptamer is locked by the single‐stranded DNA with a photocleavable group and a disulfide bond. This locked CD63 aptamer is connected with the aptamer targeting the protein of interest via double‐stranded DNA linkages to form the logic‐gate activated lysosome targeting DNA nanodevice (LALTD). With the UV light and endogenous GSH as inputs, an AND logic gate is built to efficiently manipulate the protein delivery processes by LALTD. The protein of interest can be degraded via efficient lysosome hydrolysis. Further studies show that the logic‐gate operation can be used for modulating T cell‐mediated antitumor immunity. The modularly designed activatable lysosome targeting DNA nanodevices exhibits good stability, controllability, programmability, and universality, providing a new prospect for accurate protein degradation and precise therapy. [ABSTRACT FROM AUTHOR]

The effectiveness of poly (ADP-ribose) polymerase inhibitors (PARPi) in creating single-stranded DNA gaps and inducing sensitivity requires the FANCJ DNA helicase. Yet, how FANCJ relates to PARP1 inhibition or trapping, which contribute to PARPi toxicity, remains unclear. Here, we find PARPi effectiveness hinges on S-phase PARP1 activity, which is reduced in FANCJ deficient cells as G-quadruplexes sequester PARP1 and MSH2. Additionally, loss of the FANCJMLH1 interaction diminishes PARP1 activity; however, depleting MSH2 reinstates PARPi sensitivity and gaps. Indicating sequestered and trapped PARP1 are distinct, FANCJ loss increases PARPi resistance in cells susceptible to PARP1 trapping. However, with BRCA1 deficiency, the loss of FANCJ mirrors PARP1 loss or inhibition, with the detrimental commonality being loss of S-phase PARP1 activity. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA1 deficient cells and emphasize the importance of understanding drug mechanisms for enhancing therapeutic response. [ABSTRACT FROM AUTHOR]

Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed single strand DNA (ssDNA). To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX (RPA-related RAD51-antagonist on the X chromosome) is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here, we present a structure-based investigation of RADX's mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration-dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined ab initio by cryo-electron microscopy (cryo-EM) from maps in the 2 to 4 Å range. The structure reveals the molecular basis for RADX oligomerization and the coupled multi-valent binding of ssDNA binding. The interaction of RADX with RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the end of RAD51 filaments. [ABSTRACT FROM AUTHOR]

Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 system is promising for the reversal of tumor immunosuppression in immunotherapy, but the controlled delivery of CRISPR/Cas9 remains challenging. Herein, the study reported a smart DNA nanoassembly containing multivalent aptamers, realizing the controlled delivery of Cas9/sgRNA ribonucleoprotein (RNP) for enhanced cancer immunotherapy. A single‐stranded DNA complementary to sgRNA in the Cas9/sgRNA RNP can initiate a cascade‐clamped hybridization chain reaction (C‐HCR) to wrap the Cas9/sgRNA RNP up in the DNA nanoassembly. After selective internalization of DNA nanoassembly by cancer cells, Cas9/sgRNA RNP is released to cytoplasm in response to endogenous RNase H and enters the nuclei to knock out β‐catenin. The expression of the programmed death‐ligand one gene is effectively suppressed, and the immunosuppressive tumor microenvironment is reprogrammed. Meanwhile, the migration of cancer cells is inhibited, and the apoptosis of cancer cells is promoted. In a breast cancer mouse model, the administration of DNA nanoassembly effectively increased the infiltration of CD8+ T cells, eventually achieving high therapeutic efficacy. [ABSTRACT FROM AUTHOR]

In clinical oncology, cell-free DNA (cfDNA) has shown immense potential in its ability to noninvasively detect cancer at various stages and monitor the progression of therapy. Despite the rapid improvements in cfDNA liquid biopsy approaches, achieving the required sensitivity to detect rare tumor-derived cfDNA still remains a challenge. For next-generation sequencing, the perceived presentation of cfDNA is strongly linked to the extraction and library preparation protocols. Conventional double-stranded DNA library preparation (dsDNA-LP) focuses on assessing ~167bp double-stranded mononucleosomal (mncfDNA) and its other oligonucleosomal cell-free DNA counterparts in plasma. However, dsDNA-LP methods fail to include short, single-stranded, or nicked DNA in the final library preparation, biasing the representation of the actual cfDNA populations in plasma. The emergence of single-stranded library preparation (ssDNA-LP) strategies over the past decade has now allowed these other populations of cfDNA to be studied from plasma. With the use of ssDNALP, single-stranded, nicked, and ultrashort cfDNA can be comprehensively assessed for its molecular characteristics and clinical potential. In this review, we overview the current literature on applications of ssDNA-LP on plasma cfDNA from a potential cancer liquid biopsy perspective. To this end, we discuss the molecular principles of single-stranded DNA adapter ligation, how library preparation contributes to the understanding of native cfDNA characteristics, and the potential for ssDNA-LP to improve the sensitivity of circulating tumor DNA detection. Additionally, we review the current literature on the newly reported species of plasma ultrashort single-stranded cell-free DNA plasma, which appear biologically distinct from mncfDNA. We conclude with a discussion of future perspectives of ssDNA-LP for liquid biopsy endeavors. [ABSTRACT FROM AUTHOR]