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The evolution of dynamic DNA nanostructures has propelled DNA nanotechnology into a robust and versatile field, offering groundbreaking applications in nanoscale communication, drug delivery, and molecular computing. Yet, the full potential of this technology awaits further enhancement through optimization of kinetic properties governing conformational changes. In this work, we introduce a mean-field theory to characterize the kinetic behavior of a dynamic DNA origami hinge where each arm bears complementary single-stranded DNA overhangs of different lengths, which can latch the hinge at a closed conformation. This device is currently being investigated for multiple applications, being of particular interest the development of DNA-based rapid diagnostic tests for coronavirus. Drawing from classical statistical mechanics theories, we derive analytical expressions for the mean binding time of these overhangs within a constant hinge. This analysis is then extended to flexible hinges, where the angle diffuses within a predetermined energy landscape. We validate our model by comparing it with experimental measurements of the closing rates of DNA nanocalipers with different energy landscapes and overhang lengths, demonstrating excellent agreement and suggesting fast angular relaxation relative to binding. These findings offer insights that can guide the optimization of devices for specific state lifetimes. Moreover, the framework introduced here lays the groundwork for further advancements in modeling the kinetics of dynamic DNA nanostructures. [ABSTRACT FROM AUTHOR]

ABSTRACT Metal (Co3+, Ni2+, and Pt2+)–centered and noncentered ferrocene–Schiff bases were prepared. They have been structurally characterized by spectroscopic methods, elemental analysis, conductivity, and magnetic moment. Electron spin resonance was used for the characterization of the Co3+‐centered complex. All synthesized compounds showed antibacterial activity against all tested Gram‐positive and Gram‐negative strains, as well as potent antifungal effect against Candida species. Remarkably, these compounds demonstrated greater efficacy against Gram‐positive bacteria than Gram‐negative. Without an additional oxidant or reductant, the compounds exhibited DNA cleavage capability in the dark. The development of synthesized novel chemical compounds having DNA cleavage and antimicrobial activity plays a profound role in the advancement of medical therapeutics. [ABSTRACT FROM AUTHOR]

Localized surface plasmons produced by gold and silver nanostructures have been utilized to enhance the intensity of fluorophore molecules. The issue with using nanostructure plasmons for fluorescence enhancement is their short-range nature (5–50 nm from the nanostructures), which limits accessibility to a few molecules. In addition, fluorophore dipoles needed to be aligned with the plasmon electric fields to maximize the fluorescence enhancement. To address these issues, we used low-frequency electric fields (<5 MHz) and commercially available nanorod and nanosphere samples and studied their effectiveness in enhancing the fluorescence of fluorophore-labeled short single-stranded DNA molecules (22 bases). We demonstrated that DNA molecules and nanorod particles can effectively be manipulated around the charging frequency of DNA molecules (∼3 MHz). Nanorod particles enhanced the fluorescence emission rate by ∼50-fold. When the 3 MHz electric field was introduced, the emission rate increased to over 700-fold. We also found that the introduction of a 3 MHz electric field aided the enhancement of the intrinsic quantum yield fluorophore molecules, which resulted in over a 1000-fold fluorescence enhancement. This enhancement was due to the very high electric produced by polarized DNA dipoles at 3 MHz, which resulted in a torque on fluorophore dipoles and subsequently aligning the fluorophore dipole axis with the plasmon electric field. At a fundamental level, our results demonstrate the role of the low-frequency electric field in the fluorophore–plasmon coupling. These findings can directly be applied to many fluorescence detection systems, including the development of biosensors. [ABSTRACT FROM AUTHOR]

Parvoviruses have evolved to possess a linear single-stranded DNA (ssDNA) genome ranging from 4 to 6.3 kb. Adeno-associated virus (AAV), a member of the Parvoviridae family, contains approximately 5 kb of linear ssDNA within its capsid. This ssDNA features two 145-base inverted terminal repeats (ITRs) positioned at each end. ITRs have a T-shaped hairpin secondary structure, which plays a crucial role in viral replication. To investigate the impact of ITRs on ssDNA stability, we conducted a DNA denaturation-reannealing assay in 10 mM magnesium acetate, 50 mM potassium acetate, and 20 mM Tris-acetate buffer at pH7.9. Conventional double-stranded DNA (dsDNA) fragments retain a reannealing capability of over 50% for sizes under 8.8 kb, gradually losing this capability as sizes increase; however, dsDNA fragments in rAAV ranging from 0.7 to 6.3 kb did not exhibit a reannealing profile. This suggests that the presence of ITRs at both ends hinders annealing between the complementary strands. These results indicate that ITR structures preferentially induce an ssDNA conformation less than 6.3 kb in size, and that the stability of AAV ssDNA contributes to the viral life cycle, including processes such as infection, replication, and packaging. Considering the size of parvovirus genomes, it appears that their genomes require reversible flexibility in complementary DNA strands; simultaneously, this adaptability needs to be regulated by specific palindromic ITRs at both ends. [ABSTRACT FROM AUTHOR]

Protein surfaces have pivotal roles in interactions between proteins and other biological molecules. However, the structural dynamics of protein surfaces have rarely been explored and are poorly understood. Here, the surface of a single‐stranded DNA (ssDNA) binding protein (SSB) with four DNA binding domains that bind ssDNA in binding site sizes of 35, 56, and 65 nucleotides per tetramer is investigated. Using oligonucleotides as probes to sense the charged surface, NaCl induces a two‐state structural transition on the SSB surface even at moderate concentrations. Chelation of sodium ions with charged amino acids alters the network of hydrogen bonds and/or salt bridges on the surface. Such changes are associated with changes in the electrostatic potential landscape and interaction mode. These findings advance the understanding of the molecular mechanism underlying the enigmatic salt‐induced transitions between different DNA binding site sizes of SSBs. This work demonstrates that monovalent salt is a key regulator of biomolecular interactions that not only play roles in non‐specific electrostatic screening effects as usually assumed but also may configure the surface of proteins to contribute to the effective regulation of biomolecular recognition and other downstream events. [ABSTRACT FROM AUTHOR]

Blood is a common biological fluid in forensic investigations, offering significant evidential value. Currently employed presumptive blood tests often lack specificity and are sample destructive, which can compromise downstream analysis. Within this study, the development of an optical biosensor for detecting human red blood cells (RBCs) has been explored to address such limitations. Aptamer-based biosensors, termed aptasensors, offer a promising alternative due to their high specificity and affinity for target analytes. Aptamers are short, single-stranded DNA or RNA sequences that form stable three-dimensional structures, allowing them to bind to specific targets selectively. A nanoflare design has been employed within this work, consisting of a quenching gold nanoparticle (AuNP), DNA aptamer sequences, and complementary fluorophore-labelled flares operating through a fluorescence resonance energy transfer (FRET) mechanism. In the presence of RBCs, the aptamer–flare complex is disrupted, restoring fluorescence and indicating the presence of blood. Two aptamers, N1 and BB1, with a demonstrated binding affinity to RBCs, were selected for inclusion within the nanoflare. This study aimed to optimise three features of the design: aptamer conjugation to AuNPs, aptamer hybridisation to complementary flares, and flare displacement in the presence of RBCs. Fluorescence restoration was achieved with both the N1 and BB1 nanoflares, demonstrating the potential for a functional biosensor to be utilised within the forensic workflow. It is hoped that introducing such an aptasensor could enhance the forensic workflow. This aptasensor could replace current tests with a specific and sensitive reagent that can be used for real-time detection, improving the standard of forensic blood analysis. [ABSTRACT FROM AUTHOR]

