Replication protein-A, RPA, plays a pivotal role in the maintenance of recombination checkpoint in yeast meiosis.

DNA double-strand breaks (DSBs) activate DNA damage responses (DDRs) in both mitotic and meiotic cells. A single-stranded DNA (ssDNA) binding protein, Replication protein-A (RPA) binds to the ssDNA formed at DSBs to activate ATR/Mec1 kinase for the response. Meiotic DSBs induce homologous recombination monitored by a meiotic DDR called the recombination checkpoint that blocks the pachytene exit in meiotic prophase I. In this study, we further characterized the essential role of RPA in the maintenance of the recombination checkpoint during Saccharomyces cerevisiae meiosis. The depletion of an RPA subunit, Rfa1, in a recombination-defective dmc1 mutant, fully alleviates the pachytene arrest with the persistent unrepaired DSBs. RPA depletion decreases the activity of a meiosis-specific CHK2 homolog, Mek1 kinase, which in turn activates the Ndt80 transcriptional regulator for pachytene exit. These support the idea that RPA is a sensor of ssDNAs for the activation of meiotic DDR. Rfa1 depletion also accelerates the prophase I delay in the zip1 mutant defective in both chromosome synapsis and the recombination, consistent with the notion that the accumulation of ssDNAs rather than defective synapsis triggers prophase I delay in the zip1 mutant.

The single strand template shortening strategy improves the sensitivity and specificity of solid-state nanopore detection.

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

Sequential requirements for distinct Polθ domains during theta-mediated end joining.

DNA polymerase θ (Polθ) plays a central role in a DNA double-strand break repair pathway termed theta-mediated end joining (TMEJ). TMEJ functions by pairing short-sequence "microhomologies" (MHs) in single-stranded DNA at each end of a break and subsequently initiating DNA synthesis. It is not known how the Polθ helicase domain (HD) and polymerase domain (PD) operate to bring together MHs and facilitate repair. To resolve these transient processes in real time, we utilized in vitro single-molecule FRET approaches and biochemical analyses. We find that the Polθ-HD mediates the initial capture of two ssDNA strands, bringing them in close proximity. The Polθ-PD binds and stabilizes pre-annealed MHs to form a synaptic complex (SC) and initiate repair synthesis. Individual synthesis reactions show that Polθ is inherently non-processive, accounting for complex mutational patterns during TMEJ. Binding of Polθ-PD to stem-loop-forming sequences can substantially limit synapsis, depending on the available dNTPs and sequence context.

Replication of [AT/TA]25 Microsatellite Sequences by Human DNA Polymerase δ Holoenzymes Is Dependent on dNTP and RPA Levels.

Fragile sites are unstable genomic regions that are prone to breakage during stressed DNA replication. Several common fragile sites (CFS) contain A+T-rich regions including perfect [AT/TA] microsatellite repeats that may collapse into hairpins when in single-stranded DNA (ssDNA) form and coincide with chromosomal hotspots for breakage and rearrangements. While many factors contribute to CFS instability, evidence exists for replication stalling within [AT/TA] microsatellite repeats. Currently, it is unknown how stress causes replication stalling within [AT/TA] microsatellite repeats. To investigate this, we utilized FRET to characterize the structures of [AT/TA]25 sequences and also reconstituted lagging strand replication to characterize the progression of pol δ holoenzymes through A+T-rich sequences. The results indicate that [AT/TA]25 sequences adopt hairpins that are unwound by the major ssDNA-binding complex, RPA, and the progression of pol δ holoenzymes through A+T-rich sequences saturated with RPA is dependent on the template sequence and dNTP concentration. Importantly, the effects of RPA on the replication of [AT/TA]25 sequences are dependent on dNTP concentration, whereas the effects of RPA on the replication of A+T-rich, nonstructure-forming sequences are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how [AT/TA] microsatellite repeats contribute to genome instability.

Recent advances in the synthesis of single-stranded DNA in vitro.

