ABSTRACT
Spatial visualization of single-cell transcripts is limited by signal specificity and multiplexing. Here, we report hierarchical DNA branch assembly-encoded fluorescent nanoladders, which achieve denoised and highly multiplexed signal amplification for single-molecule transcript imaging. This method first offers independent RNA-primed rolling circle amplification without nonspecific amplification based on circular DNAzyme. It then executes programmable DNA branch assembly on these amplicons to encode virtual signals for visualizing numbers of targets by FISH. In theory, more virtual signals can be encoded via the increase of detection spectral channels and repeats of the same sequences on barcode. Our method almost eliminates nonspecific amplification in fixed cells (reducing nonspecific spots of single cells from 16 to nearly zero), and achieves simultaneous quantitation of nine transcripts by using only two detection spectral channels. We demonstrate accurate RNA profiling in different cancer cells, and reveal diverse localization patterns for spatial regulation of transcripts.
Subject(s)
DNA, Catalytic , DNA , Nucleic Acid Amplification Techniques/methods , RNA , Fluorescence , Single-Cell AnalysisABSTRACT
Nucleic acids are naturally decorated with various chemical modifications at nucleobases. Most nucleic acid modifications (NAMs) do not alter Watson-Crick base pairing but can regulate gene expression known as "epigenetics". Their abundances present a very wide range, approximately 10-2 to 10-6 of total bases. Different NAMs may coexist in spatial proximity (e.g., <20 nm) in the crowded intracellular environment. Considering the highly dynamic chromatin accessibility (physical access to DNA), the NAMs in inaccessible DNA probably plays different roles. These multilayered features of NAMs vary from cell to cell. Our understanding of the function and mechanism of NAMs in biological processes and disease states has largely been driven by the expanding array of sequencing-based methodologies. However, an underexplored aspect is the measurement of the subcellular distribution, spatial proximity, and inaccessibility of NAMs in single cells. In recent years, we have developed new approaches that light up single-cell NAMs with single-site sensitivity. These methods are mainly based on the integration of chemical or chemoenzymatic tools, DNA amplification and nanotechnology, and/or microfluidics. An overview of these methods together with conventional methods such as immunofluorescence (IF) and fluorescence in situ hybridization (FISH) is provided in this Account.Our laboratory has proposed DNA-encoded amplification (DEA) as the main strategy for developing a set of single-cell NAM imaging methods. In brief, DEA transforms the different features of NAMs into unique DNA primers for rolling circle amplification (RCA) followed by FISH imaging. The first method is base-encoded amplifying FISH (BEA-FISH), in which we convert individual NAMs into RCA primers via chemoselective labeling and click bioconjugation. It enables the in situ visualization of low-abundance NAMs (e.g., 5hmU), which is impracticable by conventional methods. We subsequently developed pairwise proximity-differentiated amplifying FISH (PPDA-FISH), which integrates BEA-FISH with DNA nanotechnology. PPDA-FISH utilizes proximity ligation and toehold strand displacement to label the adjacent site of two different NAMs (one-to-one proximity) and their respective residual sites with three unique RCA probes. It achieves simultaneous counting of the above-mentioned three types of modified sites in the same cells. The third method is cellular macromolecule-tethered DNA walking indexing (Cell-TALKING) to probe more than two NAMs within the same nanoenvironments. Cell-TALKING uses dynamic DNA proximity cleavage to continuously activate different preblocked RCA primers (for each NAM) near one walking probe (for one target molecule). We have explored three NAMs around one histone (one-to-many proximity) in different cancer cell lines and clinical specimens. Then, we describe a single-cell hydrogel encoding amplification (scHEA) method by integrating droplet microfluidics with BEA-FISH. This method generates hydrogel beads that encapsulate single cells and their genomic DNA after cell lysis. It realizes the analysis of global (accessible and inaccessible) DNA from the same cells. We find that the global levels of both 5hmC and 5hmU in single cells can distinguish different breast cancer cells. Finally, the current limitations of these strategies and the future development directions are also discussed. We hope that this Account can spark new ideas and invite new efforts from different disciplines for single-cell NAM analysis.