Therapies that modulate and appropriately direct the immune response are promising candidates for the treatment of infectious diseases. One such candidate therapeutic is DZ13, a short, synthetic, single-stranded DNA molecule. This molecule has enzymatic activity and can modulate the immune response by binding to and degrading the mRNA encoding a key immunoregulatory molecule. Originally developed and entering clinical trials as an anticancer agent, DZ13 has also been evaluated as a treatment for viral infections, and has been shown to provide protection against infection with influenza virus in a mouse model of infection. In this work, we evaluated whether the immunomodulatory properties of DZ13 could provide protection against the potential biothreat pathogen Burkholderia pseudomallei which causes the neglected tropical disease melioidosis. Treatment of mice infected with B. pseudomallei demonstrated that DZ13 did indeed provide excellent protection after only two post-exposure treatments. However, our data indicated that the enzymatic activity contained in DZ13 was not required for protection, with control oligonucleotide treatments lacking activity against the target mRNA equally as protective against B. pseudomallei. We have designed new sequences to study the mechanism of protection further. These novel sequences offer enhanced protection against infection, but are not directly anti-microbial and do not appear to be stimulating the immune system via TLR9 or other key innate immune sensors, despite containing CpG motifs. The molecular mechanism of these novel sequences remains to be elucidated, but the data highlights that these oligonucleotide-sensing pathways are attractive and relevant targets to modulate during bacterial and viral infections. [ABSTRACT FROM AUTHOR]

Precise and efficient identification of single‐stranded nucleic acids is crucial for both pathological research and early diagnosis of diseases, such as cancers. Therefore, we have devised a novel biosensor that utilizes an elegantly designed handgrip‐ring structure sensing probe to enhance the detection sensitivity and reduce background signals. The handgrip‐ring structure sensing probe combines ring padlock‐based target recognition and hairpin structure probe‐based signal amplification. The target sequences form a binding interaction with the ring padlock in the sensing probe, leading to the elongation of the single‐stranded chain with the assistance of polymerase. This elongation step results in the release of the hairpin probe, triggering a signal amplification process. This design significantly minimized the potential discrepancies that may occur during the signal amplification process, hence bestowing the approach with a low level of background signals. By utilizing this innovative design, the current biosensor demonstrates a remarkable ability to detect miRNA with a limit as low as 376 aM and single‐stranded DNA sequences with a limit as low as 45.3 aM. In addition, it possesses exceptional discrimination capabilities. The efficacy of this approach in diagnosing targets was also effectively proved by the rational redesign of the ring padlock. [ABSTRACT FROM AUTHOR]

The detection of circulating tumor DNA (ctDNA) in blood is significant for non‐invasive cancer diagnosis and treatment monitoring. Herein, MBene nanosheets is synthesized and compared with MXene via Density Functional Theory (DFT) calculations and fluorescence kinetic evaluations, for first time, revealing MBene's exceptional DNA adsorbability and discrimination to single‐stranded DNA (ssDNA) and double‐stranded DNA (dsDNA). Then, a sensitive fluorescence biosensor for ctDNA detection is developed, demonstrating impressive performance. To facilitate point‐of‐care testing (POCT) ctDNA, a paper‐based microfluidic chip incorporated a delay area and a mixing channel is designed. The constricted‐expanded structure channel optimization is guided by numerical simulations and experiments. A WeChat mini program named “ctDNA Detection” is designed for readout assay. Furthermore, cell and mice serum samples are analyzed, with Magnetic Bead@Graphene Oxide (MB@GO) and clutch probes for magnetic pre‐enrichment. The results accuracy is confirmed by its consistency with standard qPCR results (AUC = 1). The successful detection of ctDNA in post‐surgery mouse models underscored the biosensor's potential for cancer treatment monitoring. Thus, this research not only advanced the understanding of the MBene‐DNA interaction in biosensing, but also can pave the way for novel applications in bioimaging and nanopore‐based nucleic acid sequencing, leveraging the digital transformation of DNA base‐MBene adsorption differences. [ABSTRACT FROM AUTHOR]

In eukaryotic cell nuclei, specific sets of proteins gather in nuclear bodies and facilitate distinct genomic processes. The nucleolus, a nuclear body, functions as a factory for ribosome biogenesis by accumulating constitutive proteins, such as RNA polymerase I and nucleophosmin 1 (NPM1). Although in vitro assays have suggested the importance of liquid--liquid phase separation (LLPS) of constitutive proteins in nucleolar formation, how the nucleolus is structurally maintained with the intranuclear architecture remains unknown. This study revealed that the nucleolus is encapsulated by a single-stranded (ss)DNA-based molecular complex inside the cell nucleus. Super-resolution lattice-structured illumination microscopy (lattice-SIM) showed that there was a high abundance of ssDNA beyond the 'outer shell' of the nucleolus. Nucleolar disruption and the release of NPM1 were caused by in situ digestion of ssDNA, suggesting that ssDNA has a structural role in nucleolar encapsulation. Furthermore, we identified that ssDNA forms a molecular complex with histone H1 for nucleolar encapsulation. Thus, this study illustrates how an ssDNA-based molecular complex upholds the structural integrity of nuclear bodies to coordinate genomic processes such as gene transcription and replication. [ABSTRACT FROM AUTHOR]

The presence of phenol in coal-chemical engineering wastewater is considered a serious environmental issue. The use of enzymes to degrade phenol has received wide attention. G-quadruplex can combine with hemin to form G-quadruplex/hemin, which shows peroxidase-like activity and can be used to degrade phenol. In this work, a novel CNTs-NH2@G-quadruplex/hemin was designed using a single-stranded DNA (ssDNA) that contained G-rich fragments and T-rich fragments. The G-rich fragments assembled with the hemin to form G-quadruplex/hemin; the pyrimidine group of the T-rich fragment could form stable chemical bonds with the amino group, which could be used for immobilization. Characterization analysis of FTIR, XRD and TEM showed that the CNTs-NH2@G-quadruplex/hemin was successfully prepared. Meanwhile, the activity and phenol degradation ability for CNTs-NH2@G-quadruplex/hemin were studied in detail, and the effects of pH, temperature, initial phenol concentration, H2O2 concentration and reusability on the degradation efficiency of phenol were also explored. The results showed that when immobilized, the CNTs-NH2@G-quadruplex/hemin presented good activity, especially under conditions of temperature 30 °C and pH 7.0. Notably, the maximum reaction velocity of CNTs-NH2@G-quadruplex/hemin was increased from 0.016 mM mg−1 min−1 (free enzyme) to 0.056 mM mg−1 min−1. For phenol degradation ability, it was observed that under the conditions of temperature 30 °C, pH 7.0, initial phenol concentration 40 mg L−1, H2O2 concentration 120 mM, enzyme dose 40 mg L−1, more than 90% of phenol could be degraded within 600 min. In addition, the CNTs-NH2@G-quadruplex/hemin still maintained high activity after 10 cycles of phenol removal, demonstrating its appealing potential for practical applications. [ABSTRACT FROM AUTHOR]

Break-induced replication (BIR) is mutagenic, and thus its use requires tight regulation, yet the underlying mechanisms remain elusive. Here we uncover an important role of 53BP1 in suppressing BIR after end resection at double strand breaks (DSBs), distinct from its end protection activity, providing insight into the mechanisms governing BIR regulation and DSB repair pathway selection. We demonstrate that loss of 53BP1 induces BIR-like hyperrecombination, in a manner dependent on Polα-primase-mediated end fill-in DNA synthesis on single-stranded DNA (ssDNA) overhangs at DSBs, leading to PCNA ubiquitination and PIF1 recruitment to activate BIR. On broken replication forks, where BIR is required for repairing single-ended DSBs (seDSBs), SMARCAD1 displaces 53BP1 to facilitate the localization of ubiquitinated PCNA and PIF1 to DSBs for BIR activation. Hyper BIR associated with 53BP1 deficiency manifests template switching and large deletions, underscoring another aspect of 53BP1 in suppressing genome instability. The synthetic lethal interaction between the 53BP1 and BIR pathways provides opportunities for targeted cancer treatment. Break-induced replication (BIR) mechanism is mutagenic, and thus needs to be tightly regulated. Here the authors identify an important role of 53BP1 in suppressing mutagenic BIR, providing insights into DSB repair pathway selection and BIR regulation. [ABSTRACT FROM AUTHOR]

