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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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

During meiosis, nucleoprotein filaments of the strand exchange proteins RAD51 and DMC1 are crucial for repairing SPO11-generated DNA double-strand breaks (DSBs) by homologous recombination (HR). A balanced activity of positive and negative RAD51/DMC1 regulators ensures proper recombination. Fidgetin-like 1 (FIGNL1) was previously shown to negatively regulate RAD51 in human cells. However, FIGNL1's role during meiotic recombination in mammals remains unknown. Here, we decipher the meiotic functions of FIGNL1 and FIGNL1 Interacting Regulator of Recombination and Mitosis (FIRRM) using male germline-specific conditional knock-out (cKO) mouse models. Both FIGNL1 and FIRRM are required for completing meiotic prophase in mouse spermatocytes. Despite efficient recruitment of DMC1 on ssDNA at meiotic DSB hotspots, the formation of late recombination intermediates is defective in Firrm cKO and Fignl1 cKO spermatocytes. Moreover, the FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independently from the formation of SPO11-catalyzed DSBs. Purified human FIGNL1ΔN alters the RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro. Thus, this complex might regulate RAD51 and DMC1 association at sites of meiotic DSBs to promote proficient strand invasion and processing of recombination intermediates. Successful meiotic homologous recombination relies on the tight regulation of RAD51 and DMC1 strand exchange proteins. This study shows that the conserved FIGNL1-FIRRM complex plays a critical role for fertility by restraining uncontrolled assembly of RAD51 and DMC1 filament during meiosis. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas12a system is emerging as a promising candidate for next-generation diagnostic biosensing platforms, with the discovery of new activation modes greatly expanding its applications. Here, we have identified two novel CRISPR/Cas12a system activation modes: PAM- and toehold-free DNA hairpins, and DNA-RNA hybrid strands. Utilizing a well-established real-time fluorescence method, we have demonstrated a strong correlation between DNA hairpin structures and Cas12a activation. Compared with previously reported activation modes involving single-stranded DNA and PAM-contained double-stranded DNA, the DNA hairpin activation way exhibits similar specificity and generality. Moreover, our findings indicate that increasing the number of RNA bases in DNA-RNA hybrid strands can decelerate the kinetics of Cas12a-triggered trans -cleavage of reporter probes. These newly discovered CRISPR/Cas12a activation ways hold significant potential for the development of high-performance biosensing strategies. • Two new CRISPR/Cas12a system activation modes were discovered. • The DNA hairpin activation way has better specificity and generality. • The increasing number of RNA bases in hybrid strands could slow the kinetics of Cas12a-triggered trans- cleavage. [ABSTRACT FROM AUTHOR]

Anti-cancer therapy is crucial in cancer prevention and anti-cancer, and thus, highly sensitive methods for detecting cancer biomarkers are essential for cancer early diagnosis. Herein, an electrochemical aptamer biosensor based on the CRISPR-Cas12a system was constructed for the detection of cancer tumor biomarker MUC1 mucin. The sensitivity was significantly prompted by enzyme-catalyzed signal amplification, and the selectivity was improved by the dual recognition of the aptamer to MUC1 and crRNA-Cas12a system to the aptamer. Glucose oxidase (GOD) was loaded on the surface of magnetic Fe3O4@Au (MGNP) via probe single-stranded DNA (pDNA) with the terminal modification of mercapto (–SH) to form GOD-pDNA/MGNP. The corresponding aptamer of MUC1 (MUC1 Apt) binds to its complementary ssDNA (cDNA) to form the activator Apt/cDNA, which is specifically recognized by crRNA-Cas12a and excites the trans-cleavage function of Cas12a, thus in turn trans-cleaves pDNA and detaches GOD from the magnetic particles. The magnetic beads were separated and transferred into a glucose solution, and the oxidation current of H2O2 produced by the catalytic reaction of GOD was measured on a Pt-modified magnetically-controlled glassy carbon electrode, resulting in an indirect determination of MUC1. The current change was linear with the logarithm of MUC1 concentration in the range from 1.0 × 10−17 g mL−1 to 1.0 × 10−10 g mL−1. The detection limit was as low as 7.01 × 10−18 g mL−1. The method was applied for the detection of MUC1 in medical samples. [ABSTRACT FROM AUTHOR]

