Single-stranded DNA Research Articles

Ingenious entropy-driven DNA circuit intercommunicating with DNAzyme-powered DNA walker for dual-mode biosensing

Conventional DNA motor-based cascaded amplification methods have relative low DNA utilization efficiency and lack of self-feedback. Herein, a novel dual-mode biosensor of miRNA-155 was constructed based on a waste-free entropy-driven DNA circuit (EDC) cascaded with a self-feedback DNAzyme-powered DNA walker (DNAzyme Walker). The target (T)-triggered EDC generates two same single-stranded DNA (ssDNA) labelled with CdTe QDs as the signal probes (CdTe-O) and one double-stranded DNA (dsDNA) S/F to unlock the downstream blocked DNAzyme (D), which could then be activated in the presence of Mn2+ ions cofactor, and stochastically walk on the surfaces of SiO2 nanospheres, producing a lot of target analogue (T*) for self-feedback. With the assistance of Fe3O4@SiO2-capture DNA, the released CdTe-O can be rapidly extracted for the following self-validating dual-mode biosensing. The autocatalytic EDC module possesses a high operation efficiency and atomic economy, since it is not only hairpin- and leak-free, but also enzyme- and waste-free. The interactive EDC-DNAzyme Walker network-based fluorescent and colorimetric biosensor has a limit of detection low to 0.35 amol L−1 and 1.87 fmol L−1 at 3σ/S, respectively. The as-designed biosensor not only enables reliable and robust detection of miRNA expression levels, but also provides a remarkable signal cascaded amplification platform with self-feedback and high atom economy.

Sodium Ion-Induced Structural Transition on the Surface of a DNA-Interacting Protein.

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

CRISPR/Cas12a-Triggered Visible-Light-Driven Photoelectrochemical Assay with Single-Nucleotide Resolution for Drug-Resistant Foodborne Salmonella Detection.

The prevalence of foodborne pathogenic bacteria, especially drug-resistant strains, such as Salmonella enterica, poses serious threats to public health, highlighting the requirement for the development of rapid and precise detection methods. Herein, a CRISPR/Cas12a-triggered visible-light-driven photoelectrochemical (PEC) assay (CasPEC) was developed using a SiO2-quenched BiVO4/MoS2 p/n-type heterojunction as the photoactive material. The CRISPR/Cas12a recognition endowed the CasPEC assay with high specificity capable of resolving single-nucleotide polymorphisms (SNPs) and identifying SNP-involved drug-resistant bacteria. SiO2 was linked to the surface of the BiVO4/MoS2 heterojunction by single-stranded DNA (ssDNA), which would be cleaved by target-activated CRISPR/Cas12a. This cleavage of ssDNA resulted in the detachment of SiO2, thereby achieving a "signal-on" PEC output. Leveraging the multiple-turnover CRISPR cleavage and the outstanding photoactive performance of PEC signaling, the CasPEC assay for S. enterica showed a detection limit of 103 colony-forming units (CFU)/mL and the ability to detect as few as 0.01% drug-resistant strains. The CasPEC assay can accurately sense the S. enterica contamination in complex food matrices, including beef and milk. These findings demonstrated the great potential of the CasPEC assay for detecting pathogenic bacterial contamination in food, particularly concerning food safety related to SNP-involved drug-resistant bacteria.

Electrochemiluminescence Biosensor Based on a Duplex-Specific Nuclease and Dual-Output Toehold-Mediated Strand Displacement Cascade Amplification Strategy for Sensitive Detection of MicroRNA-499.

The timely and accurate diagnosis of acute myocardial infarction (AMI) is of great significance to reduce mortality and morbidity associated with the condition. Herein, we developed an electrochemiluminescence (ECL) biosensor for the detection of the potential AMI biomarker microRNA-499 (miRNA-499), which was based on duplex-specific nuclease-assisted target recycling and dual-output toehold-mediated strand displacement (TMSD). First, miRNA-499 was converted into a large amount of single-stranded DNA through the DSN-assisted target recycling, which was further incubated with the DNA triple-stranded complex (S) to implement TMSD cycles. Thus, the Ru(bpy)32+-labeled signal strands were released and captured by the capture probe on the electrode surface, resulting in an intense ECL signal. Owing to the prominent cascade signal amplification, the constructed biosensor exhibited a good linear response to miRNA-499 within the range of 100 aM-100 pM with a detection limit of 69.99 aM. Furthermore, it demonstrated superior selectivity, stability, and reproducibility. In addition, the biosensor was successfully applied to detect miRNA-499 in real human serum samples, demonstrating its potential for nucleic acid detection in the early diagnosis of diseases.