Subject(s)
Nucleic Acid Amplification Techniques , Nucleic Acids , DNA/analysis , Hydrogels , In Situ Hybridization, Fluorescence , Nucleic Acid Amplification Techniques/methods , Nucleic Acid HybridizationABSTRACT
Assembling DNA on solid surfaces is fundamental to surface-based DNA technology. However, precise control over DNA conformation and organization at solid-liquid interfaces remains a challenge, resulting in limited stability and sensitivity in biosensing applications. We herein communicate a simple and robust method for creating highly uniform DNA monolayers on gold surfaces by a freeze-thawing process. Using Raman spectroscopy, fluorescent imaging, and square wave voltammetry, we demonstrate that thiolated DNA is concentrated and immobilized on gold surfaces with an upright conformation. Moreover, our results reveal that the freezing-induced DNA surfaces are more uniform, leading to improved DNA stability and target recognition. Lastly, we demonstrate the successful detection of a model drug in undiluted whole blood while mitigating the effects of biofouling. Our work not only provides a simple approach to tailor the DNA-gold surface for biosensors but also sheds light on the unique behavior of DNA oligonucleotides upon freezing on the liquid-solid interface.
Subject(s)
Biosensing Techniques , Gold , Gold/chemistry , Freezing , DNA/chemistry , Oligonucleotides , Biosensing Techniques/methods , Electrochemical Techniques/methodsABSTRACT
The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has spread rapidly around the world. Accurate and scalable diagnostics are essential for immediate intervention and control of viral transmission. Currently reported diagnostics are rapid and sensitive, yet most are limited by their principle of single-locus identification and suffer from false-negative results because of the mutation-prone nature of RNA viruses. Here, we propose a multilocus detection method for SARS-CoV-2 based on a modified loop-mediated isothermal amplification with a pair of universal primers. The sequence-specific probes are designed to recognize the sequence of nucleocapsid protein (N) and the open reading frame 1ab (Orf1ab) gene from the SARS-CoV-2 genome. In the presence of a target locus, separated probes are ligated to be an intact template, the bipartite ends of which are repetitive sequences for the sequential binding of universal primers to initiate strand displacement. A kind of flap structure-dependent endonuclease is involved in cleaving multicolor TaqMan probes during multiplex amplification, realizing a real-time and multiplex analysis. We evaluated the quantitative performance of the developed method with spiked samples using synthetic target RNA, resulting in a limit of detection as low as 250 aM. Furthermore, the feasibility of multilocus detection was validated using various mutation-prone genes, demonstrating a significant potential for accurate analysis of SARS-CoV-2 and holding great promise for the clinical diagnosis of other infectious diseases.
Subject(s)
COVID-19 , Humans , Mutation , Nucleic Acid Amplification Techniques , Nucleocapsid Proteins/genetics , RNA, Viral , SARS-CoV-2 , Sensitivity and SpecificityABSTRACT
Cellular oxidative thymines, 5-hydroxymethyluracil (5hmU) and 5-formyluracil (5fU), are found in the genomes of a diverse range of organisms, the distribution of which profoundly influence biological processes and living systems. However, the distribution of cellular oxidative thymines has not been explored because of lacking both specific bioorthogonal labeling and sensitivity methods for single-cell analysis. Herein, we report a bioorthogonal chemical signature enabling amplified visualization of cellular oxidative thymines in single cells. The synthesized ATP-γ-alkyne, an ATP analogue with bioorthogonal tag modified on γ-phosphate can be specifically linked to cellular 5hmU by chemoenzymatic labeling. DNA with 5-alkynephosphomethyluracil were then clicked with azide (N3)-modified 5hmU-primer. Identification of 5fU is based on selective reduction from 5fU to 5hmU, subsequent chemoenzymatic labeling of the newly generated 5hmU, and cross-linking with N3-modified 5fU-primer via click chemistry. Then, all of the 5hmU and 5fU sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. The above two kinds of barcodes can be simultaneously amplified for differentiated visualization of 5hmU and 5fU in single cells. We find these two kinds of cellular oxidative thymines are spatially organized in a cell-type-dependent style with cell-to-cell heterogeneity. We also investigate their multilevel subcellular information and explore their dynamic changes during cell cycles. Further, using DNA sequencing instead of fluorescence imaging, our proposed bioorthogonal chemical signature holds great potential to offer the sequence information of these oxidative thymines in cells and may provide a reliable chemical biology approach for studying the whole-genome oxidative thymines profiles and insights into their functional role and dynamics in biology.
