-
Mikrochimica Acta Jun 2024With their regulated Boolean logic operations in vitro and in vivo, DNA logic circuits have shown great promise for target recognition and disease diagnosis. However,...
With their regulated Boolean logic operations in vitro and in vivo, DNA logic circuits have shown great promise for target recognition and disease diagnosis. However, significant obstacles must be overcome to improve their operational efficiency and broaden their range of applications. In this study, we propose an Exo III-powered closed-loop DNA circuit (ECDC) architecture that integrates four highly efficient AND logic gates. The ECDC utilizes Exo III as the sole enzyme-activated actuator, simplifying the circuit design and ensuring optimal performance. Moreover, the use of Exo III enables a self-feedback (autocatalytic) mechanism in the dynamic switching between AND logic gates within this circulating logic circuit. After validating the signal flow and examining the impact of each AND logic gate on the regulation of the circuit, we demonstrate the intelligent determination of miR-21 using the carefully designed ECDC architecture in vitro. The proposed ECDC exhibits a linear detection range for miR-21 from 0 to 300 nM, with a limit of detection (LOD) of approximately 0.01 nM, surpassing most reported methods. It also shows excellent selectivity for miR-21 detection and holds potential for identifying and imaging live cancer cells. This study presents a practical and efficient strategy for monitoring various nucleic acid-based biomarkers in vitro and in vivo through specific sequence modifications, offering significant potential for early cancer diagnosis, bioanalysis, and prognostic clinical applications.
Topics: Humans; MicroRNAs; Biosensing Techniques; Exodeoxyribonucleases; Limit of Detection; DNA
PubMed: 38877347
DOI: 10.1007/s00604-024-06476-0 -
Mikrochimica Acta Jun 2024A fluorescence biosensor for determination of aflatoxin B (AFB) based on polydiacetylene (PDA) liposomes and exonuclease III (EXO III)-assisted recycling amplification...
A fluorescence biosensor for determination of aflatoxin B (AFB) based on polydiacetylene (PDA) liposomes and exonuclease III (EXO III)-assisted recycling amplification was developed. The AFB aptamer partially hybridizes with complementary DNA (cDNA), which is released upon recognition of AFB by the aptamer. Subsequently, the cDNA hybridizes with hairpin H to form double-stranded DNA that undergoes digestion by EXO III, resulting in the cyclic release of cDNA and generation of capture DNA for further reaction. The capture DNA then hybridizes with probe modified on PDA liposomes, leading to aggregation of liposomes and subsequent fluorescence production. This strategy exhibited a limit of detection of 0.18 ng/mL within the linear range 1-100 ng/mL with a determination coefficient > 0.99. The recovery ranged from 92.81 to 106.45%, with relative standard deviations (RSD) between 1.73 and 4.26%, for corn, brown rice, peanut butter, and wheat samples. The stability, accuracy, and specificity of the method demonstrated the applicability for real sample analysis.