Bacteriophage λ exonuclease and a 5'-phosphorylated DNA guide allow PAM-independent targeting of double-stranded nucleic acids.

Sequence-specific recognition of double-stranded nucleic acids is essential for molecular diagnostics and in situ imaging. Clustered regularly interspaced short palindromic repeats and Cas systems rely on protospacer-adjacent motif (PAM)-dependent double-stranded DNA (dsDNA) recognition, limiting the range of targetable sequences and leading to undesired off-target effects. Using single-molecule fluorescence resonance energy transfer analysis, we discover the enzymatic activity of bacteriophage λ exonuclease (λExo). We show binding of 5'-phosphorylated single-stranded DNA (pDNA) to complementary regions on dsDNA and DNA-RNA duplexes, without the need for a PAM-like motif. Upon binding, the λExo-pDNA system catalytically digests the pDNA into nucleotides in the presence of Mg2+. This process is sensitive to mismatches within a wide range of the pDNA-binding region, resulting in exceptional sequence specificity and reduced off-target effects in various applications. The absence of a requirement for a specific motif such as a PAM sequence greatly broadens the range of targets. We demonstrate that the λExo-pDNA system is a versatile tool for molecular diagnostics, DNA computing and gene imaging applications.

Biochemical reconstitution of heat-induced mutational processes.

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.

Label-Free and Universal CRISPR/Cas12a-Based Detection Platform for Nucleic Acid Biomarkers.

CRISPR/Cas12a has been widely used in molecular diagnostics due to its excellent trans-cleavage activity. However, conventional reporters, such as F/Q-labeled single-stranded DNA (ssDNA) reporters, enzyme-labeled reporters, and spherical nucleic acid reporters, require complex modification or labeling processes. In this study, we have developed a rapid, universal, and label-free CRISPR/Cas12a-based biomarker detection platform via designing a G-quadruplex (G4) containing a hairpin structure as the reporter. The hairpin loop design of hairpin G4 improves the cleavage efficiency of Cas12a and the signal strength of the G4 binding ligand. Meanwhile, the incorporation of a G4 binding dye (protoporphyrin IX) eliminates the need for complex modifications. The CRISPR-hairpin G4 detection platform is capable of detecting ssDNA, double-stranded DNA, genetic RNAs, and miRNAs. Moreover, this platform achieves label-free detection in clinical samples, demonstrating its practical applicability and efficiency.

Multi-sensor integration on one microfluidics chip for single-stranded DNA detection

Multi-sensor integration on one microfluidics chip for single-stranded DNA detection

Mei5-Sae3 stabilizes Dmc1 nucleating clusters for efficient Dmc1 assembly on RPA-coated single-stranded DNA.

Interhomolog recombination in meiosis requires a meiosis-specific recombinase, Dmc1. In Saccharomyces cerevisiae, the Mei5-Sae3 complex facilitates the loading of Dmc1 onto the replication protein A (RPA)-coated single-stranded DNA (ssDNA) to form nucleoprotein filaments. In vivo, Dmc1 and Mei5-Sae3 are interdependent in their colocalization on the chromosomes. However, the mechanistic role of Mei5-Sae3 in mediating Dmc1 activity remains unclear. We used single-molecule fluorescence resonance energy transfer and colocalization single-molecule spectroscopy experiments to elucidate how Mei5-Sae3 stimulates Dmc1 assembly on ssDNA and RPA-coated ssDNA. We showed that Mei5-Sae3 stabilized Dmc1 nucleating clusters with two to threemolecules on naked DNA by preferentially reducing Dmc1 dissociation rates. Mei5-Sae3 also stimulated Dmc1 assembly on RPA-coated DNA. Using green fluorescent protein-labeled RPA, we showed the coexistence of an intermediate with Dmc1 and RPA on ssDNA before RPA dissociation. Moreover, the displacement efficiency of RPA depended on Dmc1 concentration, and its dependence was positively correlated with the stability of Dmc1 clusters on short ssDNA. These findings suggest a molecular model that Mei5-Sae3 mediates Dmc1 binding on RPA-coated ssDNA by stabilizing Dmc1 nucleating clusters, thus altering RPA dynamics on DNA to promote RPA dissociation.

Elucidating sequence-function relationships in a template-independent polymerase to enable novel DNA recording applications.