Single-stranded DNA (ssDNA) is the foundation of modern biology, with wide applications in gene editing, sequencing, DNA information storage, and materials science. However, synthesizing ssDNA with high efficiency, high throughput, and low error rate in vitro remains a major challenge. Various methods have been developed for ssDNA synthesis, and some significant results have been achieved. In this review, six main methods were introduced, including solid-phase oligonucleotide synthesis, terminal deoxynucleotidyl transferase-based ssDNA synthesis, reverse transcription, primer exchange reaction, asymmetric polymerase chain reaction, and rolling circle amplification. The advantages and limitations of each method were compared, as well as illustrate their representative achievements and applications. Especially, rolling circle amplification has received significant attention, including ssDNA synthesis, assembly, and application based on recent work. Finally, the future challenges and opportunities of ssDNA synthesis were summarized and discussed. Envisioning the development of new methods and significant progress will be made in the near future with the efforts of scientists around the world.

An Innovative and Efficient Fluorescent Detection Technique for Salmonella in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA.

Here, we present a method for Salmonella detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the Salmonella invA gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the Salmonella concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 × 101-2 × 109 CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid Salmonella detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive Salmonella detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.

Linear dichroism reveals the perpendicular orientation of DNA bases in the RecA and Rad51 recombinase filaments: A possible mechanism for the strand exchange reaction.

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

Common occurrence of hotspots of single strand DNA breaks at transcriptional start sites.

BACKGROUND: We recently developed two high-resolution methods for genome-wide mapping of two prominent types of DNA damage, single-strand DNA breaks (SSBs) and abasic (AP) sites and found highly complex and non-random patterns of these lesions in mammalian genomes. One salient feature of SSB and AP sites was the existence of single-nucleotide hotspots for both lesions. RESULTS: In this work, we show that SSB hotspots are enriched in the immediate vicinity of transcriptional start sites (TSSs) in multiple normal mammalian tissues, however the magnitude of enrichment varies significantly with tissue type and appears to be limited to a subset of genes. SSB hotspots around TSSs are enriched on the template strand and associate with higher expression of the corresponding genes. Interestingly, SSB hotspots appear to be at least in part generated by the base-excision repair (BER) pathway from the AP sites. CONCLUSIONS: Our results highlight complex relationship between DNA damage and regulation of gene expression and suggest an exciting possibility that SSBs at TSSs might function as sensors of DNA damage to activate genes important for DNA damage response.

Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein.

Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found that Marinomonas mediterranea RT-Cas1/Cas2 adds short 3'-DNA (dN) tails to RNA protospacers, enabling their direct integration into CRISPR arrays as 3'-dN-RNAs or 3'-dN-RNA/cDNA duplexes at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers is initiated at 3' proximal sites by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of near full-length cDNAs of diverse RNAs without fixed sequence requirements. The integration of 3'-dN-RNAs or single-stranded DNAs (ssDNAs) is favored over duplexes at higher protospacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage defense nucleases. Our findings reveal mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications.

Tris buffer-accelerated ligand exchange rate for instant fluorescence detection of trivalent chromium ion.

Functional nucleic acids (FNAs) have attracted a lot of attention for the rapid detection of metal ions. Cr3+ is one of the major heavy metal ions in natural waters. Due to the slow ligand exchange rate of Cr3+, the FNA-based Cr3+ sensors require long assay times, limiting the on-site applications. In this study, we report that the good's buffers containing amino and polyhydroxy groups greatly increase the ligand exchange rate of Cr3+. Using EDTA as a model coordinate ligand, the Tris buffer (100 mM, pH 7.0) showed the best acceleration effect among the eight buffers. It improved the rate constant ∼20-fold, shorten the half-time 19-fold, and lowered the activation energy ∼70% at 40 °C. The Tris buffer was then applied for sensor based on the Cr3+-binding induced fluorescence quenching of fluorescein (FAM)-labeled and single-stranded DNA (ssDNA), which shortened the assay time from 1 h to 1 min. The Tris buffer also ∼100% enhanced the fluorescence intensity of FAM, achieving the 11.4-fold lower limit of detection (LOD = 6.97 nM, S/N = 3). By the combination use of the Tris buffer and ascorbic acid, the strong interference from Cu2+, Pb2+, and Fe3+ suffered in many previous reported Cr3+ sensors was avoided. The practical application of the sensor for the detection of Cr3+ spiked in the real water samples were demonstrated with high recovery percentages. The Tris buffer could be applied for other metal ions with slow ligand exchange rate (such as V2+, Co3+ and Fe2+) to solve diverse issues such as long assay time and low synthesis yield of metal complexes, without the need of heating treatment.

Split activator of CRISPR/Cas12a for direct and sensitive detection of microRNA.

