RESUMEN
Elucidating spatiotemporal changes in gene expression has been an essential goal in studies of health, development, and disease. In the emerging field of spatially resolved transcriptomics, gene expression profiles are acquired with the tissue architecture maintained, sometimes at cellular resolution. This has allowed for the development of spatial cell atlases, studies of cell-cell interactions, and in situ cell typing. In this review, we focus on padlock probe-based in situ sequencing, which is a targeted spatially resolved transcriptomic method. We summarize recent methodological and computational tool developments and discuss key applications. We also discuss compatibility with other methods and integration with multiomic platforms for future applications.
Asunto(s)
Comunicación Celular , Perfilación de la Expresión Génica , Humanos , Multiómica , TranscriptomaRESUMEN
Although RNA plays a vital role in gene expression, it is less used as an in situ biomarker for clinical diagnostics than DNA and protein. This is mainly due to technical challenges caused by the low expression level and easy degradation of RNA molecules. To tackle this issue, methods that are sensitive and specific are needed. Here, we present an RNA single-molecule chromogenic in situ hybridization assay based on DNA probe proximity ligation and rolling circle amplification. When the DNA probes hybridize into close proximity to the RNA molecules, they form a V-shape structure and mediate the circularization of circle probes. Thus, our method was termed vsmCISH. We successfully applied our method to assess HER2 mRNA expression status in invasive breast cancer tissue and investigated the utility of albumin mRNA ISH for differentiating primary from metastatic liver cancer. The promising results on clinical samples indicate that our method has great potential for application in diagnosing diseases using RNA biomarkers.
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ADN , ARN , ARN/genética , Hibridación in Situ , ARN Mensajero/genética , Sondas de ADNRESUMEN
We present a combined amplification-based single-molecule fluorescence in situ hybridization and immunofluorescence (asmFISH-IF) method for the detection of multiple RNAs and proteins simultaneously in cells and formaldehyde-fixed and paraffin-embedded tissue sections. We showed that performing asmFISH before immunofluorescence gives a better IF signal than the opposite. Our asmFISH-IF method could help study the interplay of RNA and protein, helping to understand their functions.
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Formaldehído , ARN , Hibridación Fluorescente in Situ/métodos , Adhesión en Parafina , Fijación del Tejido , ARN/genética , Técnica del Anticuerpo Fluorescente , ProteínasRESUMEN
DNA hydrogels hold significant promise for biomedical applications and can be synthesized through enzymatic Rolling Circle Amplification (RCA). Due to the exploratory nature of this emerging field, standardized RCA protocols specifying the impact of reaction parameters are currently lacking. This study varied template sequences and reagent concentrations, evaluating RCA synthesis efficiency and hydrogel mechanical properties through quantitative PCR (qPCR) and indentation measurements, respectively. Primer concentration and stabilizing additives showed minimal impact on RCA efficiency, while changes in polymerase and nucleotide concentrations had a stronger effect. Concentration of the circular template exerted the greatest influence on RCA productivity. An exponential correlation between hydrogel viscosity and DNA amplicon concentration was observed, with nucleobase sequence significantly affecting both amplification efficiency and material properties, particularly through secondary structures. This study suggests that combining high-throughput experimental methods with structural folding prediction offers a viable approach for systematically establishing structure-property relationships, aiding the rational design of DNA hydrogel material systems.
RESUMEN
Cytosolic DNA sensors (CDSs) recognize DNA molecules that are abnormally located in the cytosol, thus leading to the activation of the stimulator of interferon genes (STING) and the induction of type 1 interferon. In turn, type 1 interferon evokes defensive reactions against viral infections and activates the immune system; therefore, the use of agonists of CDSs as cancer therapeutics and vaccine adjuvants is expected. Double-stranded DNA molecules with dozens to thousands of bases derived from bacteria and viruses are agonists of CDSs. However, DNA is a water-soluble molecule with a high molecular weight, resulting in poor cellular uptake and endosomal escape. In contrast, long single-stranded DNA (lssDNA) obtained by rolling circle amplification is efficiently taken up and localized to endosomes. Here we constructed a CDS-targeting lssDNA via the facilitation of its intracellular transport from endosomes to the cytosol. An endosome-disrupting GALA peptide was used to deliver the lssDNA to the cytosol. A peptide-oligonucleotide conjugate (POC) was successfully obtained via the conjugation of the GALA peptide with an oligonucleotide complementary to the lssDNA. By hybridization of the POC to the complementary lssDNA (POC/lssDNA), the CDS-STING pathway in dendritic cells was efficiently stimulated. GALA peptide-conjugated DNA seems to be a helpful tool for the delivery of DNA to the cytosol.