Harnessing DNA as a high-density storage medium for information storage and molecular recording of signals has been of increasing interest in the biotechnology field. Recently, progress in enzymatic DNA synthesis, DNA digital data storage, and DNA-based molecular recording has been made by leveraging the activity of the template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT). TdT adds deoxyribonucleotides to the 3' end of single-stranded DNA, generating random sequences of single-stranded DNA. TdT can use several divalent cations for its enzymatic activity and exhibits shifts in deoxyribonucleotide incorporation frequencies in response to changes in its reaction environment. However, there is limited understanding of sequence-structure-function relationships regarding these properties, which in turn limits our ability to modulate TdT to further advance TdT-based tools. Most TdT literature to-date explores the activity of murine, bovine or human TdTs; studies probing TdT sequence and structure largely focus on strictly conserved residues that are functionally critical to TdT activity. Here, we explore non-conserved TdT sequence space by surveying the natural diversity of TdT. We characterize a diverse set of TdT homologs from different organisms and identify several TdT residues/regions that confer differences in TdT behavior between homologs. The observations in this study can design rules for targeted TdT libraries, in tandem with a screening assay, to modulate TdT properties. Moreover, the data can be useful in guiding further studies of potential residues of interest. Overall, we characterize TdTs that have not been previously studied in the literature, and we provide new insights into TdT sequence-function relationships.

53BP1 deficiency leads to hyperrecombination using break-induced replication (BIR).

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.

Perylene Diimide-based Fluorescent Aptasensor for Quantitative Analysis of Pb2+ Based on Terminal Deoxynucleotidyl Transferase-assisted Formation of Elongated Aptamer and Gold Nanoparticles.

Due to the exceedingly poisonous properties of Pb2+, it is imperative to conduct a thorough assessment of its quantity in both biological and environmental samples, as this is crucial for safeguarding public health. This study describes an economic turn-off fluorescent aptasensor for the quantitative analysis of Pb2+ employing 3,4,9,10-perylenetetracarboxylic acid diimide (PTCDI) as a cost-effective fluorophore, gold nanoparticles (AuNPs) as separating agent and an elongated aptamer as both targeting agent and PTCDI loading site. The fundamental principle of the suggested fluorescent aptasensor, which is based on PTCDI, relies on detecting variations in the fluorescence intensity of PTCDI when an elongated aptamer (as single-stranded DNA) is present or absent. The advanced aptasensor is advantageous due to the elongation of the lead aptamer sequence length induced by terminal deoxynucleotidyl transferase (TdT), resulting in enhanced sensitivity. The presence of Pb2+ and the centrifugation process causes the separation of the poly A-modified aptamer/Pb2+ conjugate from the poly T sequence. Hence, the interaction of PTCDI with the poly A moiety in the modified aptamer leads to a decrease in its fluorescence emission. The findings showcased that the fluorescent aptasensor exhibited exceptional specificity towards Pb2+ ions, while the biosensing platform accomplished an impressive detection limit of 3.7pM. Moreover, the suggested aptasensor utilizing PTCDI exhibits a commendable capability in quantitatively analyzing Pb2+ within human serum samples and mineral water.

DNA template triggered copper nanoparticles generation coupled with hybridization chain reaction amplification for simultaneous detection of three mycotoxins in food samples

The co-occurrence of various mycotoxins in food, resulting from fungal infections, can lead to increased toxicity and enhanced harm through synergistic effects. In this work, a fluorescent assay was developed for simultaneous detection of zearalenone (ZEN), ochratoxin A (OTA) and aflatoxin B1 (AFB1) to address the issue of multiple mycotoxins co-contamination. Specific DNA hairpin-based aptamers were employed to recognize the mycotoxins and form a double-stranded DNA structure. Exonuclease III was introduced for the DNA cleavage, releasing single-stranded DNA triggers that could activate the assembling of DNA quadrilateral template. Copper nanoparticles were generated on the frame of DNA quadruplex template and produced fluorescent detection signals, while two hybridization chain reactions occurring on the different branches of the template and provided two additional detection signals. Inadequately reacted DNA chains were efficiently adsorbed and eliminated by Fe3O4@graphene oxide through magnetic separation. The limits of detection and linear ranges for ZEN, AFB1 and OTA were determined as 0.008 ng/mL, 0.003 ng/mL, 0.006 ng/mL and 0.010–100.000 ng/mL, 0.010–100.000 ng/mL, 0.010–50.000 ng/mL, respectively. Moreover, successful verification for real samples yielded recoveries from 99.2% to 102.7%, with a relative standard deviation of <5.23%. Artificially prepared toxic fungus infected samples were also analyzed. This method has demonstrated practical implications for the monitoring of food contaminants.