Amid global health concerns and the constant threat of zoonotic diseases, this study delves into the diversity of circular replicase-encoding single-stranded DNA (CRESS-DNA) viruses within Chinese wild bird populations. Employing viral metagenomics to tackle the challenge of "viral dark matter," the research collected and analyzed 3,404 cloacal swab specimens across 26 bird families. Metagenomic analysis uncovered a rich viral landscape, with 67.48% of reads classified as viral dark matter, spanning multiple taxonomic levels. Notably, certain viral families exhibited host-specific abundance patterns, with Galliformes displaying the highest diversity. Diversity analysis categorized samples into distinct groups, revealing significant differences in viral community structure, particularly noting higher diversity in terrestrial birds compared to songbirds and unique diversity in migratory birds versus perching birds. The identification of ten novel Circoviridae viruses, seven Smacoviridae viruses, and 167 Genomoviridae viruses, along with 100 unclassified CRESS-DNA viruses, underscores the expansion of knowledge on avian-associated circular DNA viruses. Phylogenetic and structural analyses of Rep proteins offered insights into evolutionary relationships and potential functional variations among CRESS-DNA viruses. In conclusion, this study significantly enhances our understanding of the avian virome, shedding light on the intricate relationships between viral communities and host characteristics in Chinese wild bird populations. The diverse array of CRESS-DNA viruses discovered opens avenues for future research into viral evolution, spread factors, and potential ecosystem impacts. [ABSTRACT FROM AUTHOR]

The s-subunit of the clamp loader complex physically interacts with both the DnaB helicase and the polymerase III (Pol III) core a-subunit through domains IV and V, respectively. This interaction is proposed to help maintain rapid and efficient DNA synthesis rates with high genomic fidelity and plasticity, facilitating enzymatic coupling within the replisome. To test this hypothesis, CRISPR-Cas9 editing was used to create site-directed genomic mutations within the dnaX gene at the C terminus of s predicted to interact with the a-subunit of Pol III. Perturbation of the a-s binding interaction in vivo resulted in cellular and genomic stress markers that included reduced growth rates, fitness, and viabilities. Specifically, dnaX:mut strains showed increased cell filamentation, mutagenesis frequencies, and activated SOS. In situ fluorescence flow cytometry and microscopy quantified large increases in the amount of ssDNA gaps present. Removal of the C terminus of s (I618X) still maintained its interactions with DnaB and stimulated unwinding but lost its interaction with Pol III, resulting in significantly reduced rolling circle DNA synthesis. Intriguingly, dnaX:L635P/D636G had the largest induction of SOS, high mutagenesis, and the most prominent ssDNA gaps, which can be explained by an impaired ability to regulate the unwinding speed of DnaB resulting in a faster rate of in vitro rolling circle DNA replication, inducing replisome decoupling. Therefore, s-stimulated DnaB unwinding and physical coupling with Pol III acts to enforce replisome plasticity to maintain an efficient rate of synthesis and prevent genomic instability. [ABSTRACT FROM AUTHOR]

In eubacteria, Holliday junction (HJ) resolvases (HJRs) are crucial for faithful segregation of newly replicated chromosomes, homologous recombination, and repair of stalled/collapsed DNA replication forks. However, compared with the Escherichia coli HJRs, little is known about their orthologs in mycobacterial species. A genome-wide analysis of Mycobacterium smegmatis identified two genes encoding putative HJRs, namely RuvC (MsRuvC) and RuvX (MsRuvX); but whether they play redundant, overlapping, or distinct roles remains unknown. Here, we reveal that MsRuvC exists as a homodimer while MsRuvX as a monomer in solution, and both showed high-binding affinity for branched DNAs compared with unbranched DNA species. Interestingly, the DNA cleavage specificities of MsRuvC and MsRuvX were found to be mutually exclusive: the former efficiently promotes HJ resolution, in a manner analogous to the Escherichia coli RuvC, but does not cleave other branched DNA species; whereas the latter is a versatile DNase capable of cleaving a variety of branched DNA structures, including 30 and 50 flap DNA, splayed-arm DNA and dsDNA with 30 and 50 overhangs but lacks the HJ resolution activity. Point mutations in the RNase H-like domains of MsRuvC and MsRuvX pinpointed critical residues required for their DNA cleavage activities and also demonstrated uncoupling between DNA-binding and DNA cleavage activities. Unexpectedly, we found robust evidence that MsRuvX possesses a double-strand/single-strand junction-specific endonuclease and ssDNA exonucleolytic activities. Combined, our findings highlight that the RuvC and RuvX DNases play distinct complementary, and not redundant, roles in the processing of branched DNA structures in M. smegmatis. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a refractory neurodegenerative disease characterized by the degeneration and loss of motor neurons, typically resulting in death within five years of onset. There have been few effective treatments, making the development of robust therapies an urgent challenge. Genetic mutations have been identified as contributors to ALS, with mutations in superoxide dismutase 1 (SOD1), which neutralizes the harmful reactive oxygen species superoxide, accounting for approximately 2% of all ALS cases. To counteract the toxic gain of function caused by SOD1 mutations, therapeutic strategies aimed at suppressing SOD1 gene expression have shown promise. Antisense oligonucleotide (ASO) is an artificially synthesized, short, single-stranded DNA/RNA molecule that binds to target RNA to alter gene expression, representing a next-generation therapeutic approach. In 2023, tofersen became the first ASO drug approved by the FDA for ALS. Administered intrathecally, tofersen specifically binds to SOD1 mRNA, inhibiting the production of toxic SOD1 protein, thereby improving biomarkers of ALS. The long-term efficacy and safety of tofersen require further validation, and the development of more optimized treatment protocols is essential. A series of studies and therapeutic developments related to SOD1 mutations have advanced the understanding of ALS pathophysiology and significantly contributed to treatment strategies for central nervous system disorders. This review focuses on an overview of SOD1 mutations and the development process of tofersen, aiming to deepen the understanding of advancements in ALS research and discuss future challenges and directions for ASO therapy. [ABSTRACT FROM AUTHOR]

The mechanism used by polyomavirus and other viral SF3 helicases to unwind DNA at replication forks remains unknown. Using AlphaFold2, we have determined the structure of a representative SF3 helicase, the SV40 T-antigen (T-ag). This model has been analyzed in terms of the features of T-ag required for helicase activity, particularly the proximity of the T-ag origin binding domain (OBD) to the replication fork and the distribution of basic residues on the surface of the OBD that are known to play roles in DNA unwinding. These and related studies provide additional evidence that the T-ag OBDs have a role in the unwinding of DNA at the replication fork. Nuclear magnetic resonance and modeling experiments also indicate that protonated histidines on the surface of the T-ag OBD play an important role in the unwinding process, and additional modeling studies indicate that protonated histidines are essential in other SF3 and SF6 helicases. Finally, a model for T-ag's helicase activity is presented, which is a variant of the "rope climber." According to this model, the hands are the N-terminal OBD domains that interact with the replication fork, while the C-terminal helicase domains contain the feet that bind to single-stranded DNA. [ABSTRACT FROM AUTHOR]

A fluorescent and photoelectrochemical (PEC) dual-mode biosensor based on target biorecognition-triggered cyclic amplification was constructed for Kana detection. With the assistance of the catalyzed reaction of exonuclease III, a Kana-aptamer DNA duplex was designed for conducting the cyclic release of G-rich DNA sequence as well as output DNA S2. The released G-rich sequence triggers the fluorescence (FL) of thioflavin T (ThT), the intensity of which is positively correlated with the Kana concentration. The linear range is 0.2 to 30 nM, and the detection limit reaches 0.07 nM. Simultaneously, the released output DNA S2 was captured by Fe3O4@CdTe-probe ssDNA and then combined with methylene blue to realize the transduction of polarity-reversed PEC signal, leading to the sensitive detection of Kana with a linear range of 0.2 to 40 nM and a calculated detection limit of 0.2 nM. The outstanding performance endows the dual-mode biosensor a promising prospect for practical application. [ABSTRACT FROM AUTHOR]

Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils (U's) and function in antiviral innate immunity and RNA editing and can cause hypermutation in chromosomes. The resulting U's can be directly replicated, resulting in C to T mutations, or U–DNA glycosylase can convert the U's to abasic (AP) sites which are then fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers, contributions of C to T and C to G mutations depend on the origin of ssDNA mutagenized by APOBECs. Since ssDNA in eukaryotic genomes readily binds to replication protein A (RPA) we asked if RPA could affect APOBEC-induced mutation spectrum in yeast. For that purpose, we expressed human APOBECs in the wild-type (WT) yeast and in strains carrying a hypomorph mutation rfa1-t33 in the large RPA subunit. We confirmed that the rfa1-t33 allele can facilitate mutagenesis by APOBECs. We also found that the rfa1-t33 mutation changed the ratio of APOBEC3A-induced T to C and T to G mutations in replicating yeast to resemble a ratio observed in long persistent ssDNA in yeast and in cancers. We present the data suggesting that RPA may shield APOBEC formed U's in ssDNA from Ung1, thereby facilitating C to T mutagenesis through the accurate copying of U's by replicative DNA polymerases. Unexpectedly, we also found that for U's shielded from Ung1 by WT RPA, the mutagenic outcome is reduced in the presence of translesion DNA polymerase zeta. [ABSTRACT FROM AUTHOR]