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ADN de Cadena Simple , Péptidos , Citosol/metabolismo , ADN de Cadena Simple/metabolismo , Péptidos/química , ADN/genética , Interferones/genética , Interferones/metabolismo , Oligonucleótidos/metabolismoRESUMEN
Multiplexed in situ RNA imaging offers new opportunities for gene expression profiling by providing high-throughput spatial information. In this work, we present a cyclic combinatorial fluorescent in situ hybridization (combFISH) assay to achieve multiplexed detection of RNA in cell cultures and tissues. Specifically, multiplexing is achieved through cyclic interrogation of barcode sequences on the rolling circle amplicons generated from the padlock probe assay by using sets of combinatorial detection probes. Theoretically, combFISH can detect 64 genes in three hybridization cycles by combinatorial barcoding using 12 fluorescently labeled detection probes. Our method eliminates sequencing-by-ligation (SBL) chemistry in the in situ sequencing protocol and directly uses RNA as targets for ligation, making it more straightforward. We showed that our method works in fresh-frozen and formalin-fixed paraffin-embedded tissue sections. With its straightforward protocols, we expect our method to be adopted by the scientific community and extended to clinical settings.
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Hibridación Fluorescente in Situ , ARN , Hibridación Fluorescente in Situ/métodos , ARN/análisis , Humanos , Animales , Colorantes Fluorescentes/química , Perfilación de la Expresión Génica/métodosRESUMEN
Extrachromosomal circular DNAs (eccDNAs) carrying random genomic segments are broadly found across different cancer types, but their molecular functions and impact in gastric cancer (GC) are rarely known. In this study, we aimed to investigate the potential role of eccDNA in GC. Using the Circle-seq strategy, we observed the eccDNA abundance in gastric cancer tissues (GCT) was aberrantly higher than that of normal adjacent tissues (NAT). The high abundance of eccDNAs carrying oncogene-segments in GCT may represent the DNA damage products of amplified oncogenes. Analysis of GCT over-represented eccDNA carrying enhancer (eccEnhancer) based on data from FANTOM5 project combined with TCGA database suggested the GC over-represented eccEnhancers may contribute to development of GC. GC over-represented eccDNAs carrying pre-miRNA (eccMIR) were enriched to multiple cancer-relevant signal pathways by KEGG analysis. We then synthesized the top six GC over-represented eccMIRs and found four of them enabled high expression of miRNAs and down-regulation of miRNA-target genes in MGC803 cells. Furthermore, we observed the inheritance of GC over-represented eccMIRs benefited host cell proliferation and promoted the aggressive features of host cells. Altogether, this study revealed the GC over-represented eccDNAs carrying functional genomic segments were related to the carcinogenesis of GC and presented the capability to facilitate cancer progression, suggesting the cancerous eccDNAs may serve as a dynamic reservoir for genome plasticity and rapid adaptive evolution of cancer. Therefore, blocking the pathways for eccDNAs generation may provide a novel therapeutic strategy for the treatment of gastric cancer.
