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1.
PLoS One ; 19(5): e0301234, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38728290

RESUMO

Nucleic Acid Lateral Flow Assays (NALFAs) are a promising solution for the point-of-care detection of viruses like SARS-CoV-2. However, they show some drawbacks, such as the great dependency on the use of antibodies and the need for post-amplification protocols that enable the preparation of amplicons for effective readings, as well as low sensitivity. Here, we developed amplicons of a specific SARS-CoV-2 gene tailed with single-strand DNA (ssDNA) sequences to hybridize with DNA probes immobilized on the NALFA strips, thus overcoming the aforementioned problems. Results have shown that tailed primers have not compromised the amplification efficiency and allowed the correct detection of the amplicons in the lateral flow strip. This approach has presented a limit of detection (LOD) of 25 RNA copies /reaction mix (1 copy/µL) and the test of cross-reactivity with other related viruses has not shown any cross-reactivity. Twenty clinical samples were evaluated by NALFA and simultaneously compared with the gold standard RT-qPCR protocol, originating equal results. Although the number of clinical specimens tested being relatively small, this indicates a sensitivity and specificity both of 100%. In short, an alternative NALFA was successfully implemented, rendering an accurate route for SARS-CoV-2 diagnosis, compatible with low-resource settings.


Assuntos
COVID-19 , RNA Viral , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Humanos , COVID-19/diagnóstico , COVID-19/virologia , RNA Viral/genética , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Sensibilidade e Especificidade , Teste de Ácido Nucleico para COVID-19/métodos , DNA de Cadeia Simples/genética , Primers do DNA/genética , Sondas de DNA
2.
J Nanobiotechnology ; 22(1): 237, 2024 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-38735920

RESUMO

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) promote tumor growth, metastasis, and lead to immunotherapy resistance. Studies revealed that miRNAs are also expressed in MDSCs and promote the immunosuppressive function of MDSCs. Currently, few studies have been reported on inducible cellular microvesicle delivery of nucleic acid drugs targeting miRNA in MDSCs for the treatment of malignant tumors. RESULTS AND CONCLUSION: In this study, we designed an artificial DNA named G-quadruplex-enhanced circular single-stranded DNA-9 (G4-CSSD9), that specifically adsorbs the miR-9 sequence. Its advanced DNA folding structure, rich in tandem repeat guanine (G-quadruplex), also provides good stability. Mesenchymal stem cells (MSCs) were prepared into nanostructured vesicles by membrane extrusion. The MSC microvesicles-encapsulated G4-CSSD9 (MVs@G4-CSSD9) was delivered into MDSCs, which affected the downstream transcription and translation process, and reduced the immunosuppressive function of MDSCs, so as to achieve the purpose of treating melanoma. In particular, it provides an idea for the malignant tumor treatment.


Assuntos
DNA de Cadeia Simples , Quadruplex G , Células-Tronco Mesenquimais , MicroRNAs , Células Supressoras Mieloides , Animais , Células Supressoras Mieloides/metabolismo , Camundongos , DNA de Cadeia Simples/química , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/metabolismo , DNA Circular/química , Humanos , Melanoma/tratamento farmacológico
3.
ACS Nano ; 18(19): 12401-12411, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38701333

RESUMO

Accurate identification of single nucleotide variants (SNVs) in key driver genes holds a significant value for disease diagnosis and treatment. Fluorescent probes exhibit tremendous potential in specific, high-resolution, and rapid detection of SNVs. However, additional steps are required in most post-PCR assays to convert double-stranded DNA (dsDNA) products into single-stranded DNA (ssDNA), enabling them to possess hybridization activity to trigger subsequent reactions. This process not only prolongs the complexity of the experiment but also introduces the risk of losing target information. In this study, we proposed two strategies for enriching active double-stranded DNA, involving PCR based on obstructive groups and cleavable units. Building upon this, we explored the impact of modified units on the strand displacement reaction (SDR) and assessed their discriminatory efficacy for mutations. The results showed that detection of low variant allele frequencies (VAF) as low as 0.1% can be achieved. The proposed strategy allowed orthogonal identification of 45 clinical colorectal cancer tissue samples with 100% specificity, and the results were generally consistent with sequencing results. Compared to existing methods for enriching active targets, our approach offers a more diverse set of enrichment strategies, characterized by the advantage of being simple and fast and preserving original information to the maximum extent. The objective of this study is to offer an effective solution for the swift and facile acquisition of active double-stranded DNA. We anticipate that our work will facilitate the practical applications of SDR based on dsDNA.


Assuntos
DNA , Polimorfismo de Nucleotídeo Único , Polimorfismo de Nucleotídeo Único/genética , Humanos , DNA/genética , DNA/química , Neoplasias Colorretais/genética , Reação em Cadeia da Polimerase , Corantes Fluorescentes/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/química
4.
Proc Natl Acad Sci U S A ; 121(19): e2318438121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38696464

RESUMO

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.