Topics: Polyacetylene Polymer; Liposomes; Exodeoxyribonucleases; Biosensing Techniques; Aflatoxin B1; Limit of Detection; Aptamers, Nucleotide; Nucleic Acid Amplification Techniques; Polyynes; Spectrometry, Fluorescence; Zea mays; Triticum; Oryza; Polymers; Food Contamination
PubMed: 38877314
DOI: 10.1007/s00604-024-06482-2 -
Medicine Jun 2024In Algeria, the issue of antibiotic resistance is on the rise, being the Staphylococcus aureus infection as a significant concern of hospital-acquired infections. The... (Observational Study)
Observational Study
In Algeria, the issue of antibiotic resistance is on the rise, being the Staphylococcus aureus infection as a significant concern of hospital-acquired infections. The emergence of antibiotic resistance in this bacterium poses a worldwide challenge. The aim of this study aims to establish the incidence of S aureus strains in Algeria as well as identify phenotypic and genotypic resistance based on the "mecA" and "nuc" genes. From 2014 to 2017, a total of 185 S aureus strains were isolated from patients at a hospital in the city of Rouïba, Algiers the number of isolates was slightly higher in males at 58.06% compared to females at 41.94%, resulting in a sex ratio of 1.38. the Oxacillin and Cefoxitin DD test (1 μg oxacillin disk and 30 μg cefoxitin disk) identified 42 strains as resistant. The results indicated high resistance to lactam antibiotics, with penicillin having a 100% resistance rate. There was also significant resistance to oxacillin (51.25%) and cefoxitin (50%). This resistance was frequently associated with resistance to other antibiotic classes, such as aminoglycosides (50%) and Macrolides (28.29%). To confirm methicillin-resistant characteristics, a polymerase chain reaction (PCR) multiplex was conducted on 10 isolates (6 SARM; 4 MSSA) on a phenotypic level. Three isolates tested positive for "mecA," while 7 were negative. All strains carry the nuc gene, which is specific to S aureus. In Algeria, the incidence of S aureus resistance is slightly lower compared to other countries, but it is increasing over time. It is now more crucial than ever to restrict the proliferation of multidrug-resistant strains and reduce undue antibiotic prescriptions. To achieve this, it is vital to keep updated on the epidemiology of this bacterium and its antibiotic susceptibility. This will enable the formulation of appropriate preventive control measures to manage its progression.
Topics: Humans; Anti-Bacterial Agents; Female; Male; Staphylococcus aureus; Staphylococcal Infections; Algeria; Prevalence; Microbial Sensitivity Tests; Bacterial Proteins; Oxacillin; Adult; Penicillin-Binding Proteins; Cefoxitin; Middle Aged; Micrococcal Nuclease; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Methicillin-Resistant Staphylococcus aureus
PubMed: 38875387
DOI: 10.1097/MD.0000000000038562 -
Analytical Chemistry Jul 2024The trans-cleavage properties of Cas12a make it important for gene editing and disease diagnosis. In this work, the effect of spatial site resistance on the...
The trans-cleavage properties of Cas12a make it important for gene editing and disease diagnosis. In this work, the effect of spatial site resistance on the trans-cleavage activity of Cas12a was studied. First, we have explored the cutting effect of Cas12a when different-sized nanoparticles are linked with various spacings of DNA strands using the fluorescence method. The minimum spacing with different-sized nanoparticles that cas12a can cut was determined. We found that when the size of the nanoparticles increases, the minimum spacing that cas12a can cut gradually increases. Subsequently, we verified the conclusion using the surface-enhanced Raman scattering (SERS) method, and at the same time, we designed a SERS biosensor that can achieve ultrasensitive detection of P53 DNA with a linear range of 1 fM-10 nM and a limit of detection of 0.40 fM. Our work develops a deep study of the trans-cleavage activity of Cas12a and gives a guide for DNA design in cas12a-related studies, which can be applied in biomedical analysis and other fields.
Topics: Spectrum Analysis, Raman; Tumor Suppressor Protein p53; DNA; CRISPR-Cas Systems; Humans; Biosensing Techniques; Metal Nanoparticles; Endodeoxyribonucleases; CRISPR-Associated Proteins; Limit of Detection; Bacterial Proteins
PubMed: 38875020
DOI: 10.1021/acs.analchem.4c01347 -
Open Forum Infectious Diseases Jun 2024A future (Strep A) vaccine will ideally prevent a significant burden of lower limb cellulitis; however, natural immune responses to proposed vaccine antigens following...
BACKGROUND
A future (Strep A) vaccine will ideally prevent a significant burden of lower limb cellulitis; however, natural immune responses to proposed vaccine antigens following an episode of cellulitis remain uncharacterized.