Subject(s)
Azides , Thymine , Click Chemistry , DNA , Oxidative StressABSTRACT
Dynamic information of intracellular transcripts is essential to understand their functional roles. Routine RNA-sequencing (RNA-seq) methods only measure RNA species at a steady state and do not provide RNA dynamic information. Here, we develop addition-elimination mechanism-activated nucleotide transition sequencing (AENT-seq) for transcriptome-wide profiling of RNA dynamics. In AENT-seq, nascent transcripts are metabolically labeled with 4-thiouridine (4sU). The total RNA is treated with N2H4·H2O under aqueous conditions. N2H4·H2O is demonstrated to convert 4sU to 4-hydrazino cytosine (C*) based on an addition-elimination chemistry. C* is regarded as cytosine (C) during the DNA extension process. This 4sU-to-C transition marks nascent transcripts, so it enables sequencing analysis of RNA dynamics. We apply our AENT-seq to investigate transcript dynamic information of several genes involved in cancer progression and metastasis. This method uses a simple chemical reaction in aqueous solutions and will be rapidly disseminated with extensive applications.
Subject(s)
RNA , Thiouridine , Base Sequence , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Nucleotides , RNA/genetics , Sequence Analysis, RNAABSTRACT
Spatially resolved visualization of RNA processing and structures is important for better studying single-cell RNA function and landscape. However, currently available RNA imaging methods are limited to sequence analysis, and not capable of identifying RNA processing events and structures. Here, we developed click-encoded rolling FISH (ClickerFISH) for visualizing RNA polyadenylation and structures in single cells. In ClickerFISH, RNA 3' polyadenylation tails, single-stranded and duplex regions are chemically labeled with different clickable DNA barcodes. These barcodes then initiate DNA rolling amplification, generating repetitive templates for FISH to image their subcellular distributions. Combined with single-molecule FISH, the proposed strategy can also obtain quantitative information of RNA of interest. Finally, we found that RNA poly(A) tailing and higher-order structures are spatially organized in a cell type-specific style with cell-to-cell heterogeneity. We also explored their spatiotemporal patterns during cell cycle stages, and revealed the highly dynamic organization especially in S phase. This method will help clarify the spatiotemporal architecture of RNA polyadenylation and structures.
Subject(s)
Click Chemistry/methods , In Situ Hybridization, Fluorescence/methods , Polyadenylation , RNA, Messenger/chemistry , Single-Cell Analysis/methods , Cell Line, Tumor , DNA Barcoding, Taxonomic/methods , Gene Expression Profiling/methods , Humans , MCF-7 Cells , Mass Spectrometry , Poly A/chemistry , Sequence Analysis, RNA/methods , Spatio-Temporal AnalysisABSTRACT
Spatial positioning and proximity of relevant biomolecules such as DNA epigenetic marks are fundamental to a deeper understanding of life. However, it remains poorly explored and technically challenging. Here we report the pairwise proximity-differentiated visualization of single-cell 5-formylcytosine (5fC) and 5-hydroxymethylcytosine (5hmC). These two marks on chromatin in fixed cells are successively labeled and crosslinked with their DNA primer probes via click chemistry. Based on a pairwise proximity-differentiated mechanism, proximal 5fC/5hmC sites and residual 5fC or 5hmC sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. We thus demonstrate the differentiated visualization of 5fC or 5hmC spatial positioning and their pairwise proximity in single cells. Such multi-level subcellular information may provide insights into regulation functions and mechanisms of chromatin modifications, and the spatial proximity can expose the potential crosstalk or interaction between their reader proteins.
Subject(s)
5-Methylcytosine/analogs & derivatives , Cytosine/analogs & derivatives , DNA/chemistry , Single-Cell Analysis , 5-Methylcytosine/chemistry , Cell Line , Chromatin/chemistry , Chromatin/metabolism , Cytosine/chemistry , Humans , Molecular StructureABSTRACT
5-Hydroxymethyluracil ( 5hmU ) is found in the genomes of a diverse range of organisms as another kind of 5-hydroxymethylpyrimidine, with the exception of 5-hydroxymethylcytosine ( 5hmC ). The biological function of 5hmU has not been well explored due to lacking both specific 5hmU recognition and single-cell analysis methods. Here we report differentiated visualization of single-cell 5hmU and 5hmC with microfluidic hydrogel encoding (sc 5hmU / 5hmC -microgel). Single cells and their genomic DNA after cell lysis can be encapsulated in individual agarose microgels. The 5hmU sites are then specifically labeled with thiophosphate for the first time, followed by labeling 5hmC with azide glucose. These labeled bases are each encoded into respective DNA barcode primers by chemical cross-linking. In situ amplification is triggered for single-molecule fluorescence visualization of single-cell 5hmU and 5hmC . On the basis of the sc 5hmU / 5hmC -microgel, we reveal cell type-specific molecular signatures of these two bases with remarkable single-cell heterogeneity. Utilizing machine learning algorithms to decode four-dimensional signatures of 5hmU / 5hmC , we visualize the discrimination of nontumorigenic, carcinoma and highly invasive breast cell lines. This strategy provides a new route to analyze and decode single-cell DNA epigenetic modifications.
