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The Analyst Jun 2024Single nucleotide variants (SNVs) play a crucial role in understanding genetic diseases, cancer development, and personalized medicine. However, existing ligase-based...
Single nucleotide variants (SNVs) play a crucial role in understanding genetic diseases, cancer development, and personalized medicine. However, existing ligase-based amplification and detection techniques, such as Rolling Circle Amplification and Ligase Detection Reaction, suffer from low efficiency and difficulties in product detection. To address these limitations, we propose a novel approach that combines Ligase Chain Reaction (LCR) with acoustic detection using highly dissipative liposomes. In our study, we are using LCR combined with biotin- and cholesterol-tagged primers to produce amplicons also modified at each end with a biotin and cholesterol molecule. We then apply the LCR mix without any purification directly on a neutravidin modified QCM device Au-surface, where the produced amplicons can bind specifically through the biotin end. To improve sensitivity, we finally introduce liposomes as signal enhancers. For demonstration, we used the detection of the BRAF V600E point mutation the wild-type allele, achieving an impressive detection limit of 220 aM of the mutant target in the presence of the same amount of the wild type. Finally, we combined the assay with a microfluidic fluidized bed DNA extraction technology, offering the potential for semi-automated detection of SNVs in patients' crude samples. Overall, our LCR/acoustic method outperforms other LCR-based approaches and surface ligation biosensing techniques in terms of detection efficiency and time. It effectively overcomes challenges related to DNA detection, making it applicable in diverse fields, including genetic disease and pathogen detection.
Topics: Liposomes; Humans; Limit of Detection; Ligase Chain Reaction; Proto-Oncogene Proteins B-raf; Polymorphism, Single Nucleotide; Biotin; Acoustics; Avidin; Quartz Crystal Microbalance Techniques; Gold; DNA; Cholesterol; Point Mutation
PubMed: 38758167
DOI: 10.1039/d3an02142d -
Scientific Reports May 2024Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of...
Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5'- and 3'-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.
Topics: SARS-CoV-2; Software; Polymerase Chain Reaction; Oligonucleotides; COVID-19; Computational Biology; DNA, Single-Stranded
PubMed: 38750104
DOI: 10.1038/s41598-024-59497-3 -
Scientific Reports May 2024MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal...
MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-β (PDGFR-β)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-β. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-β. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p.
Topics: Animals; MicroRNAs; Fibrosis; Mice; Mice, Knockout; Receptor, Platelet-Derived Growth Factor beta; Kidney; Mesenchymal Stem Cells; Ribonuclease III; Signal Transduction; Kidney Diseases; DEAD-box RNA Helicases; Male
PubMed: 38745066
DOI: 10.1038/s41598-024-61560-y -
Mutagenesis May 2024DNA ligase (LIG) I and IIIα finalize base excision repair (BER) by sealing a nick product after nucleotide insertion by DNA polymerase (pol) β at the downstream steps....
DNA ligase (LIG) I and IIIα finalize base excision repair (BER) by sealing a nick product after nucleotide insertion by DNA polymerase (pol) β at the downstream steps. We previously demonstrated that a functional interplay between polβ and BER ligases is critical for efficient repair, and polβ mismatch or oxidized nucleotide insertions confound final ligation step. Yet, how targeting downstream enzymes with small molecule inhibitors could affect this coordination remains unknown. Here, we report that DNA ligase inhibitors, L67 and L82-G17, slightly enhance hypersensitivity to oxidative stress-inducing agent, KBrO3, in polβ+/+ cells more than polβ-/- null cells. We showed less efficient ligation after polβ nucleotide insertions in the presence of the DNA ligase inhibitors. Furthermore, the mutations at the ligase inhibitor binding sites (G448, R451, A455) of LIG1 significantly affect nick DNA binding affinity and nick sealing efficiency. Finally, our results demonstrated that the BER ligases seal a gap repair intermediate by the effect of polβ inhibitor that diminishes gap filling activity. Overall, our results contribute to understand how the BER inhibitors against downstream enzymes, polβ, LIG1, and LIGIIIα, could impact the efficiency of gap filling and subsequent nick sealing at the final steps leading to the formation of deleterious repair intermediates.
PubMed: 38736258
DOI: 10.1093/mutage/geae013 -
Cancers Apr 2024Gene rearrangements affecting are frequent in acute myeloid leukemia (AML) and are often associated with a poor prognosis. gene fusions are often detected by...
BACKGROUND
Gene rearrangements affecting are frequent in acute myeloid leukemia (AML) and are often associated with a poor prognosis. gene fusions are often detected by chromosome banding analysis and confirmed by fluorescence in situ hybridization. However, small intragenic insertions, termed partial tandem duplication (KMT2A-PTD), are particularly challenging to detect using standard molecular and cytogenetic approaches.
METHODS
We have validated the use of a custom hybrid-capture-based next-generation sequencing (NGS) panel for comprehensive profiling of AML patients seen at our institution. This NGS panel targets the entire consensus coding DNA sequence of . To deduce the presence of a KMT2A-PTD, we used the relative ratio of exons coverage. We sought to corroborate the KMT2A-PTD NGS results using (1) multiplex-ligation probe amplification (MLPA) and (2) optical genome mapping (OGM).