METHODS
We enrolled 63 patients with cellulitis and 26 with invasive beta hemolytic streptococci infection, using a multiplexed assay to measure immunoglobulin G against Strep A vaccine candidate antigens, including: streptolysin O (SLO), deoxyribonuclease B (DNB), group A carbohydrate (GAC), C5a peptidase (ScpA), cell envelope proteinase (SpyCEP), and adhesion and division protein (SpyAD). Responses in the invasive cohort were used to predict the infecting etiology in the cellulitis cohort.
RESULTS
Of 41 patients with cellulitis and paired serological samples, 68.3% had evidence of beta hemolytic streptococci infection by conventional anti-SLO and/or anti-DNB criteria. A positive serological response to at least 1 of the tested antigens was seen in 78.0% of the cellulitis cohort. Individually, anti-SLO (58.5%), anti-SpyAD (46.3%), and anti-ScpA (39.0%) were the most common. Based on principal component analysis, increases in these 3 antibodies, without responses to DNB, GAC, and SpyCEP characterized (SDSE) infection.
CONCLUSIONS
SDSE appears to be the predominant cause of lower limb cellulitis. Effective Strep A vaccines incorporating antigens that provide additional cross protection against SDSE may prevent a significant burden of lower limb cellulitis.
PubMed: 38872850
DOI: 10.1093/ofid/ofae272 -
Molecular Cell Jun 2024In Saccharomyces cerevisiae (S. cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or...
In Saccharomyces cerevisiae (S. cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or protein blocks, while in humans, the MRE11-RAD50-NBS1 (MRN) homolog with CtIP is needed to initiate DNA end resection of all breaks. Phosphorylated Sae2/CtIP stimulates the endonuclease activity of MRX/N. Structural insights into the activation of the Mre11 nuclease are available only for organisms lacking Sae2/CtIP, so little is known about how Sae2/CtIP activates the nuclease ensemble. Here, we uncover the mechanism of Mre11 activation by Sae2 using a combination of AlphaFold2 structural modeling of biochemical and genetic assays. We show that Sae2 stabilizes the Mre11 nuclease in a conformation poised to cleave substrate DNA. Several designs of compensatory mutations establish how Sae2 activates MRX in vitro and in vivo, supporting the structural model. Finally, our study uncovers how human CtIP, despite considerable sequence divergence, employs a similar mechanism to activate MRN.
Topics: Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; Endonucleases; DNA-Binding Proteins; Endodeoxyribonucleases; Humans; Exodeoxyribonucleases; Models, Molecular; Phosphorylation; DNA Repair Enzymes; DNA Breaks, Double-Stranded; Acid Anhydride Hydrolases; Mutation; MRE11 Homologue Protein; DNA Repair; Enzyme Activation
PubMed: 38870937
DOI: 10.1016/j.molcel.2024.05.019 -
Methods in Molecular Biology (Clifton,... 2024Nucleosome occupancy plays an important role in chromatin compaction, affecting biological processes by hampering the binding of cis-acting elements such as...
Nucleosome occupancy plays an important role in chromatin compaction, affecting biological processes by hampering the binding of cis-acting elements such as transcription factors, RNA polymerase machinery, and coregulatory. Accessible regions allow for cis-acting elements to bind DNA and regulate transcription. Here, we detail our protocol to profile nucleosome occupancy and chromatin structure dynamics under drought stress at the genome-wide scale using micrococcal nuclease (MNase) digestion. Combining variable MNase concentration treatments and high-throughput sequencing, we investigate the changes in the overall chromatin state using bread wheat samples from an exemplary drought experiment.
Topics: Nucleosomes; Micrococcal Nuclease; High-Throughput Nucleotide Sequencing; Stress, Physiological; Triticum; Droughts; Gene Expression Regulation, Plant; Chromatin; Plants
PubMed: 38869785
DOI: 10.1007/978-1-0716-3973-3_2 -
Mikrochimica Acta Jun 2024The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the...