CRISPR/Cas12a-based nucleic acid assays have been increasingly used for molecular diagnostics. However, most current CRISPR/Cas12a-based RNA assays require the conversion of RNA into DNA by preamplification strategies, which increases the complexity of detection. Here, we found certain chimeric DNA-RNA hybrid single strands could activate the trans-cleavage activity of Cas12a, and then discovered the activating effect of split ssDNA and RNA when they are present simultaneously. As proof of concept, split nucleic acid-activated Cas12a (SNA-Cas12a) strategy was developed for direct detection of miR-155. By adding a short ssDNA to the proximal end of the crRNA spacer sequence, we realized the direct detection of RNA targets using Cas12a. With the assistance of ssDNA, we extended the limitation that CRISPR/Cas12a cannot be activated by RNA targets. In addition, by taking advantage of the programmability of crRNA, the length of its binding to DNA and RNA was optimized to achieve the optimal efficiency in activating Cas12a. The SNA-Cas12a method enabled sensitive miR-155 detection at pM level. This method was simple, rapid, and specific. Thus, we proposed a new Cas12a-based RNA detection strategy that expanded the application of CRISPR/Cas12a.

Novel Enzyme-Assisted Recycle Amplification Strategy for Tetracycline Detection Based on Oxidized Single-Walled Carbon Nanohorns.

In this study, oxidized single-walled carbon nanohorns (oxSWCNHs) were prepared using nitric acid oxidation and subsequently combined with 3'6-carboxyfluorescein through charge transfer to prepare fluorescent probes. These oxSWCNHs were used to quench fluorogen signals at short distances and dissociate ssDNA using cryonase enzymes. We established a method for rapidly detecting tetracycline (TC) in complex samples based on the amplification of cryonase enzyme signals. After optimizing the experimental conditions, our method showed a detection limit of 5.05 ng/mL, with good specificity. This method was used to determine the TC content in complex samples, yielding a recovery rate of 90.0-103.3%. This result validated the efficacy of our method in detecting TC content within complex samples.

Primer extension refractory PCR: an efficient and reliable genome walking method.

Genome walking, a molecular technique for obtaining unknown flanking genomic sequences from a known genomic sequence, has been broadly applied to determine transgenic sites, mine new genetic resources, and fill in chromosomal gaps. This technique has advanced genomics, genetics, and related disciplines. Here, an efficient and reliable genome walking technique, called primer extension refractory PCR (PER-PCR), is presented. PER-PCR uses a set of primary, secondary, and tertiary walking primers. The middle 15 nt of the primary walking primer overlaps with the 3' parts of the secondary and tertiary primers. The 5' parts of the three primers are heterologous to each other. The short overlap allows the walking primer to anneal to its predecessor only in a relaxed-stringency PCR cycle, resulting in a series of single-stranded DNAs; however, the heterologous 5' part prevents the creation of a perfect binding site for the walking primer. In the next stringent cycle, the target single strand can be extended into a double-stranded DNA molecule by the sequence-specific primer and thus can be exponentially amplified by the remaining stringent cycles. The nontarget single strand fails to be enriched due to the lack of a perfect binding site for any primer. PER-PCR was validated by extension into unknown flanking regions of the hyg gene in rice and the gadR gene in Levilactobacillus brevis CD0817. In summary, in this study, a new practical PER-PCR method was constructed as a potential alternative to existing genome walking methods.

Electrochemiluminescence aptasensing method for ultrasensitive determination of lipopolysaccharide based on CRISPR-Cas12a accessory cleavage activity.