The human primase-polymerase PrimPol is a key participant of the mechanism of DNA synthesis restart during replication fork stalling at sites of DNA damage. PrimPol has DNA primase activity and synthesizes DNA primers that are used by processive DNA polymerases to continue replication. Recruitment of PrimPol to the sites of DNA damage, as well as stimulation of catalytic activity, depends on interaction with the replicative protein RPA, which binds single-stranded DNA. The C-terminal domain of PrimPol contains a negatively charged RPA-binding motif (RBM), mutations in which disrupt the interaction between two proteins. The RBM motif also plays a role in the negative regulation of PrimPol interaction with DNA. Deletion of the RBM dramatically increases PrimPol affinity to DNA and stimulates PrimPol activity. The mechanism of RBM-mediated regulation of PrimPol activity is unclear. The relatively strong negative charge of RBM potentially may contribute to the interaction of PrimPol with RPA and DNA. RBM contains two negatively charged regions RBM-A and RBM-B. In this work, we additionally added (substitution V546E) or decreased (substitution D547H) a negative charge in RBM-B PrimPol and characterized these mutant variants biochemically. It was shown that the local change in the RBM-B charge has no effect on the interaction of PrimPol with DNA and RPA, or of the catalytic activity of the enzyme. [ABSTRACT FROM AUTHOR]

CRISPR diagnostics are effective but suffer from low signal transduction efficiency, limited sensitivity, and poor stability due to their reliance on the trans-cleavage of single-stranded nucleic acid fluorescent reporters. Here, we present CrisprAIE, which integrates CRISPR/Cas reactions with "one to more" aggregation-induced emission luminogen (AIEgen) lighting-up fluorescence generated by the trans-cleavage of Cas proteins to AIEgen-incorporated double-stranded DNA labeled with single-stranded nucleic acid linkers and Black Hole Quencher groups at both ends (Q-dsDNA/AIEgens-Q). CrisprAIE demonstrates superior performance in the clinical nucleic acid detection of norovirus and SARS-CoV-2 regardless of amplification. Moreover, the diagnostic potential of CrisprAIE is further enhanced by integrating it with spherical nucleic acid-modified AIEgens (SNA/AIEgens) and a portable cellphone-based readout device. The improved CrisprAIE system, utilizing Q-dsDNA/AIEgen-Q and SNA/AIEgen reporters, exhibits approximately 80- and 270-fold improvements in sensitivity, respectively, compared to conventional CRISPR-based diagnostics. We believe CrisprAIE can be readily extended as a universal signal generation strategy to significantly enhance the detection efficiency of almost all existing CRISPR-based diagnostics. Current CRISPR diagnostic approaches can be hampered by several limitations, including low signal transduction efficiency and limited sensitivity. Here, the authors present CrisprAIE, an approach based on CRISPR/Cas-mediated "one to more" lighting-up nucleic acid detection using aggregation-induced emission luminogens. [ABSTRACT FROM AUTHOR]

The detection of lipopolysaccharide (LPS) has important value for the monitoring of diseases such as sepsis and the impurity control of drugs. In this work, we prepared guanidinylated carbon dots (GQ-CDs) and used them to adsorb 5-carboxyfluorescein (FAM)-labeled single-stranded DNA (ssDNA) to become GQ-CDs/FAM-DNA, resulting in quenched FAM. The quenching efficiency of the FAM-DNA by GQ-CDs in the GQ-CDs/FAM-DNA system was 91.95%, and this quenching was stable over the long term. Upon the addition of LPS, the quenched FAM-DNA in the GQ-CDs/FAM-DNA system regained fluorescence at 520 nm. The mechanism studies found that the addition of LPS promoted the dissociation of FAM-DNA adsorbed on GQ-CDs, thereby restoring fluorescence. The degree of fluorescence recovery was closely related to the content of LPS. Under optimized conditions, the fluorescence recovery was linearly related to LPS concentrations ranging from 5 to 90 μg/mL, with a detection limit of 0.75 μg/mL. The application of this method to plasma samples and trastuzumab injections demonstrated good spiked recoveries and reproducibility. This platform, based on GQ-CDs for the adsorption and quenching of FAM-DNA, enables the detection of LPS through relatively simple mixing operations, showing excellent competitiveness for the determination of actual samples under various conditions. [ABSTRACT FROM AUTHOR]

Deoxyribonucleic Acid (DNA) chip technology is of interest in the development of diagnostic devices and drug delivery applications. Silicon has been widely used for the fabrication of micro-electro-mechanical system (MEMS) and smart sensors, which are now commercially available. The aim of this work is to develop a sensor or chip for specific DNA sequences. The high sensitivity of the biochip is achieved by increasing the specific surface area of the device. Nanostructured porous silicon has high surface area that can be prepared to have large variety of pore morphologies by electrochemical etching of silicon wafer. In the present work, the immobilization of single stranded DNA and its hybridization on modified porous silicon as well as on flat silicon surface has been demonstrated. DNA immobilization on modified silicon surfaces and its subsequent hybridization was studied using gP32 labelled DNA, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectronspectroscopy (XPS). More than 70% of DNA binding was achieved on the porous silicon in comparison to the flat silicon surface which has less than 20% of DNA binding. Enhanced DNA binding on nanostructured porous silicon as compared to flat silicon surface is due to its large surface area and good molecular penetration into its pores. The high fidelity of nanostructured porous silicon surface promises substantial benefit to the emerging technology for nucleic acid-based biosensors and diagnostic chips. [ABSTRACT FROM AUTHOR]

Aptamer-based biosensors have been widely constructed and applied to detect diverse targets. Glutathione S-transferase (GST), a pivotal phase II metabolic enzyme, plays a critical role in biotransformation in vivo, and aberrant GST expression is associated with various health risks. Herein, aptamers targeting GST were systematically selected from a randomized single-stranded DNA (ssDNA) library of 79 nucleotides (nt) using a biotinylated GST-immobilized streptavidin agarose (SA) bead SELEX technology. Following rigorous screening across eight rounds, four aptamers with strikingly similar secondary structures emerged. Among these, Seq3 exhibited the highest affinity towards GST and was selected for further optimization. A semi-rational post-SELEX truncation strategy was then employed based on base composition analysis, secondary structure analysis and affinity assessment. This strategy enabled the systematic removal of redundant nucleotides in Seq3 without compromising its affinity, ultimately yielding a truncated aptamer, Seq3-3, which retains its specificity with a compact 39nt length. Building upon Seq3-3, a double-stranded fluorescent aptamer probe was ingeniously designed for the in vitro detection of GST. The detection mechanism hinges on the competitive displacement of the complementary chain from the probe, mediated by the target protein, leading to the separation of the antisense oligonucleotide from the double-stranded complex. This process triggers the restoration of the fluorescence signal, enabling sensitive detection, and the probe exhibits excellent response within a linear range of GST activity ranging from 0 to 1500 U/L. The results show that not only an efficient strategy for screening robust and practicable aptamers but also an ultrahighly sensitive detection platform for GST was established. [ABSTRACT FROM AUTHOR]