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Neoplasias Gástricas , Humanos , Neoplasias Gástricas/genética , ADN Circular/genética , Genoma , ADN , Carcinogénesis/genéticaRESUMEN
Tumor-associated antigen (TAA)-based diagnosis has gained prominence for early tumor screening, treatment monitoring, prognostic assessment, and minimal residual disease detection. However, limitations such as low sensitivity and difficulty in extracting non-specific binding membrane proteins still exist in traditional detection methods. Upconversion luminescence (UCL) exhibits unique physical and chemical properties under wavelength near-infrared light excitation. Rolling circle amplification (RCA) is an efficient DNA amplification technique with amplification factors as high as 105. Therefore, the above two excellent techniques can be employed for highly accurate imaging analysis of tumor cells. Herein, we developed a novel nanoplatform for TAA-specific cell imaging based on UCL and RCA technology. An aptamer-primer complex selectively binds to Mucin 1 (MUC1), one of TAA on cell surface, to trigger RCA reaction, generating a large number of repetitive sequences. These sequences provide lots of binding sites for complementary signal probes, producing UCL from lanthanide-doped upconversion nanoparticles (UCNPs) after releasing quencher group. The experimental results demonstrate the specific attachment of upconversion nanomaterials to cancer cells which express a high level of MUC1, indicating the potential of UCNPs and RCA in tumor imaging.
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Luminiscencia , Ácidos Nucleicos , Diagnóstico por Imagen , Membrana Celular , Técnicas de Amplificación de Ácido NucleicoRESUMEN
A label-free immunoassay based on rolling circle amplification (RCA) and G-quadruplex/Thioflavin T (G4/ThT) is proposed to realize the sensitive detection of carboxy-terminal cross-linked fragment of type I collagen (CTX I) for bone loss. Under the optimal conditions, as low as 38.02 pg/mL of CTX I can be detected. To improve the detecting throughput and simplify the operation, a microfluidic chip was designed, fabricated, and used for CTX I detection based on the proposed assay. The detection can be completed with only a single on-chip magnetic separation step, which was easy to operate, less time-consuming, and has only low reagent consumption. The limit of detection was 131.83 pg/mL by observing with fluorescence microscope. With further improvement of detection equipment, the sensitivity of on-chip detection can be improved. It can be expected that the proposed RCA/G4/ThT immunoassay for sensitive and high-throughput automated detection of CTX I might be chosen as a potential analytical tool for clinical osteoporosis diagnosis and in-orbit bone loss detection.
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G-Cuádruplex , Microfluídica , Benzotiazoles , BioensayoRESUMEN
A novel functional nucleic acid (FNA) nanomaterial based on hybrid chain reaction (HCR) nanoscaffolds is proposed to solve the problem of time superposition and repeated primer design in sensitive miRND detection using cascade amplification technique. Rolling circle amplification (RCA) was cascaded with the prepared FNA nanomaterials for miRNA let-7a (as a model target) sensitive detection by lateral flow assay (LFA). Under the optimal conditions, the proposed RCA-FNA-LFA assay demonstrated the specificity and accuracy for miRNA let-7a detection with a detection limit of 1.07 pM, which increased sensitivity by nearly 20 times compared with that of RCA -LFA assay. It is worth noting that the non-target-dependent self-assembly process of HCR nanoscaffolds does not take up the whole detection time, thus, less time is taken than that of the conventional cascaded method. Moreover, the proposed assay does not need to consider the system compatibility between two kinds of isothermal amplification techniques. As for detection of different miRNAs, only the homologous arm of the padlock probe of RCA needs to be changed, while the FNA nanomaterial does not need any change, which greatly simplifies the primer design of the cascaded amplification techniques. With further development, the proposed RCA-FNA-LFA assay might achieve more sensitive and faster results to better satisfy the requirements of clinical diagnosis combing with more sensitive labels or small strip reader.
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Límite de Detección , MicroARNs , Nanoestructuras , Técnicas de Amplificación de Ácido Nucleico , Técnicas de Amplificación de Ácido Nucleico/métodos , MicroARNs/análisis , Humanos , Nanoestructuras/química , Técnicas Biosensibles/métodosRESUMEN
Rolling circle amplification (RCA) enables the facile construction of compact and versatile DNA nanoassemblies which are yet rarely explored for intracellular analysis. This is might be ascribed to the uncontrollable and inefficient probe integration/activation. Herein, by encoding with tandem allosteric deoxyribozyme (DNA-cleaving DNAzyme), a multifunctional RCA nanogel was established for realizing the efficient intracellular microRNA imaging via the successive activation of the RCA-disassembly module and signal amplification module. The endogenous microRNA stimulates the precise degradation of DNA nanocarriers, thus leading to the efficient exposure of RCA-entrapped DNAzyme biocatalyst for an amplified readout signal. Our bioorthogonal DNAzyme disassembly strategy achieved the robust analysis of intracellular biomolecules, thus showing more prospects in clinical diagnosis.