The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the duplex protospacer adjacent motif (PAM) in the activator of CRISPR/Cas12a was used as a molecular switch, and a sensitive adenosine triphosphate (ATP) detection biosensor was constructed using an allosteric probe-conjugated PAM site formation in hybridization chain reaction (HCR) integrated with the CRISPR/Cas12a system (APF-CRISPR). In the absence of ATP, an aptamer-containing probe (AP) is in a stem-loop structure, which blocks the initiation of HCR. In the presence of ATP, the structure of AP is changed upon ATP binding, resulting in the release of the HCR trigger strand and the production of long duplex DNA with many PAM sites. Since the presence of a duplex PAM site is crucial for triggering the cleavage activity of CRISPR/Cas12a, the ATP-dependent formation of the PAM site in HCR products can initiate the FQ-reporter cleavage, allowing ATP quantification by measuring the fluorescent signals. By optimizing the sequence elements and detection conditions, the aptasensor demonstrated superior detection performance. The limit of detection (LOD) of the assay was estimated to be 1.16 nM, where the standard deviation of the blank was calculated based on six repeated measurements. The dynamic range of the detection was 25-750 nM, and the whole workflow of the assay was approximately 60 min. In addition, the reliability and practicability of the aptasensor were validated by comparing it with a commercially available chemiluminescence kit for ATP detection in serum. Due to its high sensitivity, specificity, and reliable performance, the APF-CRISPR holds great potential in bioanalytical studies for ATP detection. In addition, we have provided a proof-of-principle for constructing a CRISPR/Cas12a-based aptasensor, in which the PAM is utilized to regulate Cas12a cleavage activity.
Topics: Adenosine Triphosphate; Biosensing Techniques; Aptamers, Nucleotide; CRISPR-Cas Systems; CRISPR-Associated Proteins; Limit of Detection; Bacterial Proteins; Nucleic Acid Hybridization; Endodeoxyribonucleases
PubMed: 38867016
DOI: 10.1007/s00604-024-06477-z -
Nature Communications Jun 2024Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT...
Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT's adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.
Topics: Humans; Leukemia, Myeloid, Acute; INDEL Mutation; CRISPR-Cas Systems; Nucleophosmin; Neoplasm, Residual; Nuclear Proteins; Proto-Oncogene Proteins B-raf; Genetic Testing; ErbB Receptors; Bacterial Proteins; Endodeoxyribonucleases; CRISPR-Associated Proteins
PubMed: 38866774
DOI: 10.1038/s41467-024-49414-7 -
PLoS Biology Jun 2024CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new...
CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new Cas12a nucleases for mammalian genome editing, successfully identifying 9 active ones. Notably, these Cas12a nucleases prefer pyrimidine-rich PAMs. Among these nucleases, we extensively characterized Mb4Cas12a obtained from Moraxella bovis CCUG 2133, which recognizes a YYN PAM (Y = C or T). Our biochemical analysis demonstrates that Mb4Cas12a can cleave double-strand DNA across a wide temperature range. To improve specificity, we constructed a SWISS-MODEL of Mb4Cas12a based on the FnCas12a crystal structure and identified 8 amino acids potentially forming hydrogen bonds at the target DNA-crRNA interface. By replacing these amino acids with alanine to disrupt the hydrogen bond, we tested the influence of each mutation on Mb4Cas12a specificity. Interestingly, the F370A mutation improved specificity with minimal influence on activity. Further study showed that Mb4Cas12a-F370A is capable of discriminating single-nucleotide polymorphisms. These new Cas12a orthologs and high-fidelity variants hold substantial promise for therapeutic applications.
Topics: Gene Editing; CRISPR-Cas Systems; CRISPR-Associated Proteins; Alleles; Humans; Endodeoxyribonucleases; Animals; Protein Engineering; Bacterial Proteins; Polymorphism, Single Nucleotide; Mutation; DNA; HEK293 Cells
PubMed: 38865309
DOI: 10.1371/journal.pbio.3002680