RESULTS
We analyzed 932 AML cases and identified 41 individuals harboring a KMT2A-PTD. MLPA, NGS, and OGM confirmed the presence of a KMT2A-PTD in 22 of the cases analyzed where orthogonal testing was possible. The two false-positive KMT2A-PTD calls by NGS could be explained by the presence of cryptic structural variants impacting and interfering with KMT2A-PTD analysis. OGM revealed the nature of these previously undetected gene rearrangements in , while MLPA yielded inconclusive results. MLPA analysis for KMT2A-PTD is limited to exon 4, whereas NGS and OGM resolved KMT2A-PTD sizes and copy number levels.
CONCLUSIONS
KMT2A-PTDs are complex gene rearrangements that cannot be fully ascertained using a single genomic platform. MLPA, NGS panels, and OGM are complementary technologies applied in standard-of-care testing for AML patients. MLPA and NGS panels are designed for targeted copy number analysis; however, our results showed that integration of concurrent genomic alterations is needed for accurate KMT2A-PTD identification. Unbalanced chromosomal rearrangements overlapping with can interfere with the diagnostic sensitivity and specificity of copy-number-based KMT2A-PTD detection methodologies.
PubMed: 38730645
DOI: 10.3390/cancers16091693 -
European Journal of Ophthalmology May 2024Leber hereditary optic neuropathy (LHON) is an inherited progressive optic neuropathy usually caused by mitochondrial DNA mutations. Recently, autosomal recessive...
BACKGROUND
Leber hereditary optic neuropathy (LHON) is an inherited progressive optic neuropathy usually caused by mitochondrial DNA mutations. Recently, autosomal recessive (arLHON), which is caused by biallelic mutations in the gene (usually c.152A > G), has been described. The onset of LHON before the age of 12 is uncommon and it is typically associated with a more variable clinical course and a more favorable visual prognosis than adult-onset LHON.
MATERIALS AND METHODS
Detailed clinical findings of a female child with vision loss due to arLHON together with choroideremia (CHM) carrier state are presented.
RESULTS
Genetic testing for the three most common mitochondrial LHON pathogenic variants was negative. On suspicion of arLHON, genetic testing was continued with the next-generation sequencing (NGS) of the nuclear DNA, identifying a homozygous pathogenic variant in c.152A > G, p.(Tyr51Cys), but no alterations in the gene. Idebenone treatment was started 4.5 months after the first evaluation. Clinical diagnosis of the CHM carrier state was confirmed by multiplex ligation-dependent probe amplification (MLPA) assay, which revealed a heterozygous deletion of all exons of the .
CONCLUSIONS
In children with acute or subacute, simultaneous, or sequential vision loss that is unresponsive to immunomodulatory treatment, LHON should be considered as a possible diagnosis. Our case emphasizes the diagnostic advantage of sequencing in parallel with the mitochondrial DNA, especially in Eastern European descent patients. Genomic rearrangement testing should be considered for patients with a CHM carrier phenotype who have negative results on sequencing tests.
PubMed: 38715355
DOI: 10.1177/11206721241254408 -
BMC Pregnancy and Childbirth May 2024This study aims to perform a prenatal genetic diagnosis of a high-risk fetus with trisomy 7 identified by noninvasive prenatal testing (NIPT) and to evaluate the...
OBJECTIVE
This study aims to perform a prenatal genetic diagnosis of a high-risk fetus with trisomy 7 identified by noninvasive prenatal testing (NIPT) and to evaluate the efficacy of different genetic testing techniques for prenatal diagnosis of trisomy mosaicism.
METHODS
For prenatal diagnosis of a pregnant woman with a high risk of trisomy 7 suggested by NIPT, karyotyping and chromosomal microarray analysis (CMA) were performed on an amniotic fluid sample. Low-depth whole-genome copy number variation sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) were used to clarify the results further. In addition, methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was performed to analyze the possibility of uniparental disomy(UPD).
RESULTS
Amniotic fluid karyotype analysis revealed a 46, XX result. Approximately 20% mosaic trisomy 7 was detected according to the CMA result. About 16% and 4% of mosaicism was detected by CNV-seq and FISH, respectively. MS-MLPA showed no methylation abnormalities. The fetal ultrasound did not show any detectable abnormalities except for mild intrauterine growth retardation seen at 39 weeks of gestation. After receiving genetic counseling, the expectant mother decided to continue the pregnancy, and follow-up within three months of delivery was normal.
CONCLUSION
In high-risk NIPT diagnosis, a combination of cytogenetic and molecular genetic techniques proves fruitful in detecting low-level mosaicism. Furthermore, the exclusion of UPD on chromosome 7 remains crucial when NIPT indicates a positive prenatal diagnosis of trisomy 7.