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Técnicas Biosensibles , ADN Catalítico , MicroARNs , MicroARNs/análisis , Nanogeles , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/análisis , Técnicas Biosensibles/métodos , Límite de DetecciónRESUMEN
Next-generation sequencing has transformed the acquisition of vast amounts of genomic information, including the rapid identification of target gene sequences in metagenomic databases. However, dominant species can sometimes hinder the detection of rare bacterial species. Therefore, a highly sensitive amplification technique that can selectively amplify bacterial genomes containing target genes of interest was developed in this study. The rolling circle amplification (RCA) method can initiate amplification from a single locus using a specific single primer to amplify a specific whole genome. A mixed cell suspension was prepared using Pseudomonas fluorescens ATCC17400 (targeting nonribosomal peptide synthetase [NRPS]) and Escherichia coli (non-target), and a specific primer designed for the NRPS was used for the RCA reaction. The resulting RCA product (RCP) amplified only the Pseudomonas genome. The NRPS was successfully amplified using RCP as a template from even five cells, indicating that the single-priming RCA technique can specifically enrich the target genome using gene-specific primers. Ultimately, this specific genome RCA technique was applied to metagenomes extracted from sponge-associated bacteria, and NRPS sequences were successfully obtained from an unknown sponge-associated bacterium. Therefore, this method could be effective for accessing species-specific sequences of NRPS in unknown bacteria, including viable but non-culturable bacteria.
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Genoma Bacteriano , Técnicas de Amplificación de Ácido Nucleico , Péptido Sintasas , Péptido Sintasas/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Escherichia coli/genética , Pseudomonas fluorescens/genética , Análisis de Secuencia de ADN/métodos , Metagenoma/genéticaRESUMEN
Rapid detection of heparin-binding protein (HBP) is essential for timely intervention in sepsis cases. Current detection techniques are usually antibody-based immunological methods, which have certain problems, such as complexity and slow detection, and fall short in meeting the urgency of clinical needs. The application of an aptamer can address these concerns well. In this study, HBP-specific DNA aptamers were screened first. Among which, Apt-01, Apt-02, and Apt-13 had a high affinity for HBP, exhibiting impressive KD values of 3.42, 1.44, and 1.04 nmol/L, respectively. Then, the aptamer of HBP and its partially complementary primer probe were combined to form double-stranded DNA (dsDNA) and synthesize a circular DNA template. The template is complementary to the primer probe, but due to the presence of dsDNA, ExoIII cleaves C2-13 as an RCA primer probe, rendering the template unable to recognize the primer probe and preventing the RCA reaction from proceeding. When the target is present, it competes with the adapter for recognition and releases C2-13, exposing its 3' end. After initiating the RCA at room temperature and reacting with SYBR GreenII at 37 °C for 20 min, fluorescence changes can be observed and quantitatively analyzed at a 530 nm wavelength, achieving quantitative biological analysis. Apt-01 was used to develop a fluorescent biosensor for HBP detection, which exhibited a good linear range (0.01 nmol/L to 10 nmol/L) and detection limit (0.0056 nmol/L). This advancement holds the potential to lay a solid groundwork for pioneering sensitive and specific methods for HBP detection and to significantly enhance the diagnostic processes for sepsis.