Mechanism of BCDX2-mediated RAD51 nucleation on short ssDNA stretches and fork DNA.

Homologous recombination (HR) factors are crucial for DSB repair and processing stalled replication forks. RAD51 paralogs, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have emerged as essential tumour suppressors, forming two subcomplexes, BCDX2 and CX3. Mutations in these genes are associated with cancer susceptibility and Fanconi anaemia, yet their biochemical activities remain unclear. This study reveals a linear arrangement of BCDX2 subunits compared to the RAD51 ring. BCDX2 shows a strong affinity towards single-stranded DNA (ssDNA) via unique binding mechanism compared to RAD51, and a contribution of DX2 subunits in binding branched DNA substrates. We demonstrate that BCDX2 facilitates RAD51 loading on ssDNA by suppressing the cooperative requirement of RAD51 binding to DNA and stabilizing the filament. Notably, BCDX2 also promotes RAD51 loading on short ssDNA and reversed replication fork substrates. Moreover, while mutants defective in ssDNA binding retain the ability to bind branched DNA substrates, they still facilitate RAD51 loading onto reversed replication forks. Our study provides mechanistic insights into how the BCDX2 complex stimulates the formation of BRCA2-independent RAD51 filaments on short stretches of ssDNA present at ssDNA gaps or stalled replication forks, highlighting its role in genome maintenance and DNA repair.

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.

Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Selection and Characterization of a DNA Aptamer Recognizing High Mobility Group Box 1 Protein (HMGB1) and Enhancing Its Pro-inflammatory Activity

Background: High mobility group box 1 (HMGB1) plays an essential role in various pathological conditions, including inflammation, fibrosis, autoimmune diseases, and carcinogenesis. The quantification of HMGB1 in body fluids holds promise for clinical applications. Objectives: This study aimed to isolate high-affinity single-stranded DNA (ssDNA) aptamers that target HMGB1. Methods: In this study, ssDNA aptamers were selected using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The affinity and specificity of the aptamers were evaluated through South-Western blot analysis, enzyme-linked aptamer sorbent assay (ELASA), and aptamer-based histochemistry staining. The impact of the aptamers on the biological activity of HMGB1 was tested in the human acute monocytic leukemia cell line, THP-1. Results: An aptamer (H-ap25, dissociation constant = 8.20 ± 0.53 nmol/L) with high affinity for the HMGB1 B box was generated. Further experiments verified that H-ap25 can be used to detect HMGB1 in South-Western blot analysis, ELASA, and aptamer-based histochemistry staining. Moreover, H-ap25 significantly augmented HMGB1-induced expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, Toll-like receptor 9 (TLR9), and activation of NF-κB in THP-1 cells. Conclusions: Our results demonstrated that H-ap25 can be used both as an enhancer of HMGB1 and as a probe in research.

Click-Chemistry-Enabled Functionalization of Cellulose Nanocrystals with Single-Stranded DNA for Directed Assembly.

Controlling the self-assembly of cellulose nanocrystals (CNCs) requires precise control over their surface chemistry for the directed assembly of advanced nanocomposites with tailored mechanical, thermal, and optical properties. In this work, in contrast to traditional chemistries, we conducted highly selective click-chemistry functionalization of cellulose nanocrystals with complementary DNA strands via a three-step hybridization-guided process. By grafting terminally functionalized oligonucleotides through copper-free click chemistry, we successfully facilitated the assembly of brushlike DNA-modified CNCs into bundled nanostructures with distinct chiral optical dichroism in thin films. The complexation behavior of grafted DNA chains during the evaporation-driven formation of ultrathin films demonstrates the potential for mediating chiral interactions between the DNA-branched nanocrystals and their assembly into chiral bundles. Furthermore, we discuss the future directions and challenges that include new avenues for the development of functional, responsive, and bioderived nanostructures capable of dynamic reconfiguration via selective complexation, further surface modification strategies, mitigating diverse CNC aggregation, and exploring environmental conditions for the CNC-DNA assembly.

Synergistic effect of split DNA activators of Cas12a with exon-unwinding and induced targeting effect.