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism for the protection of tumor cells in the bloodstream and the promotion of tumor progression and metastases. The platelet C-type lectin-like receptor 2 (CLEC-2) can bind podoplanin (PDPN) on a cancer cell surface to facilitate TCIPA. Selective blockage of PDPN-mediated platelet–tumor cell interaction is a plausible strategy for inhibiting metastases. In this study, we aimed to screen for aptamers, which are the single-stranded DNA oligonucleotides that form a specific three-dimensional structure, bind to specific molecular targets with high affinity and specificity, bind to PDPN, and interfere with PDPN/CLEC-2 interactions. The systematic evolution of ligands by exponential enrichment (SELEX) was employed to enrich aptamers that recognize PDPN. The initial characterization of ssDNA pools enriched by SELEX revealed a PDPN aptamer designated as A1 displaying parallel-type G-quadruplexes and long stem-and-loop structures and binding PDPN with a material with a dissociation constant (Kd) of 1.3 ± 1.2 nM. The A1 aptamer recognized both the native and denatured form of PDPN. Notably, the A1 aptamer was able to quantitatively detect PDPN proteins in Western blot analysis. The A1 aptamer could interfere with the interaction between PDPN and CLEC-2 and inhibit PDPN-induced platelet aggregation in a concentration-dependent manner. These findings indicated that the A1 aptamer is a candidate for the development of biosensors in detecting the levels of PDPN expression. The action by A1 aptamer could result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients. [ABSTRACT FROM AUTHOR]

Introduction: Porcine Parvovirus (PPV) is a significant pathogen in the pig industry, with eight genotypes, including PPV7, identified since its emergence in 2016. Co-infections with viruses such as Porcine Circovirus 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) pose serious risks to swine health. Thus, there is an urgent need for rapid, sensitive, and specific detection methods suitable for use in field settings or laboratories with limited resources. Methods: We developed a CRISPR/Cas12a-based assay combined with recombinase polymerase amplification (RPA) for the rapid detection of PPV7. Specific RPA primers and five CRISPR RNAs (crRNAs) were designed to target a highly conserved region within the NS1 gene of PPV7. Optimization of crRNA and single-stranded DNA (ssDNA) concentrations was performed to enhance the assay's performance. Results: CrRNA optimization identified crRNA-05 as the optimal candidate for Cas12a-based detection of PPV7, as all synthesized crRNAs demonstrated similar performance. The optimal crRNA concentration was determined to be 200 nM, yielding consistent results across tested concentrations. For ssDNA optimization, the strongest fluorescence signal was achieved with 500 nM of the FAM-BHQ ssDNA receptor. The assay showed a minimal detection limit of 100copies/μl for PPV7, confirmed through fluorescence and lateral flow detection methods. Specificity testing indicated that only PPV7 DNA samples returned positive results, confirming the assay's accuracy. In tests of 50 lung tissue samples from diseased pigs, the RPA-Cas12a assay identified 29 positive samples (58%), surpassing the 22 positive samples (44%) detected by conventional PCR. This highlights the RPA-Cas12a method's enhanced detection capability and its potential utility in clinical surveillance and management of PPV7 in swine populations. Discussion: The RPA-Cas12a assay effectively detects PPV7 in clinical samples, enhancing disease surveillance and control in pigs. Its adaptability to resourcelimited settings significantly improves PPV7 management and prevention strategies, thereby supporting the overall health and development of the pig industry. [ABSTRACT FROM AUTHOR]

Cyclic nucleic acids are biologically stable against nucleic acid exonucleases due to the absence of 5′ and 3′ termini. Studies of cyclic nucleic acids mainly focus on cyclic single-stranded nucleic acids. Cyclic single-stranded nucleic acids are further divided into circular RNA (circRNA) and circular single-stranded DNA (cssDNA). The synthesis methods of circRNA include lasso-driven cyclization, intron-paired cyclization, intron cyclization, intron complementary pairing-driven cyclization, RNA-binding protein-driven cyclization, and artificial synthesis depending on the source. Its main role is to participate in gene expression and the treatment of some diseases. Circular single-stranded DNA is mainly synthesized by chemical ligation, template-directed enzyme ligation, and new techniques for the efficient preparation of DNA single loops and topologies based on CircLigase. It is mainly used in rolling circle amplification (RCA) technology and in the bioprotection of circular aptamers and second messengers. This review focuses on the types, synthesis methods, and applications of cyclic single-stranded nucleic acids, providing a reference for further research on cyclic single-stranded nucleic acids. • Circular single-stranded nucleic acids can be divided into circular single-stranded DNA (cssDNA) and circular RNA (circRNA). • Using circRNA is a new intervention of PKR related autoimmune diseases. • Natural circRNA can act as a miRNA sponge to regulate its activity. • Golden Gate assembly can generate user-defined cssDNA. • CssDNA can be used as a biosensor to detect Marine toxicity. [ABSTRACT FROM AUTHOR]

Non-enzymatic spontaneous deamination of 5-methylcytosine, producing thymine, is the proposed etiology of cancer mutational signature 1, which is the most predominant signature in all cancers. Here, the proposed mutational process was reconstituted using synthetic DNA and purified proteins. First, single-stranded DNA containing 5-methylcytosine at CpG context was incubated at an elevated temperature to accelerate spontaneous DNA damage. Then, the DNA was treated with uracil DNA glycosylase to remove uracil residues that were formed by deamination of cytosine. The resulting DNA was then used as a template for DNA synthesis by yeast DNA polymerase δ. The DNA products were analyzed by next-generation DNA sequencing, and mutation frequencies were quantified. The observed mutations after this process were exclusively C>T mutations at CpG context, which was very similar to signature 1. When 5-methylcytosine modification and uracil DNA glycosylase were both omitted, C>T mutations were produced on C residues in all sequence contexts, but these mutations were diminished by uracil DNA glycosylase-treatment. These results indicate that the CpG>TpG mutations were produced by the deamination of 5-methylcytosine. Additional mutations, mainly C>G, were introduced by yeast DNA polymerase ζ on the heat-damaged DNA, indicating that G residues of the templates were also damaged. However, the damage on G residues was not converted to mutations with DNA polymerase δ or ε. [ABSTRACT FROM AUTHOR]

A Ca2+ sensor based on DNA nanospheres was developed. The DNA nanospheres were obtained by complementary pairing of Y-shaped DNA and DNAzyme, in which Y-shaped DNA is a triple-branched DNA with three sticky ends formed by three partially complementary single-stranded DNAs. DNAzyme is a recently reported RNA-cleaving DNAzyme, EtNa, that has high selectivity for Ca2+ and was used as linker in DNA nanosphere preparation. When EtNa DNAzyme was formed by enzyme chain (E) and substrate chain (S), the fluorescence signal was quenched significantly due to the proximity of fluorophore and quencher. In the presence of Ca2+, it bound to the E strand of EtNa DNAzyme, catalyzing the cleavage of S strand, which led to the separation of S from E and then the DNA nanosphere was disintegrated. At the same time, the fluorescence signal was restored and Ca2+ assay was realized. This detection system based on DNA nanospheres determined Ca2+ across a linear range from 20 to 400 μM with a detection limit of 1.6 μM and excellent selectivity. In addition, this proposed Ca2+ assay has been successfully applied to the assay of Ca2+ in human serum, thus demonstrating clinical application. [ABSTRACT FROM AUTHOR]

In this study, we developed an isothermal fluorometric diagnostic method for DNA virus-generating disorders such as Mpox. Our results showed that the release of a large number of protons during multiplex-LAMP markedly lowered the pH level, which transformed the retinoblastoma (Rb) linear ssDNA into i-motifs. Consequently, thiazole orange (TO; a fluorometric probe sensitive to the i-motif) boosted the signal-on fluorescence because of its ability to bind selectively to i-motifs. This multiplex-LAMP/i-motif-TO system enabled simultaneous detection aimed at numerous potential targets with remarkable sensitivity (1.47 pg per mL) and efficiency (30 minutes). Our method is expected to enable DNA-virus-related diseases to be efficiently and accurately assessed. [ABSTRACT FROM AUTHOR]

The development of ultrasensitive and visual methods is of great significance for molecular diagnosis at the point-of-care. In this study, we have integrated recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system to design an ultrasensitive strategy for visual nucleic acid testing. RPA is utilized to amplify the target nucleic acid, producing amplicons that activate the single-stranded DNase property of CRISPR-Cas12a. The activated CRISPR-Cas12a then degrades the single-stranded DNA on magnetic nanoparticles (MNPs), releasing immobilized GOx from the MNPs which catalyses the chromogenic substrate. The developed method exhibits remarkable sensitivity, successfully detecting as low as 10 aM (∼6 copies per μL) of the target nucleic acid by visual colour changes in solution. The instrumental limit of detection is calculated to be 2.86 aM (∼2 copies per μL), comparable to the sensitivity of polymerase chain reaction (PCR). Importantly, this approach only requires isothermal incubation operation and does not involve costly instruments. The method has been validated by visually detecting the SARS-CoV-2 RNA gene fragment within 50 minutes. With its ultrasensitivity, simplicity of operation, and potential for integration into a point-of-care detection kit, this strategy holds great promise for nucleic acid testing in various settings. [ABSTRACT FROM AUTHOR]