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Péptidos Catiónicos Antimicrobianos , Aptámeros de Nucleótidos , Técnicas Biosensibles , Proteínas Sanguíneas , Humanos , Péptidos Catiónicos Antimicrobianos/química , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Proteínas Sanguíneas/química , ADN/química , Límite de DetecciónRESUMEN
Immunotherapy faces insufficient immune activation and limited immune effectiveness. Herein, we report a smart DNA hydrogel that enables the release of multivalent functional units at the tumor site to enhance the efficacy of immunotherapy. The smart DNA hydrogel was assembled from two types of ultra-long DNA chains synthesized via rolling circle amplification. One DNA chain contained immune adjuvant CpG oligonucleotides and polyaptamers for loading natural killer cell-derived exosomes; the other chain contained multivalent G-quadruplex for loading photodynamic agents. DNA chains formed DNA hydrogel through base-pairing. HhaI restriction endonuclease sites were designed between functional units. Upon stimuli in the tumor sites, the hydrogel was effectively cleaved by the released HhaI and disassembled into functional units. Natural killer cell-derived exosomes played an anti-tumor role, and the CpG oligonucleotide activated antigen-presenting cells to enhance the immunotherapy. Besides the tumor-killing effect of photodynamic therapy, the generated cellular debris acted as an immune antigen to further enhance the immunotherapeutic effect. In a mouse melanoma orthotopic model, the smart DNA hydrogel as a localized therapeutic agent, achieved a remarkable tumor suppression rate of 91.2 %. The smart DNA hydrogel exhibited enhanced efficacy of synergistic immunotherapy and photodynamic therapy, expanding the application of DNA materials in biomedicine.
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Melanoma , Fotoquimioterapia , Animales , Ratones , Melanoma/tratamiento farmacológico , Hidrogeles , ADN , Inmunoterapia , Modelos Animales de Enfermedad , Línea Celular TumoralRESUMEN
Multimeric aptamers have gained more attention than their monomeric counterparts due to providing more binding sites for target analytes, leading to increased affinity. This work attempted to engineer the surface-based generation of multimeric aptamers by employing the room temperature rolling circle amplification (RCA) technique and chemically modified primers for developing a highly sensitive and selective electrochemical aptasensor. The multimeric aptamers, generated through surface RCA, are hybridized to modified spacer primers, facilitating the positioning of the aptamers in the proximity of sensing surfaces. These multimeric aptamers can be used as bio-receptors for capturing specific targets. The surface amplification process was fully characterized, and the optimal amplification time for biosensing purposes was determined, using SARS-CoV-2 spike protein (SP). Interestingly, multimeric aptasensors produced considerably higher response signals and affinity (more than 10-fold), as well as higher sensitivity (almost 4-fold) compared to monomeric aptasensors. Furthermore, the impact of surface structures on the response signals was studied by utilizing both flat working electrodes (WEs) and nano-/microislands (NMIs) WEs. The NMIs multimeric aptasensors showed significantly higher sensitivity in buffer and saliva media with the limit of detection less than 2â fg/ml. Finally, the developed NMIs multimeric aptasensors were clinically challenged with several saliva patient samples.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Técnicas Electroquímicas , Técnicas de Amplificación de Ácido Nucleico , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Conformación de Ácido Nucleico , Propiedades de Superficie , ADN Circular/química , ADN Circular/genética , Amplificación de Genes , Humanos , COVID-19/diagnóstico , COVID-19/virología , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Técnicas de Amplificación de Ácido Nucleico/métodos , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single-mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.