Subject(s)
5-Methylcytosine/analogs & derivatives , Hydrogels/chemistry , Microfluidics , Pentoxyl/analogs & derivatives , Single-Cell Analysis/methods , 5-Methylcytosine/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , DNA/genetics , Epigenesis, Genetic , Female , Humans , Neoplasm Invasiveness , Pentoxyl/metabolismABSTRACT
Splice variants visualization is pivotal for a deeper understanding of cell growth and development. However, it remains technically challenging due to short lengths, similar sequences, and low abundance. The existing single-cell imaging strategies suffer from nonspecific amplification that causes considerable noise during visualization of the splice variants. Herein we develop a new RNA-primed amplification strategy for noise-suppressed visualization of single-cell splice variants. Block probes were designed to specifically identify the conjugated region of exons in mRNA, which was then digested by endonuclease and provided a hydroxyl group at the 3' terminal. The RNA target can act as primer to trigger rolling circle amplification, achieving visualization of splice variants with noise suppressed to nearly zero. We further explored the expression and distribution of BRCA1 splice variants in three breast cell lines, revealing cell-type specific mapping of this cancer suppressor gene.
Subject(s)
Nucleic Acid Amplification Techniques/methods , RNA Splicing , RNA, Messenger/metabolism , BRCA1 Protein/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , DNA Probes/chemistry , DNA Probes/metabolism , Female , Humans , Microscopy, Confocal , RNA, Messenger/analysis , Single-Cell Analysis/methodsABSTRACT
Monitoring RNA synthesis and spatial distribution can help to understand its role in physiology and diseases. However, visualizing newly synthesized RNA in single cells remains a great challenge. Here, we developed a bioorthogonal labeling-primed DNA amplification strategy to visualize newly synthesized RNA in single cells. The new bioorthogonal N6-allyladenosine nucleoside was prepared to metabolically label cellular newly synthesized RNAs. These allyl-functionalized RNAs then reacted with tetrazine-modified primers. These primers could initiate rolling circle amplification, producing tandem periodic long single DNA strands to capture hundreds of fluorescence probes for signal amplification. Using this method, we explored the subcellular distributions of newly synthesized RNAs. And we found that newly synthesized RNAs are spatially organized in a cell type-specific style with cell-to-cell heterogeneity.
Subject(s)
DNA, Neoplasm/chemistry , In Situ Hybridization, Fluorescence , Nucleic Acid Amplification Techniques , RNA, Neoplasm/analysis , Carbocyanines/chemistry , DNA Primers/chemistry , DNA, Neoplasm/genetics , Fluorescent Dyes/chemistry , Humans , Molecular Structure , Optical Imaging , RNA, Neoplasm/chemical synthesis , RNA, Neoplasm/genetics , Single-Cell Analysis , Tumor Cells, CulturedABSTRACT
Tumor progressions such as metastasis are complicated events that involve abnormal expression of different miRNAs and enzymes. Monitoring these biomolecules in live cells with computational DNA nanotechnology may enable discrimination of tumor progression via digital outputs. Herein, we report intracellular entropy-driven multivalent DNA circuits to implement multi-bit computing for simultaneous analysis of intracellular telomerase and microRNAs including miR-21 and miR-31. These three biomolecules can trigger respective DNA strand displacement recycling reactions for signal amplification. They are visualized by fluorescence imaging, and their signal outputs are encoded as multi-bit binary codes for different cell types. The results can discriminate non-tumorigenic, malignant and metastatic breast cells as well as respective tumors. This DNA computing circuit is further performed in a microfluidic chip to differentiate rare co-cultured cells, which holds a potential for the analysis of clinical samples.