Neurotransmitters are crucial for the proper functioning of neural systems, with dopamine playing a pivotal role in cognition, emotions, and motor control. Dysregulated dopamine levels are linked to various disorders, underscoring the need for accurate detection in research and diagnostics. Single-stranded DNA (ssDNA) aptamers are promising bioreceptors for dopamine detection due to their selectivity, improved stability, and synthesis feasibility. However, discrepancies in dopamine specificity have presented challenges. Here, we surface-functionalized a nano-plasmonic biosensing platform with a dopamine-specific ssDNA aptamer for selective detection. The biosensor, featuring narrowband hybrid plasmonic resonances, achieves high specificity through functionalization with aptamers and passivation processes. Sensitivity and selectivity for dopamine detection are demonstrated across a wide range of concentrations, including in diverse biological samples like protein solutions, cerebrospinal fluid, and whole blood. These results highlight the potential of plasmonic "aptasensors" for developing rapid and accurate diagnostic tools for disease monitoring, medical diagnostics, and targeted therapies. [ABSTRACT FROM AUTHOR]

Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi− assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression. [ABSTRACT FROM AUTHOR]

This article discusses the development of a rapid and portable method for detecting Epstein-Barr virus (EBV) DNA using enzymatic recombinase amplification (ERA) combined with the CRISPR-Cas12a system. EBV DNA detection is important for screening and monitoring diseases related to EBV infection, such as nasopharyngeal carcinoma. The study optimized the ERA and CRISPR-Cas12a systems to improve sensitivity and specificity. The integrated ERA-Cas12a system was able to detect EBV as low as 20 copies/µL with high specificity. This method offers a practical solution for the detection of EBV infections and may have clinical applicability for screening and diagnosing related diseases. [Extracted from the article]

DNA damage occurs when cells encounter exogenous and endogenous stresses such as long periods of desiccation, ionizing radiation and genotoxic chemicals. Efforts have been made to detect DNA damage in vivo and in vitro to characterize or quantify the damage level. It is well accepted that single‐stranded DNA (ssDNA) is one of the important byproducts of DNA damage to trigger the downstream regulation. A recent study has revealed that PprI efficiently recognizes ssDNA and cleaves DdrO at a specific site on the cleavage site region (CSR) loop in the presence of ssDNA, which enables the radiation resistance of Deinococcus. Leveraging this property, we developed a quantitative DNA damage detection method in vitro based on fluorescence resonance energy transfer (FRET). DdrO protein was fused with eYFP and eCFP on the N‐terminal and C‐terminal respectively, between which the FRET efficiency serves as an indicator of cleavage efficiency as well as the concentration of ssDNA. The standard curve between the concentration of ssDNA and the FRET efficiency was constructed, and application examples were tested, validating the effectiveness of this method. [ABSTRACT FROM AUTHOR]

Atherosclerosis cardiovascular disease (ASCVD) has become one of the leading death causes in humans. Low-density lipoprotein (LDL) is an important biomarker for assessing ASCVD risk level. Thus, monitoring LDL levels can be an important means for early diagnosis of ASCVD. Herein, a novel electrochemical aptasensor for determination LDL was designed based on nitrogen-doped reduced graphene oxide-hemin-manganese oxide nanoparticles (NrGO-H-Mn3O4 NPs) integrated with clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR/Cas12a) system. NrGO-H-Mn3O4 NPs not only have a large surface area and remarkable enhanced electrical conductivity but also the interconversion of different valence states of iron in hemin can provide an electrical signal. Nonspecific single-stranded DNA (ssDNA) was bound to NrGO-H-Mn3O4 NPs to form a signaling probe and was immobilized on the electrode surface. The CRISPR/Cas12a system has excellent trans-cleavage activity, which can be used to cleave ssDNA, thus detaching the NrGO-H-Mn3O4 NPs from the sensing interface and attenuating the electrical signal. Significant signal change triggered by the target was ultimately obtained, thus achieving sensitive detection of the LDL in range from 0.005 to 1000.0 nM with the detection limit of 0.005 nM. The proposed sensor exhibited good stability, selectivity, and stability and achieved reliable detection of LDL in serum samples, demonstrating its promising application prospects for the diagnostic application of LDL. [ABSTRACT FROM AUTHOR]

Abstract Parvoviruses have evolved to possess a linear single-stranded DNA (ssDNA) genome ranging from 4 to 6.3 kb. Adeno-associated virus (AAV), a member of the Parvoviridae family, contains approximately 5 kb of linear ssDNA within its capsid. This ssDNA features two 145-base inverted terminal repeats (ITRs) positioned at each end. ITRs have a T-shaped hairpin secondary structure, which plays a crucial role in viral replication. To investigate the impact of ITRs on ssDNA stability, we conducted a DNA denaturation-reannealing assay in 10 mM magnesium acetate, 50 mM potassium acetate, and 20 mM Tris-acetate buffer at pH7.9. Conventional double-stranded DNA (dsDNA) fragments retain a reannealing capability of over 50% for sizes under 8.8 kb, gradually losing this capability as sizes increase; however, dsDNA fragments in rAAV ranging from 0.7 to 6.3 kb did not exhibit a reannealing profile. This suggests that the presence of ITRs at both ends hinders annealing between the complementary strands. These results indicate that ITR structures preferentially induce an ssDNA conformation less than 6.3 kb in size, and that the stability of AAV ssDNA contributes to the viral life cycle, including processes such as infection, replication, and packaging. Considering the size of parvovirus genomes, it appears that their genomes require reversible flexibility in complementary DNA strands; simultaneously, this adaptability needs to be regulated by specific palindromic ITRs at both ends.

A single-stranded product scaffold obtained by RCA reaction is woven into an origami-like nano-fence-shaped product (FP) microarray superstructure by only one short bridge strand, circumventing the requirement for a very few viral genome scaffold. Using FP microarray as a template, the two-dimensional protein or nanoparticle patterns can be assembled in a precise and programmable manner, exhibiting the potential applications such as biomedical engineering, therapy, nanophotonics and electronics. [Display omitted] The universal programmed construction of patterned periodic self-assembled nanostructures is a technical challenge in DNA origami nanotechnology but has numerous potential applications in biotechnology and biomedicine. In order to circumvent the dilemma that traditional DNA origami requires a long unusual single-stranded virus DNA as the scaffold and hundreds or even thousands of short strands as staples, we report a method for constructing periodically-self-folded rolling circle amplification products (RPs). The repeating unit is designed to have 3 intra-unit duplexes (inDP1,2,3) and 2 between-unit duplexes (buDP1,2). Based on the complementary pairing of bases, RPs each can self-fold into a periodic grid-patterned ribbon (GR) without the help of any auxiliary oligonucleotide staple. Moreover, by using only an oligonucleotide bridge strand, the GRs are connected together into the larger and denser planar nano-fence-shaped product (FP), which substantially reduces the number of DNA components compared with DNA origami and eliminates the obstacles in the practical application of DNA nanostructures. More interestingly, the FP-based DNA framework can be easily functionalized to offer spatial addressability for the precise positioning of nanoparticles and guest proteins with high spatial resolution, providing a new avenue for the future application of DNA assembled framework nanostructures in biology, material science, nanomedicine and computer science that often requires the ordered organization of functional moieties with nanometer-level and even molecular-level precision. [ABSTRACT FROM AUTHOR]