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ADN Mitocondrial , Hidrogeles , Microfluídica/métodos , Sefarosa , MicroscopíaRESUMEN
The precise and accurate identification and quantification of transcriptional start sites (TSSs) is key to understanding the control of transcription. The core promoter consists of the TSS and proximal non-coding sequences, which are critical in transcriptional regulation. Therefore, the accurate identification of TSSs is important for understanding the molecular regulation of transcription. Existing protocols for TSS identification are challenging and expensive, leaving high-quality data available for a small subset of organisms. This sparsity of data impairs study of TSS usage across tissues or in an evolutionary context. To address these shortcomings, we developed Smart-Seq2 Rolling Circle to Concatemeric Consensus (Smar2C2), which identifies and quantifies TSSs and transcription termination sites. Smar2C2 incorporates unique molecular identifiers that allowed for the identification of as many as 70 million sites, with no known upper limit. We have also generated TSS data sets from as little as 40 pg of total RNA, which was the smallest input tested. In this study, we used Smar2C2 to identify TSSs in Glycine max (soybean), Oryza sativa (rice), Sorghum bicolor (sorghum), Triticum aestivum (wheat) and Zea mays (maize) across multiple tissues. This wide panel of plant TSSs facilitated the identification of evolutionarily conserved features, such as novel patterns in the dinucleotides that compose the initiator element (Inr), that correlated with promoter expression levels across all species examined. We also discovered sequence variations in known promoter motifs that are positioned reliably close to the TSS, such as differences in the TATA box and in the Inr that may prove significant to our understanding and control of transcription initiation. Smar2C2 allows for the easy study of these critical sequences, providing a tool to facilitate discovery.
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Sorghum , Transcripción Genética , Sitio de Iniciación de la Transcripción , Regiones Promotoras Genéticas/genética , TATA Box/genética , Zea mays/genética , Sorghum/genética , ARNRESUMEN
Since epigenetic modifications differ from cell to cell, detecting the DNA methylation status of individual cells is requisite. Therefore, it is important to conduct "morphology-based epigenetics research", in which the sequence-specific DNA methylation status is observed while maintaining tissue architecture. Here we demonstrate a novel histochemical technique that efficiently shows the presence of a single methylated cytosine in a sequence-dependent manner by applying ICON (interstrand complexation with osmium for nucleic acids) probes. By optimizing the concentration and duration of potassium osmate treatment, ICON probes selectively hybridize to methylated cytosine on tissue sections. Since the elongation process by rolling-circle amplification through the padlock probe and synchronous amplification by the hyperbranching reaction at a constant temperature efficiently amplifies the reaction, it is possible to specifically detect the presence of a single methylated cytosine. Since the ICON probe is cross-linked to the nuclear or mitochondrial DNA of the target cell, subsequent elongation and multiplication reactions proceed like a tree growing in soil with its roots firmly planted, thus facilitating the demonstration of methylated cytosine in situ. Using this novel ICON-mediated histochemical method, detection of the methylation of DNA in the regulatory region of the RANK gene in cultured cells and of mitochondrial DNA in paraffin sections of mouse cerebellar tissue was achievable. This combined ICON and rolling-circle amplification method is the first that shows evidence of the presence of a single methylated cytosine in a sequence-specific manner in paraffin sections, and is foreseen as applicable to a wide range of epigenetic studies.
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Citosina , Parafina , Animales , Ratones , Metilación de ADN , Epigénesis Genética , ADN MitocondrialRESUMEN
The Review by Yang, Yao and colleagues (DOI: 10.1002/chem.202202673) describes recent developments in biofunctional DNA hydrogels and DNA nanocomplexes based on rolling circle amplification (RCA) and introduces assembly strategies and functionalization methods of the ultralong single-strand DNA produced by RCA to construct biofunctional materials.
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ADN de Cadena Simple , ADN , Técnicas de Amplificación de Ácido Nucleico/métodosRESUMEN
A new method for the detection of genomic RNA combines RNA cleavage by the 10-23 DNAzyme and use of the cleavage fragments as primers to initiate rolling circle amplification (RCA). 230 different 10-23 DNAzyme variants were screened to identify those that target accessible RNA sites within the highly structured RNA transcripts of SARS-CoV-2. A total of 28 DNAzymes were identified with >20 % cleavage, 5 with >40 % cleavage and one with >60 % in 10â min. The cleavage fragments from these reactions were then screened for coupling to an RCA reaction, leading to the identification of several cleavage fragments that could efficiently initiate RCA. Using a newly developed quasi-exponential RCA method with a detection limit of 500 aM of RNA, 14 RT-PCR positive and 15 RT-PCR negative patient saliva samples were evaluated for SARS-CoV-2 genomic RNA, achieving a clinical sensitivity of 86 % and specificity of 100 % for detection of the virus in <2.5â h.