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Mikrochimica Acta Nov 2023Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive...
Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive oxygen species, to maintain genomic integrity of living organisms. Abnormal expression of hOGG1 has been proved to be associated with different diseases such as cancer and neurogenerative disorders, making it a potential biomarker and therapeutic target. In this study, we report the development of a novel strategy for detecting hOGG1 activity based on CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of a dumbbell DNA probe (DBP) designed with a 3' overhang and an 8-oxoG modification. When hOGG1 is present, it cleaves the DBP at the 8-oxoG site, forming a 5' phosphate termini and exposing a single-strand region allowing complementary to the 3' overhang. After hybridization, the 3' and 5' termini in the juxtaposition are ligated by T4 DNA ligase, leading to a closed DBP for CRISPR/Cas12a-crRNA to recognize and initiate the trans-cleavage of the surrounding ssDNAs with fluorophore and quencher. The method achieves a limit of detection (LOD) with 370 μU/mL and high selectivity. Furthermore, it demonstrates a good compatibility for detecting hOGG1 activity in cell lysates, suggesting a good performance for further application in disease diagnosis and scientific research.
Topics: Humans; CRISPR-Cas Systems; DNA Glycosylases; DNA; Guanine
PubMed: 37968435
DOI: 10.1007/s00604-023-06050-0 -
Chemical Science Nov 2023The human selenoprotein H is the only selenocysteine-containing protein that is located in the cell's nucleolus. studies have suggested that it plays some role in DNA...
The human selenoprotein H is the only selenocysteine-containing protein that is located in the cell's nucleolus. studies have suggested that it plays some role in DNA binding, consumption of reactive oxygen species, and may serve as a safeguard against cancers. However, the protein has never been isolated and, as a result, not yet fully characterized. Here, we used a semi-synthetic approach to obtain the full selenoprotein H with a S43T mutation. Using biolayer interferometry, we also show that the Cys-containing mutant of selenoprotein H is capable of binding DNA with sub-micromolar affinity. Employing state-of-the-art expressed protein ligation (EPL), our devised semi-synthetic approach can be utilized for the production of numerous, hard-to-obtain proteins of biological and therapeutic relevance.
PubMed: 38020378
DOI: 10.1039/d3sc03059h -
Analytical Biochemistry Jan 2024Aptamers are widely used in various biomedical areas as novel molecular recognition elements, however, short single-stranded DNA (ssDNA) or RNA oligonucleotides are... (Review)
Review
Aptamers are widely used in various biomedical areas as novel molecular recognition elements, however, short single-stranded DNA (ssDNA) or RNA oligonucleotides are easily degraded by nucleases in biological fluids. This problem can be solved by circularizing aptamers with circular ligases. Herein, a moderately thermostable ssDNA ligase was expressed and purified. The purified ligase showed good circularization activity for different length substrates and much higher circularization efficiency than T4 RNA ligase 1. Biochemical characterization revealed that the enzyme showed optimal circularization activity at pH 7.5 and 50 C. Mn and Mg increased enzyme circularization activity, with Mn having higher activity than Mg. The optimal concentrations of Mn and ligase were 1.25-2.5 mM and 0.02 nM, respectively. The kinetic parameters K, V and K of ssDNA ligase were 1.16 μM, 10.71 μM/min, and 10.7 min, respectively. The ssDNA ligase efficiency was nucleotide-dependent, and 5'-G and 3'-T were the most ligase-favored terminal nucleotides. In addition, the affinity and stability of the circular aptamer were determined. The affinity constant (K) was 4.9 μM, and the stability increased compared to its linear form. Molecular docking results showed that the circular aptamer bound to the target via two hydrogen bonds. This study provides a simple and efficient aptamer circularization modification method for improving aptamer stability and expanding its applications.
Topics: DNA, Single-Stranded; Ligases; Molecular Docking Simulation; RNA; Aptamers, Nucleotide
PubMed: 38006953
DOI: 10.1016/j.ab.2023.115409 -
The Analyst Nov 2023Specific recognition and sensitive quantification of mRNA alternative splice variants have been a necessity for exploring the regulatory mechanism of RNA splicing and...