PURPOSE: Neuroblastoma is the most common extracranial solid tumor in childhood. We previously showed that circulating cell-free DNA (cfDNA) and tumor biopsy derived 5-hydroxymethylcytosime (5-hmC) profiles identified patients with neuroblastoma who experienced subsequent relapse. Here, we hypothesized that 5-hmC modifications selectively enriched in cfDNA compared with tumor biopsy samples would identify epigenetic changes associated with aggressive tumor behavior and identify novel biomarkers of outcome in patients with high-risk neuroblastoma. METHODS: 5-hmC profiles from cfDNA (n = 64) and tumor biopsies (n = 48) were compared. Two neuroblastoma cell lines underwent chromatin immunoprecipitation followed by sequencing (ChIP-Seq) for H3K27me3, H3K4me3, and H3K27ac; kethoxal-associated single-stranded DNA sequencing; hmC-Seal for 5-hmC; and RNA-sequencing (RNA-Seq). Genes enriched for both H3K27me3 and H3K4me3 in the included cell lines were defined as bivalent. Using bivalent genes defined in vitro, a bivalent signature was established in three publicly available cohorts of patients with neuroblastoma through gene set variation analysis. Differences between tumors with high or low bivalent signatures were assessed by the Kaplan-Meier method and Cox proportional hazards models. RESULTS: In cfDNA compared with tumor biopsy derived 5-hmC profiles, we found increased 5-hmC deposition on Polycomb Repressive Complex 2 target genes, a finding previously described in the context of bivalent genes. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogeneous neuroblastoma cell lines. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in patients with high-risk neuroblastoma. CONCLUSION: Low expression of bivalent genes is a biomarker of worse outcome in patients with high-risk neuroblastoma. @mohansrinc1 et al develop a bivalent gene signature predictive of outcome in HR neuroblastoma [ABSTRACT FROM AUTHOR]

Two sequences of DNA (single-strand DNA (ssDNA) and double-strand DNA (dsDNA)) modified disposable electrodes were designed to investigate the effect of antitumor agent drug idarubicine (IDA) on the DNA chain structure. The effect of IDA on DNA structure was analyzed by square wave voltammetry (SWV) depending on not only the guanine signal but also IDA oxidation response. The present study included the electrochemical investigation of IDA and the investigation of the biomolecular interaction between IDA and DNA. IDA was detected in buffer and urine using disposable pencil graphite electrodes (PGE) with SWV and cyclic voltammetry (CV)). The detection limit (LOD) of IDA in urine samples was 0.089 µg mL−1 with SWV under optimum conditions. For the biomolecular interaction of IDA and DNA, all electrochemical conditions, such as the concentration of DNA, concentration of IDA, interaction phase (at electrode surface/in solution phase), the interaction pH, and the interaction time were optimized. IDA interacted biomolecularly with DNA at the electrode surface and in solution phase using two methods. There is no previous electrochemical study performed on the interaction of IDA with ssDNA. Hence, the effect of IDA on both ssDNA and dsDNA was presented for the first time. [ABSTRACT FROM AUTHOR]

Gyrovirus (GyV) is small, single-stranded circular DNA viruses that has recently been assigned to the family Anelloviridae. In the last decade, many GyVs that have an apparent pan-tropism at the host level were identified by high-throughput sequencing (HTS) technology. As of now, they have achieved global distribution. Several species of GyVs have been demonstrated to be pathogenic to poultry, particularly chicken anemia virus (CAV), causing significant economic losses to the global poultry industry. Although GyVs are highly prevalent in various birds worldwide, their direct involvement in the etiology of specific diseases and the reasons for their ubiquity and host diversity are not fully understood. This review summarizes current knowledge about GyVs, with a major emphasis on their morphofunctional properties, epidemiological characteristics, genetic evolution, pathogenicity, and immunopathogenesis. Additionally, the association between GyVs and various diseases, as well as its potential impact on the poultry industry, have been discussed. Future prevention and control strategies have also been explored. These insights underscore the importance of conducting research to establish a virus culture system, optimize surveillance, and develop vaccines for GyVs. [ABSTRACT FROM AUTHOR]

The ATR kinase responds to elevated levels of single-stranded DNA (ssDNA) to activate the G2/M checkpoint, regulate origin utilization, preserve fork stability, and allow DNA repair to ensure genome integrity. The intrinsic replication stress in cancer cells makes this pathway an attractive therapeutic target. The ssDNA that drives ATR signaling is sensed by the ssDNA-binding protein replication protein A (RPA), which acts as a platform for ATRIP recruitment and subsequent ATR activation by TopBP1. We have developed chemical RPA inhibitors (RPAi) that block RPA-ssDNA interactions (RPA-DBi) and RPA protein–protein interactions (RPA-PPIi); both activities are required for ATR activation. Here, we biochemically reconstitute the ATR kinase signaling pathway and demonstrate that RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of target proteins with selectivity advantages over active site ATR inhibitors. We demonstrate that RPA post-translational modifications (PTMs) impact ATR kinase activation but do not alter sensitivity to RPAi. Specifically, phosphorylation of RPA32 and TopBP1 stimulate, while RPA70 acetylation does not affect ATR phosphorylation of target proteins. Collectively, this work reveals the RPAi mechanism of action to inhibit ATR signaling that can be regulated by RPA PTMs and offers insight into the anti-cancer activity of ATR pathway-targeted cancer therapeutics. [ABSTRACT FROM AUTHOR]

Chemical synthesis of phosphoromonothioate oligonucleotides (PS-ONs) is not stereo-specific and produces a mixture of Rp and Sp diastereomers, whose disparate reactivity can complicate applications. Although the current methods to separate these diastereomers which rely on chromatography are constantly improving, many Rp and Sp diastereomers are still co-eluted. Here, based on sulfur-binding domains that specifically recognize phosphorothioated DNA and RNA in Rp configuration, we developed a universal separation system for phosphorothioate oligonucleotide isomers using immobilized SBD (SPOIS). With the scalable SPOIS, His-tagged SBD is immobilized onto Ni-nitrilotriacetic acid-coated magnetic beads to form a beads/SBD complex, Rp isomers of the mixture can be completely bound by SBD and separated from Sp isomers unbound in liquid phase, then recovered through suitable elution approach. Using the phosphoromonothioate single-stranded DNA as a model, SPOIS separated PS-ON diastereomers of 4 nt to 50 nt in length at yields of 60–90% of the starting Rp isomers, with PS linkage not locating at 5' or 3' end. Within this length range, PS-ON diastereomers that co-eluted in HPLC could be separated by SPOIS at yields of 84% and 89% for Rp and Sp stereoisomers, respectively. Furthermore, as each Rp phosphorothioate linkage can be bound by SBD, SPOIS allowed the separation of stereoisomers with multiple uniform Sp configurations for multiple phosphorothioate modifications. A second generation of SPOIS was developed using the thermolabile and non-sequence-specific SBDPed, enabling fast and high-yield recovery of PS substrate stereoisomers for the DNAzyme Cd16 and further demonstrating the efficiency of this method. Key points: • SPOIS allows isomer separations of the Rp and Sp isomers co-eluted on HPLC. • SPOIS can obtain Sp isomers with 5 min and Rp in 20 min from PS-ON diastereomers. • SPOIS was successfully applied to separate isomers of PS substrates of DNAzyme. [ABSTRACT FROM AUTHOR]

Here, a separation‐free and label‐free portable aptasensor is developed for rapid and sensitive analysis of tumor‐derived exosomes (TEXs). It integrated a parallel rolling circle amplification (RCA) reaction, selective binding of metal ions or small molecules to nucleic acid‐specific conformations, and a low‐cost, highly sensitive handheld fluorometer. Lung cancer, for example, is targeted with two typical biomarkers (mucin 1 and programmed cell death ligand 1 (PD‐L1)) on its exosomes. The affinity of aptamers to the targets modulated the amount of RCA products (T‐Hg2+‐T and cytosine (C)‐rich single‐stranded DNA), which in turn affected the fluorescence intensity of quantum dots (QDs) and methylene blue (MB). The results revealed that the limit of detection (LOD) of the handheld fluorometer for cell‐derived exosomes can be as low as 30 particles mL−1. Moreover, its specificity, sensitivity, and area under the curve (AUC) are 93% (14/15), 92% (23/25), and 0.956, as determined by the analysis of 40 clinical samples. Retesting 16 of these samples with the handheld fluorometer yielded strong concordance between the fluorometer results and those acquired from clinical computed tomography (CT) and pathology. [ABSTRACT FROM AUTHOR]