In this study, an ultrasensitive electrochemiluminescence (ECL) aptasensing method was developed for lipopolysaccharide (LPS) determination based on CRISPR-Cas12a accessory cleavage activity. Tris (2,2'-bipyridine) dichlororuthenium (II) (Ru(bpy)32+) was adsorbed on the surface of a glassy carbon electrode (GCE) coated with a mixture of gold nanoparticles (AuNPs) and Nafion film via electrostatic interaction. The obtained ECL platform (Ru(bpy)32+/AuNP/Nafion/GCE) exhibited strong ECL emission. Thiol-functionalized single-stranded DNA (ssDNA) was modified with a ferrocenyl (Fc) group and autonomously assembled on the ECL platform of Ru(bpy)32+/AuNP/Nafion/GCE via thiol-gold bonding, resulting in the quenching of ECL emission. After hybridization of the LPS aptamer strand (AS) with its partial complementary strand (CS), the formed double-stranded DNA (dsDNA) could activate CRISPR-Cas12a to indiscriminately cleave ssDNA-Fc on the surface of Ru(bpy)32+/AuNP/Nafion/GCE, resulting in recovery of the ECL intensity of Ru(bpy)32+ due to the increasing distance between Fc and the electrode surface. The combination of LPS and AS suppressed the formation of dsDNA, inhibited the activation of CRISPR-Cas12a, and prevented further cleavage of ssDNA-Fc. This mechanism aided in upholding the integrity of ssDNA-Fc on the surface of the electrode and was combined with ECL quenching induced by the target. The ECL intensity decreased linearly as the concentration of LPS increased from 1 to 50,000 pg/mL and followed a logarithmic relationship. This method exhibited a remarkably low detection limit of 0.24 pg/mL, which meets the requirement for low-concentration detection of LPS in the human body. The proposed method demonstrates the capacity of CRISPR-Cas12a to perform non-specific cutting of single-stranded DNA and transform the resultant cutting substances into changes in the ECL signal. By amalgamating this approach with the distinct identification abilities of LPS and its aptamers, a simple, responsive, and discriminatory LPS assay was established that holds immense significance for clinical diagnosis.

Structural basis of how MGME1 processes DNA 5' ends to maintain mitochondrial genome integrity.

Mitochondrial genome maintenance exonuclease 1 (MGME1) helps to ensure mitochondrial DNA (mtDNA) integrity by serving as an ancillary 5'-exonuclease for DNA polymerase γ. Curiously, MGME1 exhibits unique bidirectionality in vitro, being capable of degrading DNA from either the 5' or 3' end. The structural basis of this bidirectionally and, particularly, how it processes DNA from the 5' end to assist in mtDNA maintenance remain unclear. Here, we present a crystal structure of human MGME1 in complex with a 5'-overhang DNA, revealing that MGME1 functions as a rigid DNA clamp equipped with a single-strand (ss)-selective arch, allowing it to slide on single-stranded DNA in either the 5'-to-3' or 3'-to-5' direction. Using a nuclease activity assay, we have dissected the structural basis of MGME1-derived DNA cleavage patterns in which the arch serves as a ruler to determine the cleavage site. We also reveal that MGME1 displays partial DNA-unwinding ability that helps it to better resolve 5'-DNA flaps, providing insights into MGME1-mediated 5'-end processing of nascent mtDNA. Our study builds on previously solved MGME1-DNA complex structures, finally providing the comprehensive functional mechanism of this bidirectional, ss-specific exonuclease.

An autocatalytic CRISPR-Cas amplification effect propelled by the LNA-modified split activators for DNA sensing.

CRISPR-Cas systems with dual functions offer precise sequence-based recognition and efficient catalytic cleavage of nucleic acids, making them highly promising in biosensing and diagnostic technologies. However, current methods encounter challenges of complexity, low turnover efficiency, and the necessity for sophisticated probe design. To better integrate the dual functions of Cas proteins, we proposed a novel approach called CRISPR-Cas Autocatalysis Amplification driven by LNA-modified Split Activators (CALSA) for the highly efficient detection of single-stranded DNA (ssDNA) and genomic DNA. By introducing split ssDNA activators and the site-directed trans-cleavage mediated by LNA modifications, an autocatalysis-driven positive feedback loop of nucleic acids based on the LbCas12a system was constructed. Consequently, CALSA enabled one-pot and real-time detection of genomic DNA and cell-free DNA (cfDNA) from different tumor cell lines. Notably, CALSA achieved high sensitivity, single-base specificity, and remarkably short reaction times. Due to the high programmability of nucleic acid circuits, these results highlighted the immense potential of CALSA as a powerful tool for cascade signal amplification. Moreover, the sensitivity and specificity further emphasized the value of CALSA in biosensing and diagnostics, opening avenues for future clinical applications.

Engineering an Escherichia coli strain for production of long single-stranded DNA.

Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered Escherichia coli 'helper' strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20 724 nt with titers up to 250 µg/l following alkaline lysis purification. The efficacy of our system was confirmed through its application in primary T-cell genome modifications and DNA origami folding. The reliability, scalability and ease of our approach promise to unlock new experimental applications requiring large quantities of long ssDNA.