CRISPR-Cas12a, an RNA-guided nuclease, has been repurposed for genome editing and molecular diagnostics due to its capability of cis-cleavage on target DNA and trans-cleavage on non-target single-strand DNA (ssDNA). However, the mechanisms underlying the activation of trans-cleavage activity of Cas12a, particularly in the context of split DNA activators, remain poorly understood. We elucidate the synergistic effect of these activators and introduce the concepts of induced targeting effect and exon-unwinding to describe the phenomenon. We demonstrate that upon binding of split DNA activators adjacent to the Protospacer Adjacent Motif (PAM) to the Cas12a ribonucleoprotein (Cas12a-RNP), a ternary complex form that can capture and interact with distal split DNA activators to achieve synergistic effects. Notably, if the distal activator is double-strand DNA (dsDNA), the complex initiates exon-unwinding, facilitating the RNA-guide sequence's access. Our findings provide a mechanistic insight into action of Cas12a and propose a model that could significantly advance our understanding of its function.

Promotion of DNA end resection by BRCA1-BARD1 in homologous recombination.

The licensing step of DNA double-strand break repair by homologous recombination entails resection of DNA ends to generate a single-stranded DNA template for assembly of the repair machinery consisting of the RAD51 recombinase and ancillary factors1. DNA end resection is mechanistically intricate and reliant on the tumour suppressor complex BRCA1-BARD1 (ref. 2). Specifically, three distinct nuclease entities-the 5'-3' exonuclease EXO1 and heterodimeric complexes of the DNA endonuclease DNA2, with either the BLM or WRN helicase-act in synergy to execute the end resection process3. A major question concerns whether BRCA1-BARD1 directly regulates end resection. Here, using highly purified protein factors, we provide evidence that BRCA1-BARD1 physically interacts with EXO1, BLM and WRN. Importantly, with reconstituted biochemical systems and a single-molecule analytical tool, we show that BRCA1-BARD1 upregulates the activity of all three resection pathways. We also demonstrate that BRCA1 and BARD1 harbour stand-alone modules that contribute to the overall functionality of BRCA1-BARD1. Moreover, analysis of a BARD1 mutant impaired in DNA binding shows the importance of this BARD1 attribute in end resection, both in vitro and in cells. Thus, BRCA1-BARD1 enhances the efficiency of all three long-range DNA end resection pathways during homologous recombination in human cells.

Aptamer-based Strategies for COVID-19 Detection and Treatment: A Systematic Review Study

Background: Aptamer-based strategies have emerged as promising tools for the detection and treatment of COVID-19, offering advantages such as high specificity, sensitivity, and versatility. This systematic review aims to evaluate the effectiveness and innovation of aptamer-based approaches for COVID-19 detection and treatment. Methods: Following the guidelines of the Cochrane Handbook for Systematic Reviews and the PRISMA 2020 guidelines, a systematic search was conducted across multiple databases up to 2024. The search included studies that utilized aptamers for the diagnosis or therapy of COVID-19. Screening and selection of studies were performed independently by two reviewers, with any disagreements resolved by a third reviewer. Data were extracted regarding study characteristics, aptamer details, and outcomes. Results: In our systematic review, 98 studies from an initial pool of 1541 records met the inclusion criteria for analysis. Aptamers, single-stranded DNA or RNA molecules with unique threedimensional (3D) structures, were extensively explored for COVID-19 detection and treatment. Various aptamer-based assays, including electrochemical sensors, surface plasmon resonance (SPR) biosensors, and lateral flow assays, demonstrated high sensitivity and specificity in detecting SARSCoV- 2 in clinical samples such as saliva, nasal swabs, and wastewater. Several aptamer structures targeting viral proteins like the spike and nucleocapsid proteins were employed. Nucleic Acid Amplification Techniques (NAATs) utilizing aptamers, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based and Loop-mediated Isothermal Amplification (LAMP) assays, showed exceptional sensitivity in detecting viral genetic material. Aptamer-based therapeutic approaches showed potential by blocking viral protein activity or serving as delivery vehicles for therapeutic agents like small interfering RNAs (siRNAs). Despite their advantages, aptamer technologies face limitations such as susceptibility to nuclease degradation and rapid renal clearance, highlighting the need for further optimization. Conclusion: Aptamer-based strategies present promising avenues for COVID-19 detection and treatment. These approaches offer advantages such as high sensitivity, specificity, and rapid detection, making them valuable tools in combating the COVID-19 pandemic. Further research and development are warranted to optimize aptamer-based strategies for widespread application in clinical settings.