The efficient preparation of single-stranded DNA (ssDNA) rings, as a macromolecular construction approach with topological features, has aroused much interest due to the ssDNA rings' numerous applications in biotechnology and DNA nanotechnology. However, an extra splint is essential for enzymatic circularization, and by-products of multimers are usually present at high concentrations. Here, we proposed a simple and robust strategy using permuted precursor (linear ssDNA) for circularization by forming an intramolecular dynamic nick using a part of the linear ssDNA substrate itself as the template. After the simulation of the secondary structure for desired circular ssDNA, the linear ssDNA substrate is designed to have its ends on the duplex part (≥5 bp). By using this permuted substrate with 5′-phosphate, the splint-free circularization is simply carried out by T4 DNA ligase. Very interestingly, formation of only several base pairs (2–4) flanking the nick is enough for ligation, although they form only instantaneously under ligation conditions. More significantly, the 5-bp intramolecular duplex part commonly exists in genomes or functional DNA, demonstrating the high generality of our approach. Our findings are also helpful for understanding the mechanism of enzymatic DNA ligation from the viewpoint of substrate binding. [ABSTRACT FROM AUTHOR]

Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants—containing either substitution D395N or substitutions D395N+E456N—that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications. [ABSTRACT FROM AUTHOR]

A recent study showed that just one point mutation F33 to Y in the complementarity-determining region 1 of heavy chain (H-CDR1) could lead to the auto-antibody losing its DNA binding ability. However, the potential molecular mechanisms have not been well elucidated. In this study, we investigated how the antibody lost the DNA binding ability caused by mutation F33 to Y in the H-CDR1. We found that the electrostatic force was not the primary driving force for the interaction between anti-DNA antibodies and the antigen single strand DNA (ssDNA), and that the H-CDR2 largely contributed to the binding of antigen ssDNA, even larger than H-CDR1. The H-F33Y mutation could increase the hydrogen-bond interaction but impair the pi-pi stacking interaction between the antibody and ssDNA. We further found that F33H, W98H and Y95L in the wiletype antibody could form the stable pi-pi stacking interaction with the nucleotide bases of ssDNA. However, the Y33 in mutant could not form the parallel sandwich pi-pi stacking interaction with the ssDNA. To further confirm the importance of pi-pi stacking, the wildtype antibody and the mutants (F33YH, F33AH, W98AH and Y95AL) were experimentally expressed in CHO cells and purified, and the results from ELISA clearly showed that all the mutants lost the ssDNA binding ability. Taken together, our findings may not only deepen the understanding of the underlying interaction mechanism between autoantibody and antigen, but also broad implications in the field of antibody engineer. [ABSTRACT FROM AUTHOR]

Bufaviruses (BuV) are members of the Parvoviridae of the Protoparvovirus genus. They are non-enveloped, T = 1 icosahedral ssDNA viruses isolated from patients exhibiting acute diarrhea. The lack of treatment options and a limited understanding of their disease mechanisms require studying these viruses on a molecular and structural level. In the present study, we utilize glycan arrays and cell binding assays to demonstrate that BuV1 capsid binds terminal sialic acid (SIA) glycans. Furthermore, using cryo-electron microscopy (cryo-EM), SIA is shown to bind on the 2/5-fold wall of the capsid surface. Interestingly, the capsid residues stabilizing SIA binding are conserved in all human BuVs identified to date. Additionally, biophysical assays illustrate BuV1 capsid stabilization during endo–lysosomal (pH 7.4–pH 4) trafficking and capsid destabilization at pH 3 and less, which correspond to the pH of the stomach. Hence, we determined the cryo-EM structures of BuV1 capsids at pH 7.4, 4.0, and 2.6 to 2.8 Å, 3.2 Å, and 2.7 Å, respectively. These structures reveal capsid structural rearrangements during endo–lysosomal escape and provide a potential mechanism for this process. The structural insights gained from this study will add to the general knowledge of human pathogenic parvoviruses. Furthermore, the identification of the conserved SIA receptor binding site among BuVs provides a possible targetable surface-accessible pocket for the design of small molecules to be developed as anti-virals for these viruses. [ABSTRACT FROM AUTHOR]

Cancer is most frequently treated with antineoplastic agents (ANAs) that are hazardous to patients undergoing chemotherapy and the healthcare workers who handle ANAs in the course of their duties. All aspects related to hazardous oncological drugs illustrate that the monitoring of ANAs is essential to minimize the risks associated with these drugs. Among all analytical techniques used to test ANAs, electrochemistry holds an important position. This review, for the first time, comprehensively describes the progress done in electrochemistry of ANAs by means of a variety of bare or modified (bio)sensors over the last four decades (in the period of 1982–2021). Attention is paid not only to the development of electrochemical sensing protocols of ANAs in various biological, environmental, and pharmaceutical matrices but also to achievements of electrochemical techniques in the examination of the interactions of ANAs with deoxyribonucleic acid (DNA), carcinogenic cells, biomimetic membranes, peptides, and enzymes. Other aspects, including the enantiopurity studies, differentiation between single-stranded and double-stranded DNA without using any label or tag, studies on ANAs degradation, and their pharmacokinetics, by means of electrochemical techniques are also commented. Finally, concluding remarks that underline the existence of a significant niche for the basic electrochemical research that should be filled in the future are presented. [ABSTRACT FROM AUTHOR]