Specific recognition and sensitive quantification of mRNA alternative splice variants have been a necessity for exploring the regulatory mechanism of RNA splicing and revealing the association between pre-mRNA splicing and transcriptome function, as well as disease diagnosis. However, their wide abundance range and high sequence homology pose enormous challenges for high sensitivity and selectivity quantification of splice variants. Herein, taking advantage of the excellent specificity of ligation and the powerful nucleic acid replication feature of loop-mediated isothermal amplification (LAMP), we developed a one-pot method (termed one-pot ligation-LAMP) for specific recognition and sensitive quantification of mRNA splicing variants based on two splicing junction-specific stem-loop DNA probe ligation and the subsequently initiating LAMP. The one-pot ligation-LAMP can specifically detect as low as 100 aM mRNA splice variants without any nonspecific signals and quantify them with a wide dynamics range spanning at least six orders of magnitude. We have demonstrated that the one-pot ligation-LAMP is a versatile and practical strategy for accurately quantifying different splicing variants in complex biological samples with high sensitivity all in one tube within 90 min, thereby providing an attractive tool for mRNA splice variant-related studies.
Topics: RNA, Messenger; Nucleic Acid Amplification Techniques; DNA Probes; Sensitivity and Specificity
PubMed: 37818948
DOI: 10.1039/d3an01382k -
Journal of Biological Methods 2023Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a...
Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation. We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5' end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.
PubMed: 38023773
DOI: 10.14440/jbm.2023.411 -
Nature Chemistry Apr 2024The development of mirror-image biology systems and related applications is hindered by the lack of effective methods to sequence mirror-image (D-) proteins. Although...
The development of mirror-image biology systems and related applications is hindered by the lack of effective methods to sequence mirror-image (D-) proteins. Although natural-chirality (L-) proteins can be sequenced by bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS), the sequencing of long D-peptides and D-proteins with the same strategy requires digestion by a site-specific D-protease before mass analysis. Here we apply solid-phase peptide synthesis and native chemical ligation to chemically synthesize a mirror-image version of trypsin, a widely used protease for site-specific protein digestion. Using mirror-image trypsin digestion and LC-MS/MS, we sequence a mirror-image large subunit ribosomal protein (L25) and a mirror-image Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and distinguish between different mutants of D-Dpo4. We also perform writing and reading of digital information in a long D-peptide of 50 amino acids. Thus, mirror-image trypsin digestion in conjunction with LC-MS/MS may facilitate practical applications of D-peptides and D-proteins as potential therapeutic and informational tools.
Topics: Chromatography, Liquid; Trypsin; Tandem Mass Spectrometry; Proteins; Peptides; Digestion
PubMed: 38238467
DOI: 10.1038/s41557-023-01411-x -
Cancers Dec 2023Patients diagnosed with epithelial ovarian cancer may undergo reflex tumour / testing followed by germline testing in patients with a positive tumour test result. This...
Patients diagnosed with epithelial ovarian cancer may undergo reflex tumour / testing followed by germline testing in patients with a positive tumour test result. This testing model relies on tumour / tests being able to detect all types of pathogenic variant. We analysed germline and tumour test results from patients treated for epithelial ovarian cancer at our specialist oncological referral centre. Tumour testing was performed using the next-generation sequencing (NGS)-based myChoice companion diagnostic (CDx; Myriad Genetics, Inc.). Germline testing was performed in the North West Genomic Laboratory Hub using NGS and multiplex ligation-dependent probe amplification. Between 11 April 2021 and 11 October 2023, 382 patients were successfully tested for tumour and variants. Of these, 367 (96.1%) patients were tested for germline / variants. In those patients who underwent tumour and germline testing, 15.3% (56/367) had a / pathogenic variant (36 germline and 20 somatic). All germline pathogenic small sequencing variants were detected in tumour DNA. By contrast, 3 out of 8 germline pathogenic large rearrangements were not reported in tumour DNA. The overall concordance of germline pathogenic variants detected in germline and tumour DNA was clinically acceptable at 91.7% (33/36). The myChoice CDx was able to detect most germline pathogenic variants in tumour DNA, although a proportion of pathogenic large rearrangements were not reported. If Myriad's myChoice CDx is used for tumour testing, our data supports a testing strategy of germline and tumour testing in all patients diagnosed with epithelial ovarian cancer aged < 79 years old, with germline testing only necessary for patients aged ≥ 80 years old with a tumour pathogenic variant.
PubMed: 38201604
DOI: 10.3390/cancers16010177 -
Human DNA ligases I and IIIα as determinants of accuracy and efficiency of base excision DNA repair.Biochimie Apr 2024Mammalian Base Excision Repair (BER) DNA ligases I and IIIα (LigI, LigIIIα) are major determinants of DNA repair fidelity, alongside with DNA polymerases. Here we...