Assuntos
DNA de Cadeia Simples , Homeostase do Telômero , Telômero , Telômero/genética , Telômero/metabolismo , Humanos , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Replicação do DNA , DNA/genética , DNA/metabolismo , DNA Circular/genética , DNA Circular/metabolismo , Southern Blotting , DNA Polimerase III/metabolismo , DNA Polimerase III/genética
5.
Bioorg Med Chem Lett ; 106: 129774, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38688438

RESUMO

Herein, we constructed a fluorescence biosensor for the ultra-sensitive analysis of microRNAs (miRNAs) by combining DNA hairpins transition triggered strand displacement amplification (DHT-SDA) with primer exchange reaction (PER). Target miRNA initiated DHT-SDA to facilitate the generation of multiple single-stranded DNA (ssDNA) as PER primer, which was extended into a long ssDNA. The biosensor is successfully utilized in detecting miRNAs with high sensitivity (limit of detection for miRNA-21 was 58 fM) and a good linear relationship between 100 nM and 100 fM. By simply changing the DNA hairpin sequence, the constructed biosensor can be extended to analyze another miRNAs. Moreover, the biosensor has the feasibility of detecting miRNAs in real samples with satisfactory accuracy and reliability. Therefore, the fluorescent biosensor has great application potential in clinical diagnosis.


Assuntos
Técnicas Biossensoriais , MicroRNAs , Técnicas de Amplificação de Ácido Nucleico , MicroRNAs/metabolismo , MicroRNAs/análise , Humanos , DNA/química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Fluorescência , Sequências Repetidas Invertidas , Espectrometria de Fluorescência , Limite de Detecção , Primers do DNA/química
6.
Anal Chim Acta ; 1303: 342477, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38609257

RESUMO

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.


Assuntos
MicroRNAs , Ácidos Nucleicos , MicroRNAs/genética , Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , DNA de Cadeia Simples/genética
7.
Anal Chem ; 96(18): 6930-6939, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38652001

RESUMO

Circulating tumor DNA (ctDNA) holds great promise as a noninvasive biomarker for cancer diagnosis, treatment, and prognosis. However, the accurate and specific quantification of low-abundance ctDNA in serum remains a significant challenge. This study introduced, for the first time, a novel exponential amplification reaction (EXPAR)-assisted CRISPR/Cas12a-mediated ratiometric dual-signal electrochemical biosensor for ultrasensitive and reliable detection of ctDNA. To implement the dual-signal strategy, a signal unit (ssDNA-MB@Fc/UiO-66-NH2) was prepared, consisting of methylene blue-modified ssDNA as the biogate to encapsulate ferrocene signal molecules within UiO-66-NH2 nanocarriers. The presence of target ctDNA KRAS triggered EXPAR amplification, generating numerous activators for Cas12a activation, resulting in the cleavage of ssDNA-P fully complementary to the ssDNA-MB biogate. Due to the inability to form a rigid structure dsDNA (ssDNA-MB/ssDNA-P), the separation of ssDNA-MB biogate from the UiO-66-NH2 surface was hindered by electrostatic interactions. Consequently, the supernatant collected after centrifugation exhibited either no or only a weak presence of Fc and MB signal molecules. Conversely, in the absence of the target ctDNA, the ssDNA-MB biogate was open, leading to the leakage of Fc signal molecules. This clever ratiometric strategy with Cas12a as the "connector", reflecting the concentration of ctDNA KRAS based on the ratio of the current intensities of the two electroactive signal molecules, enhanced detection sensitivity by at least 60-300 times compared to single-signal strategies. Moreover, this strategy demonstrated satisfactory performance in ctDNA detection in complex human serum, highlighting its potential for cancer diagnosis.


Assuntos
Técnicas Biossensoriais , DNA Tumoral Circulante , Técnicas Eletroquímicas , Humanos , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Sistemas CRISPR-Cas/genética , DNA de Cadeia Simples/química , Limite de Detecção , Endodesoxirribonucleases/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Associadas a CRISPR/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética
8.
Langmuir ; 40(18): 9622-9629, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38652583

RESUMO

The detection and identification of nanoscale molecules are crucial, but traditional technology comes with a high cost and requires skilled operators. Solid-state nanopores are new powerful tools for discerning the three-dimensional shape and size of molecules, enabling the translation of molecular structural information into electric signals. Here, DNA molecules with different shapes were designed to explore the effects of electroosmotic forces (EOF), electrophoretic forces (EPF), and volume exclusion on electric signals within solid-state nanopores. Our results revealed that the electroosmotic force was the main driving force for single-stranded DNA (ssDNA), whereas double-stranded DNA (dsDNA) was primarily dominated by electrophoretic forces in nanopores. Moreover, dsDNA caused greater amplitude signals and moved faster through the nanopore due to its larger diameter and carrying more charges. Furthermore, at the same charge level and amount of bases, circular dsDNA exhibited a tighter structure compared to brush DNA, resulting in a shorter length. Consequently, circular dsDNA caused higher current-blocking amplitudes and faster passage speeds. The characterization approach based on nanopores allows researchers to get molecular information about size and shape in real time. These findings suggest that nanopore detection has the potential to streamline nanoscale characterization and analysis, potentially reducing both the cost and complexity.