Cooperative dynamics of DNA-grafted magnetic nanoparticles optimize magnetic biosensing and coupling to DNA origami.

Magnetic nanoparticles (MNPs) provide new opportunities for enzyme-free biosensing of nucleic acid biomarkers and magnetic actuation by patterning on DNA origami, yet how the DNA grafting density affects their dynamics and accessibility remains poorly understood. Here, we performed surface functionalization of MNPs with single-stranded DNA (ssDNA) via click chemistry with a tunable grafting density, which enables the encapsulation of single MNPs inside a functional polymeric layer. We used several complementary methods to show that particle translational and rotational dynamics exhibit a sigmoidal dependence on the ssDNA grafting density. At low densities, ssDNA strands adopt a coiled conformation that results in minor alterations to particle dynamics, while at high densities, they organize into polymer brushes that collectively influence particle dynamics. Intermediate ssDNA densities, where the dynamics are most sensitive to changes, show the highest magnetic biosensing sensitivity for the detection of target nucleic acids. Finally, we demonstrate that MNPs with high ssDNA grafting densities are required to efficiently couple to DNA origami. Our results establish ssDNA grafting density as a critical parameter for the functionalization of MNPs for magnetic biosensing and functionalization of DNA nanostructures.

Polydopamine functionalized dendritic fibrous silica nanoparticles as a generic platform for nucleic acid-based biosensing.

Accurate and rapid detection of nucleic acid sequences is of utmost importance in various fields, including disease monitoring, clinical treatment, gene analysis and drug discovery. In this study, we developed a "turn-on" fluorescence biosensor that enables simple and highly efficient detection of nucleic acid biomarkers. Our approach involves the utilization of 6-carboxyfluorescein modified single-stranded DNA (FAM-ssDNA) as molecular recognition element, along with polydopamine-functionalized dendritic fibrous nanosilica (DFNS). FAM-ssDNA serves as both specific molecular recognition element for the target analyte and reporter capable of transducing a detectable signal through Watson-Crick base pairing. The polydopamine-functionalized DFNS (DFNS@DA) exhibits strong binding to FAM-ssDNA via polyvalent metal mediated coordination leading to effective quenching by fluorescence resonance energy transfer. In the presence of a complementary target sequence, FAM-ssDNA forms hybridized structure and detaches from DFNS@DA, which causes an increased fluorescence emission. The analytical system based on FAM-ssDNA and DFNS@DA demonstrates exceptional sensitivity, selectivity, and rapid response for the detection of nucleic acid sequences, leveraging the high adsorption and quenching properties of DFNS@DA. For the first proof of concept, we demonstrated the successful detection of microRNA (miR-21) in cancer cells using the FAM-ssDNA/DFNS@DA system. Our results highlight the promising capabilities of DFNS@DA and nucleic acid-based biosensors, offering a generic and cost-effective solution for the detection of nucleic acid-related biomarkers.

Competition between Stacking and Divalent Cation-Mediated Electrostatic Interactions Determines the Conformations of Short DNA Sequences.

Interplay between divalent cations (Mg2+ and Ca2+) and single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), as well as stacking interactions, is important in nucleosome stability and phase separation in nucleic acids. Quantitative techniques accounting for ion-DNA interactions are needed to obtain insights into these and related problems. Toward this end, we created a sequence-dependent computational TIS-ION model that explicitly accounts for monovalent and divalent ions. Simulations of the rigid 24 base-pair (bp) dsDNA and flexible ssDNA sequences, dT30 and dA30, with varying amounts of the divalent cations show that the calculated excess number of ions around the dsDNA and ssDNA agree quantitatively with ion-counting experiments. Using an ensemble of all-atom structures generated from coarse-grained simulations, we calculated the small-angle X-ray scattering profiles, which are in excellent agreement with experiments. Although ion-counting experiments mask the differences between Mg2+ and Ca2+, we find that Mg2+ binds to the minor grooves and phosphate groups, whereas Ca2+ binds specifically to the minor groove. Both Mg2+ and Ca2+ exhibit a tendency to bind to the minor groove of DNA as opposed to the major groove. The dA30 conformations are dominated by stacking interactions, resulting in structures with considerable helical order. The near cancellation of the favorable stacking and unfavorable electrostatic interactions leads to dT30 populating an ensemble of heterogeneous conformations. The successful applications of the TIS-ION model are poised to confront many problems in DNA biophysics.