[Display omitted] The dysregulation of microRNA (miRNA) expression levels is intricately linked to a myriad of human diseases, and the precise and delicate detection thereof holds paramount significance in the realm of clinical diagnosis and therapy. Herein, a near-infrared (NIR) light-mediated homogeneous photoelectrochemical (PEC) biosensor was constructed for miRNA-155 detection based on NaYF 4 : Yb, Tm@ZnIn 2 S 4 (NYF@ ZIS) coupled with a three-dimensional (3D) walking nanomotor-assisted CRISPR/Cas12a strategy. The upconverted light emitted by the NYF in the visible and UV region upon NIR light excitation could be utilized to excite ZIS to produce a photocurrent response. The presence of target miRNA-155 initiated an amplification reaction within the 3D walking nanomotor, resulting in the production of multiple nucleic acid fragments. These fragments could activate the collateral cleavage capability of CRISPR/Cas12a, leading to the indiscriminate cleavage of single-stranded DNA (ssDNA) on ALP-ssDNA-modified magnetic beads and the subsequent liberation of alkaline phosphatase (ALP). The released ALP facilitated the catalysis of ascorbic acid 2-phosphate to generate ascorbic acid as the electron donor to capture the photogenerated holes on the NYF@ZIS surface, resulting in a positively correlated alteration in the photocurrent response. Under optimal conditions, the NIR light-initiated homogeneous PEC biosensor had the merits of good linear range (0.1 fM to 100 pM), an acceptable limit of detection (65.77 aM) for miRNA-155 detection. Considering the pronounced sensitivity, light stability, and low photodamage, this strategy presents a promising platform for detecting various other miRNA biomarkers in molecular diagnostic practice. [ABSTRACT FROM AUTHOR]

DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors. The authors identify a DNA-protein crosslink (DPC) repair pathway orchestrated by poly-ADP-ribosylation. In this process, PARP1 PARylates the DPC, marking it for removal by proteolysis. Consequently, PARP1 facilitates the repair of DPCs located next to DNA breaks, such as topoisomerase 1-DPCs. [ABSTRACT FROM AUTHOR]

Family Parvoviridae consists of small, non-enveloped viruses with linear, single-stranded DNA genomes of approximately 4-6 kilobases, subdivided into three subfamilies, Parvovirinae, Densovirinae, and Hamaparvovirinae, and unassigned genus Metalloincertoparvovirus. Parvoviruses of aquatic animals infect crustaceans, mollusks, and finfish. This review describes these parvoviruses, which are highly host-specific and associated with mass morbidity and mortality in both farmed and wild aquatic animals. They include Cherax quadricarinatus densovirus (CqDV) in freshwater crayfish in Queensland, Australia; sea star-associated densovirus (SSaDV) in sunflower sea star on the Northeastern Pacific Coast; Clinch densovirus 1 in freshwater mussels in the Clinch River, Virginia, and Tennessee, USA, in subfamily Densovirinae; hepatopancreatic parvovirus (HPV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) in farmed shrimp worldwide; Syngnathid ichthamaparvovirus 1 in gulf pipefish in the Gulf of Mexico and parts of South America; tilapia parvovirus (TiPV) in farmed tilapia in China, Thailand, and India, in the subfamily Hamaparvovirinae; and Penaeus monodon metallodensovirus (PmMDV) in Vietnamese P. monodon, in unassigned genus Metalloincertoparvovirus. Also included in the family Parvoviridae are novel parvoviruses detected in both diseased and healthy animals using metagenomic sequencing, such as zander parvovirus from zander in Hungary and salmon parvovirus from sockeye salmon smolts in British Columbia, Canada. [ABSTRACT FROM AUTHOR]

Transmembrane delivery of biologically active nucleic acids is an important process in cells and has inspired one to develop advanced drug delivery techniques. In this contribution, molecular‐level single‐stranded nucleic acid transmembrane carriers are reported based on 3.2 nm long Huc's foldamers (AOrnQ3Q3)8 and (mQ3Q2)8 with linearly and helically aligned positive charges, respectively. These two foldamers not only show very strong DNA affinity via electrostatic interactions but also discriminatively bind single‐stranded DNA (ss‐DNA) and double‐stranded DNA (ds‐DNA), corroborating the importance of precise charge arrangement in the electrostatic interactions. More importantly, these two foldamers are capable of efficiently transporting ss‐DNA across the lipid membranes, and the ss‐DNA transport activity of (AOrnQ3Q3)8 with linearly aligned charges is higher than that of (mQ3Q2)8 with helically aligned charges. Thus a type of novel single‐stranded nucleic acid transmembrane molecular carriers based on positively charged helical foldamers are introduced. Further, effective and enhanced expression in EGFP‐mRNA transfection experiments strongly demonstrates the potential of positively charged foldamers for RNA transmembrane transport and therapy. [ABSTRACT FROM AUTHOR]