Topics: Humans; Female; Mosaicism; Pregnancy; In Situ Hybridization, Fluorescence; Chromosomes, Human, Pair 7; Trisomy; Karyotyping; Adult; Uniparental Disomy; DNA Copy Number Variations; Prenatal Diagnosis; Microarray Analysis; Noninvasive Prenatal Testing; Multiplex Polymerase Chain Reaction; Amniotic Fluid
PubMed: 38702634
DOI: 10.1186/s12884-024-06522-y -
Analytical Methods : Advancing Methods... May 2024Small extracellular vesicles (sEVs) are a type of extracellular vesicle that carries many types of molecular information. The identification of sEVs is essential for the...
Small extracellular vesicles (sEVs) are a type of extracellular vesicle that carries many types of molecular information. The identification of sEVs is essential for the non-invasive detection and treatment of illnesses. Hence, there is a significant need for the development of simple, sensitive, and precise methods for sEV detection. Herein, a DNA tweezers-based assay utilizing a "turn-on" mechanism and proximity ligation was suggested for the efficient and rapid detection of sEVs through amplified fluorescence. The target facilitates the proximity combination of the C1 probe and C2 probe, resulting in the formation of a complete extended sequence. The elongated sequence can cyclically initiate the hairpin probe (HP), leading to the activation of DNA tweezers. An excellent linear correlation was achieved, with a limit of detection of 57 particles per μL. Furthermore, it has been effectively employed to analyze sEVs under intricate experimental conditions, demonstrating a promising and pragmatic prospect for future applications. Given that the identification of sEVs was successfully accomplished using a single-step method that exhibited exceptional sensitivity and strong resistance to interference, the proposed technique has the potential to provide a beneficial platform for accurate recognition of sEVs and early detection of diseases.
Topics: Extracellular Vesicles; Nucleic Acid Hybridization; Humans; DNA; Limit of Detection; Biosensing Techniques
PubMed: 38699853
DOI: 10.1039/d4ay00487f -
Journal of the American Chemical Society May 2024Cisplatin, a cornerstone in cancer chemotherapy, is known for its DNA-binding capacity and forms lesions that lead to cancer cell death. However, the repair of these...
Cisplatin, a cornerstone in cancer chemotherapy, is known for its DNA-binding capacity and forms lesions that lead to cancer cell death. However, the repair of these lesions compromises cisplatin's effectiveness. This study investigates how phosphorylation of HMGB1, a nuclear protein, modifies its binding to cisplatin-modified DNA (CP-DNA) and thus protects it from repair. Despite numerous methods for detecting protein-DNA interactions, quantitative approaches for understanding their molecular mechanism remain limited. Here, we applied click chemistry-based single-molecule force spectroscopy, achieving high-precision quantification of the interaction between phosphorylated HMGB1 and CP-DNA. This method utilizes a synergy of click chemistry and enzymatic ligation for precise DNA-protein immobilization and interaction in the system. Our results revealed that HMGB1 binds to CP-DNA with a significantly high rupture force of ∼130 pN, stronger than most natural DNA-protein interactions and varying across different DNA sequences. Moreover, Ser14 is identified as the key phosphorylation site, enhancing the interaction's kinetic stability by 35-fold. This increase in stability is attributed to additional hydrogen bonding suggested by molecular dynamics (MD) simulations. Our findings not only reveal the important role of phosphorylated HMGB1 in potentially improving cisplatin's therapeutic efficacy but also provide a precise method for quantifying protein-DNA interactions.
Topics: HMGB1 Protein; Cisplatin; Phosphorylation; Click Chemistry; Molecular Dynamics Simulation; DNA; Humans; Protein Binding; Antineoplastic Agents
PubMed: 38696488
DOI: 10.1021/jacs.4c00224 -
Biosensors & Bioelectronics Aug 2024The emergence and rapid spread of Mpox (formerly monkeypox) have caused significant societal challenges. Adequate and appropriate diagnostics procedures are an urgent...
The emergence and rapid spread of Mpox (formerly monkeypox) have caused significant societal challenges. Adequate and appropriate diagnostics procedures are an urgent necessity. Herein, we discover a pair of aptamers through the systematic evolution of ligands by exponential enrichment (SELEX) that exhibit high affinity and bind to different sites towards the A29 protein of the Mpox virus. Subsequently, we propose a facile, sensitive, convenient CRISPR/Cas12a-mediated aptasensor for detecting the A29 antigen. The procedure employs the bivalent aptamers recognition, which induces the formation of a proximity switch probe and initiates subsequent cascade strand displacement reactions, then triggers CRISPR/Cas12a DNA trans-cleavage to achieve the sensitive detection of Mpox. Our method enables selective and ultrasensitive evaluation of the A29 protein within the range of 1 ng mL to 1 μg mL, with a limit of detection (LOD) at 0.28 ng mL. Moreover, spiked A29 protein recovery exceeds 96.9%, while the detection activity remains above 91.9% after six months of storage at 4 °C. This aptasensor provides a novel avenue for exploring clinical diagnosis in cases involving Mpox as facilitating development in various analyte sensors.
Topics: Biosensing Techniques; Aptamers, Nucleotide; CRISPR-Cas Systems; Limit of Detection; Humans; SELEX Aptamer Technique; Antigens, Viral; CRISPR-Associated Proteins; Bacterial Proteins; Endodeoxyribonucleases
PubMed: 38688229
DOI: 10.1016/j.bios.2024.116313