Assuntos
DNA , Nanoporos , DNA/química , Conformação de Ácido Nucleico , DNA de Cadeia Simples/química , Eletro-Osmose/métodos
9.
Mol Cell ; 84(9): 1684-1698.e9, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38593805

RESUMO

The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.


Assuntos
Dano ao DNA , Replicação do DNA , RecQ Helicases , Homeostase do Telômero , Telômero , RecQ Helicases/metabolismo , RecQ Helicases/genética , Humanos , Telômero/metabolismo , Telômero/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Síndrome de Bloom/enzimologia , Síndrome de Bloom/patologia , Linhagem Celular Tumoral
10.
Int J Biol Macromol ; 267(Pt 2): 131509, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38608978

RESUMO

Giardia intestinalis is one of the most widespread intestinal parasites and is considered a major cause of epidemic or sporadic diarrhea worldwide. In this study, we aimed to develop a rapid aptameric diagnostic technique for G. intestinalis infection. First, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process generated DNA aptamers specific to a recombinant protein of the parasite's trophozoite. Ten selection rounds were performed; each round, the DNA library was incubated with the target protein conjugated to Sepharose beads. Then, the unbound sequences were removed by washing and the specific sequences were eluted and amplified by Polymerase Chain Reaction (PCR). Two aptamers were selected, and the dissociation constants (Kd), were determined as 2.45 and 16.95 nM, showed their high affinity for the G. intestinalis trophozoite protein. Subsequently, the aptamer sequence T1, which exhibited better affinity, was employed to develop a label-free electrochemical biosensor. A thiolated aptamer was covalently immobilized onto a gold screen-printed electrode (SPGE), and the binding of the targeted protein was monitored using square wave voltammetry (SWV). The developed aptasensor enabled accurate detection of the G. intestinalis recombinant protein within the range of 0.1 pg/mL to 100 ng/mL, with an excellent sensitivity (LOD of 0.35 pg/mL). Moreover, selectivity studies showed a negligible cross-reactivity toward other proteins such as bovine serum albumin, globulin, and G. intestinalis cyst protein.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Eletroquímicas , Giardia lamblia , Proteínas de Protozoários , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Técnica de Seleção de Aptâmeros/métodos , Técnicas Eletroquímicas/métodos , Proteínas de Protozoários/química , DNA de Cadeia Simples/química , Giardíase/diagnóstico , Giardíase/parasitologia
11.
Sci Adv ; 10(15): eadk8791, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38608016

RESUMO

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.


Assuntos
DNA Polimerase Dirigida por RNA , RNA , DNA Complementar/genética , RNA/genética , DNA Polimerase Dirigida por RNA/genética , DNA/genética , DNA de Cadeia Simples
12.
Molecules ; 29(7)2024 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-38611724

RESUMO

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.


Assuntos
Compostos Heterocíclicos , Tetraciclina , Antibacterianos , Reciclagem , Carbono , DNA de Cadeia Simples
13.
Mol Cell ; 84(8): 1460-1474.e6, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38640894

RESUMO

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.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA , DNA Polimerase Dirigida por DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples/genética , DNA Helicases/genética , Reparo do DNA por Junção de Extremidades
14.
Chem Commun (Camb) ; 60(35): 4723-4726, 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38597243

RESUMO

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.


Assuntos
DNA de Cadeia Simples , Nanoporos , Técnicas de Amplificação de Ácido Nucleico , DNA de Cadeia Simples/química
15.
Biochemistry ; 63(8): 969-983, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38623046

RESUMO

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.


Assuntos
DNA Polimerase III , Replicação do DNA , Humanos , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA de Cadeia Simples/genética , Holoenzimas/genética , Repetições de Microssatélites , Nucleotídeos
16.
Sci Rep ; 14(1): 9550, 2024 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-38664461

RESUMO

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.


Assuntos
Quebras de DNA de Cadeia Dupla , Meiose , Proteína de Replicação A , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Transcrição , Proteína de Replicação A/metabolismo , Proteína de Replicação A/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Genética , Recombinação Homóloga , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 1/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética
17.
BMC Genomics ; 25(1): 368, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38622509

RESUMO

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.


Assuntos
Quebras de DNA de Cadeia Simples , Reparo do DNA , Animais , Reparo do DNA/genética , Dano ao DNA , DNA de Cadeia Simples , Mamíferos
18.
J Agric Food Chem ; 72(15): 8831-8839, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38575365

RESUMO

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.


Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , Animais , Bovinos , Corantes , DNA de Cadeia Simples , Recombinases , Salmonella/genética , Reação em Cadeia da Polimerase
19.
Biotechnol J ; 19(4): e2400026, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38622795

RESUMO

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.


Assuntos
DNA de Cadeia Simples , DNA , DNA de Cadeia Simples/genética , Reação em Cadeia da Polimerase/métodos , DNA Polimerase Dirigida por DNA , Oligonucleotídeos , Técnicas de Amplificação de Ácido Nucleico/métodos
20.
Chirality ; 36(4): e23664, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38561319

RESUMO

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.


Assuntos
DNA , Rad51 Recombinase , Rad51 Recombinase/química , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Estereoisomerismo , DNA/química , DNA de Cadeia Simples
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