Mammalian Base Excision Repair (BER) DNA ligases I and IIIα (LigI, LigIIIα) are major determinants of DNA repair fidelity, alongside with DNA polymerases. Here we compared activities of human LigI and LigIIIα on specific and nonspecific substrates representing intermediates of distinct BER sub-pathways. The enzymes differently discriminate mismatches in the nicked DNA, depending on their identity and position, but are both more selective against the 3'-end non-complementarity. LigIIIα is less active than LigI in premature ligation of one-nucleotide gapped DNA and more efficiently discriminates misinsertion products of DNA polymerase β-catalyzed gap filling, that reinforces a leading role of LigIIIα in the accuracy of short-patch BER. LigI and LigIIIα reseal the intermediate of long-patch BER containing an incised synthetic AP site (F) with different efficiencies, depending on the DNA sequence context, 3'-end mismatch presence and coupling of the ligation reaction with DNA repair synthesis. Processing of this intermediate in the absence of flap endonuclease 1 generates non-canonical DNAs with bulged F site, which are very inefficiently repaired by AP endonuclease 1 and represent potential mutagenic repair products. The extent of conversion of the 5'-adenylated intermediates of specific and nonspecific substrates is revealed to depend on the DNA sequence context; a higher sensitivity of LigI to the sequence is in line with the enzyme structural feature of DNA binding. LigIIIα exceeds LigI in generation of potential abortive ligation products, justifying importance of XRCC1-mediated coordination of LigIIIα and aprataxin activities for the efficient DNA repair.
Topics: Animals; Humans; DNA Repair; DNA; DNA Polymerase beta; DNA-Directed DNA Polymerase; DNA Ligases; Excision Repair; Mammals; X-ray Repair Cross Complementing Protein 1
PubMed: 37573020
DOI: 10.1016/j.biochi.2023.08.007 -
Hematology (Amsterdam, Netherlands) Dec 2023-thalassemia is a common inherited hemolytic disorder caused by mutations in the HBB gene. Genetic analysis of 2 new beta-thalassemia patients with deletion mutations in...
BACKGROUND
-thalassemia is a common inherited hemolytic disorder caused by mutations in the HBB gene. Genetic analysis of 2 new beta-thalassemia patients with deletion mutations in the HBB gene and their family members.
METHODS
Their clinical presentation and blood phenotypic tests were analyzed. We detected the approximate degree of deletion of these two new HBB gene deletion mutants and analyzed their specific deletion locations by multiplex ligation-dependent probe amplification (MLPA), reverse breakpoint polymerase chain reaction (GAP-PCR), and sanger DNA sequencing.
RESULTS
Two new deletion mutants of the HBB gene were identified. First, a 49% decrease in the expression of the third exon of the HBB gene was detected by MLPA testing, and then proband 1 and her mother were found to have HBB: exon3del and proband 2 and her mother to have HBB: c.-81A > C by GAP-PCR and sanger sequencing.
CONCLUSION
When the blood phenotype and clinical manifestations do not match the genotype, the presence of new mutants should be considered, and attention should be paid to further testing to avoid missing the diagnosis, which can help in clinical diagnosis and treatment, prenatal diagnosis and genetic counseling.
Topics: Pregnancy; Female; Humans; beta-Thalassemia; Genotype; Mutation; Phenotype; alpha-Thalassemia; beta-Globins
PubMed: 37815396
DOI: 10.1080/16078454.2023.2265723 -
Scientific Reports Sep 2023DNA is a promising candidate for long-term data storage due to its high density and endurance. The key challenge in DNA storage today is the cost of synthesis. In this...
DNA is a promising candidate for long-term data storage due to its high density and endurance. The key challenge in DNA storage today is the cost of synthesis. In this work, we propose composite motifs, a framework that uses a mixture of prefabricated motifs as building blocks to reduce synthesis cost by scaling logical density. To write data, we introduce Bridge Oligonucleotide Assembly, an enzymatic ligation technique for synthesizing oligos based on composite motifs. To sequence data, we introduce Direct Oligonucleotide Sequencing, a nanopore-based technique to sequence short oligos, eliminating common preparatory steps like DNA assembly, amplification and end-prep. To decode data, we introduce Motif-Search, a novel consensus caller that provides accurate reconstruction despite synthesis and sequencing errors. Using the proposed methods, we present an end-to-end experiment where we store the text "HelloWorld" at a logical density of 84 bits/cycle (14-42× improvement over state-of-the-art).
Topics: DNA; Oligonucleotides; Consensus; Nanopores; Nutritional Status
PubMed: 37749195
DOI: 10.1038/s41598-023-43172-0