Single‐walled carbon nanotubes (SWCNTs) have gained significant interest for their potential in biomedicine and nanoelectronics. The functionalization of SWCNTs with single‐stranded DNA (ssDNA) enables the precise control of SWCNT alignment and the development of optical and electronic biosensors. This study addresses the current gaps in the field by employing high‐throughput systematic selection, enriching high‐affinity ssDNA sequences from a vast random library. Specific base compositions and patterns are identified that govern the binding affinity between ssDNA and SWCNTs. Molecular dynamics simulations validate the stability of ssDNA conformations on SWCNTs and reveal the pivotal role of hydrogen bonds in this interaction. Additionally, it is demonstrated that machine learning could accurately distinguish high‐affinity ssDNA sequences, providing an accessible model on a dedicated webpage (http://service.k‐medai.com/ssdna4cnt). These findings open new avenues for high‐affinity ssDNA‐SWCNT constructs for stable and sensitive molecular detection across diverse scientific disciplines. [ABSTRACT FROM AUTHOR]

Plant pathogens are causing substantial economic losses and thus became a significant threat to global agriculture. Effective and timely detection methods are prerequisite for combating the damages caused by the plant pathogens. In the realm of plant pathogen detection, the isothermal amplification techniques, e.g., recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), have emerged as a fast, precise, and most sensitive alternative to conventional PCR but they often comprise high rates of non-specific amplification and operational complexity. In recent advancements, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems, particularly Cas12, have emerged as powerful tools for highly sensitive, specific, and rapid pathogen detection. Exploiting the collateral activities of Cas12, which selectively cleaves single-stranded DNA (ssDNA), novel detection platforms have been developed. The mechanism employs the formation of a triple complex molecule comprising guide RNA, Cas12 enzyme, and the substrate target nucleotide sequence. Upon recognition of the target, Cas12 indiscriminately cleaves the DNA strand, leading to the release of fluorescence from the cleaved ssDNA reporter. Integration of isothermal amplification methods with CRISPR/Cas12 enables one-step detection assays, facilitating rapid pathogen identification within 30 min at a single temperature. This integrated RPA-CRISPR/Cas12a approach eliminates the need for RNA extraction and cDNA conversion, allowing direct use of crude plant sap as a template. With an affordable fluorescence visualization system, this portable method achieves 100-fold greater sensitivity than conventional techniques. This review summarizes recent advances in RPA-CRISPR/Cas12a for detecting plant pathogens, covering primer design, field-level portability, and enhanced sensitivity. [ABSTRACT FROM AUTHOR]

The FRG1(FSHD region gene 1) gene has emerged as a pivotal tumor suppressor in both breast and prostate cancer. HPF1 (Histone PARylation Factor 1), a gene crucial in the base excision repair (BER) mechanism for single-stranded DNA (ssDNA) lesions, showcases a robust correlation with FRG1. This implies that FRG1 might have the capacity to influence BER via HPF1, potentially playing a role in tumorigenesis. Using a comprehensive approach that integrates in-silico analyses involving differential gene expression, KEGG (Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology), and STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) databases, we unravelled the intricate network of genes and pathways influenced by FRG1, which includes BER. Our linear regression analysis unveiled a positive relationship between FRG1 and key genes crucial for BER. Notably, breast cancer patients with low FRG1 expression exhibited a significantly higher frequency of mutation in TP53. To enhance the accuracy of our analysis, we conducted qRT-PCR assays, which demonstrated that FRG1 affects the transcription of DNA base excision repair genes, showing differential expression in breast cancer cells. Moreover, through the Alkaline Comet Assay, a technique that quantifies DNA damage at the single-cell level, we observed diminished DNA repair capabilities when FRG1 levels are low. Risk scores were calculated using the Cox regression coefficients, and we found notable differences in Overall Survival (OS) and mRNA expression of DEGs in the low and high-risk groups. In summary, our findings shed light on the pivotal role of FRG1 in maintaining DNA repair efficiency within breast cancer cells. [ABSTRACT FROM AUTHOR]

Homologous recombination (HR) is essential for the maintenance of genome stability. During HR, Replication Protein A (RPA) rapidly coats the 3'-tailed single-strand DNA (ssDNA) generated by end resection. Then, the ssDNA-bound RPA must be timely replaced by Rad51 recombinase to form Rad51 nucleoprotein filaments that drive homology search and HR repair. How cells regulate Rad51 assembly dynamics and coordinate RPA and Rad51 actions to ensure proper HR remains poorly understood. Here, we identified that Rtt105, a Ty1 transposon regulator, acts to stimulate Rad51 assembly and orchestrate RPA and Rad51 actions during HR. We found that Rtt105 interacts with Rad51 in vitro and in vivo and restrains the adenosine 5' triphosphate (ATP) hydrolysis activity of Rad51. We showed that Rtt105 directly stimulates dynamic Rad51-ssDNA assembly, strand exchange, and D-loop formation in vitro. Notably, we found that Rtt105 physically regulates the binding of Rad51 and RPA to ssDNA via different motifs and that both regulations are necessary and epistatic in promoting Rad51 nucleation, strand exchange, and HR repair. Consequently, disrupting either of the interactions impaired HR and conferred DNA damage sensitivity, underscoring the importance of Rtt105 in orchestrating the actions of Rad51 and RPA. Our work reveals additional layers of mechanisms regulating Rad51 filament dynamics and the coordination of HR. [ABSTRACT FROM AUTHOR]

Templated DNA repair that occurs during homologous recombination and replication stress relies on RAD51. RAD51 activity is positively regulated by BRCA2 and the RAD51 paralogs. The Shu complex is a RAD51 paralog-containing complex consisting of SWSAP1, SWS1, and SPIDR. We demonstrate that SWSAP1-SWS1 binds RAD51, maintains RAD51 filament stability, and enables strand exchange. Using single-molecule confocal fluorescence microscopy combined with optical tweezers, we show that SWSAP1-SWS1 decorates RAD51 filaments proficient for homologous recombination. We also find SWSAP1-SWS1 enhances RPA diffusion on ssDNA. Importantly, we show human sgSWSAP1 and sgSWS1 knockout cells are sensitive to pharmacological inhibition of PARP and APE1. Lastly, we identify cancer variants in SWSAP1 that alter Shu complex formation. Together, we show that SWSAP1-SWS1 stimulates RAD51-dependent high-fidelity repair and may be an important new cancer therapeutic target. The human Shu complex promotes homologous recombination by regulating RAD51. Here the authors reveal that the Shu complex proteins, SWSAP1-SWS1, decorate the RAD51 filament on ssDNA and facilitate its strand exchange reaction by stimulating RPA diffusion on ssDNA. Lastly, that SWSAP1-SWS1 knockouts are Olaparib sensitive. [ABSTRACT FROM AUTHOR]

DSS1, essential for BRCA2-RAD51 dependent homologous recombination (HR), associates with the helical domain (HD) and OB fold 1 (OB1) of the BRCA2 DSS1/DNA-binding domain (DBD) which is frequently targeted by cancer-associated pathogenic variants. Herein, we reveal robust ss/dsDNA binding abilities in HD-OB1 subdomains and find that DSS1 shuts down HD-OB1's DNA binding to enable ssDNA targeting of the BRCA2-RAD51 complex. We show that C-terminal helix mutations of DSS1, including the cancer-associated R57Q mutation, disrupt this DSS1 regulation and permit dsDNA binding of HD-OB1/BRCA2-DBD. Importantly, these DSS1 mutations impair BRCA2/RAD51 ssDNA loading and focus formation and cause decreased HR efficiency, destabilization of stalled forks and R-loop accumulation, and hypersensitize cells to DNA-damaging agents. We propose that DSS1 restrains the intrinsic dsDNA binding of BRCA2-DBD to ensure BRCA2/RAD51 targeting to ssDNA, thereby promoting optimal execution of HR, and potentially replication fork protection and R-loop suppression. DSS1 is crucial for BRCA2 dependent genome repair. Here, the authors reveal how DSS1 restricts the dsDNA binding ability of BRCA2 to ensure BRCA2/RAD51 loading on ssDNA. Mutations in DSS1 disrupt this regulation, leading to impaired DNA repair, replication fork protection and R-loop suppression. [ABSTRACT FROM AUTHOR]