Subject(s)
Breast Neoplasms/metabolism , Computers, Molecular , DNA/chemistry , Disease Progression , Entropy , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Separation/methods , Coculture Techniques , DNA Probes/chemistry , Humans , Lab-On-A-Chip Devices , MicroRNAs/analysis , MicroRNAs/metabolism , Tamoxifen/pharmacology , Telomerase/chemistryABSTRACT
Multifunctional DNA nanodevices perform ever more tasks with applications ranging from in vitro biomarker detection to in situ cell imaging. However, most developed ones consist of a series of split building blocks, which suffer from asynchronous behaviors in complicated cellular microenvironments (endocytosis pathway, diffusion-limited cytoplasm, etc.), causing the loss of stoichiometric information and additional postassembly processes. Herein, we constructed all-in-one DNA nanodevices to achieve synchronous multiplexed imaging. All DNA components, including two sets of probe modules (each containing target-specific walkers, i.e., hairpin tracks with chemically damaged bases), are modified on individual gold nanoparticles. This design not only enables their integrated internalization into cells, circumventing inhomogeneous distribution of different building blocks and increasing the local concentrations of the interacting modules, but also avoids the impact of stochastic diffusion in viscous cytoplasm. A couple of intracellular enzymes in situ actuate the synchronized motion of the modules, all on-particle, after specific recognition of intracellular targets (such as RNAs and proteins), thus facilitating synchronized, multiplexed cell imaging. Finally, the proposed all-in-one nanodevices were successfully applied to monitor intracellular microRNA-21 and telomerase expression levels. The flexible design can be extended to detect other cytoplasmic molecules and monitor related pathways by simply changing the sequences.
Subject(s)
DNA, Neoplasm/chemistry , Nanostructures/chemistry , Optical Imaging , HeLa Cells , Humans , MicroRNAs/genetics , Telomerase/geneticsABSTRACT
Both biomarker and probe degradations cause serious false assay results. However, protecting a target or a target and a probe simultaneously has rarely been explored. Herein, MnO2-nanosheet-powered target- and probe-protective Janus DNA nanomachines are reported. It is formed in living cells by an RNA-responsive assembly of two chemically modified DNA partzymes and one substrate probe. MnO2 nanosheets are used to facilitate the cellular uptake of DNA reagents and generate Mn2+, which are indispensable DNAzyme cofactors for efficient catalytic cleavage. We find that DNA partzymes with modified sugar moieties (e.g., LNA or ones with 2'-O-methylation) protect the RNA of RNA-DNA hybrids from RNase H degradation. LNA blocks RNase H recruitment on the hybrid best because of its 2'-O, 4'-C methylene bridge structure. In contrast, modifications at DNA phosphate moieties fail to protect the RNA. RNA protection can exclude target-degradation-induced false negative results. In addition, the phosphorothioate-modified substrate probe is known to resist nuclease degradation, which minimizes false positive interference. Compared to canonical DNA systems without chemical modifications, the protective Janus nanomachine avoids false results and supports robust RNA imaging.
Subject(s)
DNA, Catalytic/chemistry , Manganese Compounds/chemistry , MicroRNAs/metabolism , Nanostructures/chemistry , Oxides/chemistry , Cell Line, Tumor , DNA Probes/chemistry , DNA Probes/genetics , DNA, Catalytic/genetics , Fluorescence , HEK293 Cells , Humans , Hydrolysis/drug effects , MicroRNAs/chemistry , MicroRNAs/genetics , Microscopy, Confocal/methods , Nanostructures/toxicity , Nucleic Acid Hybridization , Oxides/toxicity , Ribonuclease H/chemistryABSTRACT
Surface-enhanced Raman scattering (SERS) is a promising analysis technique for detecting various analytes in complex samples due to its unique vibrational fingerprints and high signal enhancement. However, impurity interference and substrate unreliability are direct suppression factors for practical application. Herein, we synthesize polydopamine@gold (PDA@Au) nanowaxberry, where Au nanoparticles are deposited on the surface of PDA sphere with high density and uniformity. Seed-mediated synthesis is used for fabrication of nanowaxberry. Au seeds are deposited on the surface of PDA sphere, then I ion coordinating ligand is employed to form stable AuI4- complex with AuCl4-, which decreases reduction potential of AuCl4- and avails formation of shell structure. Such nanowaxberry has high density of voids and gaps in three-dimensional space, which could absorb analytes and benefit practical SERS detection. Using malachite green as a model analyte, nanowaxberry realizes highly sensitive detection with low limit of detection (1 pM) and good reproducibility (relative standard deviation of about 10%). Meanwhile, the nanowaxberry is employed for practical detection of thiram, benzidine, and 2,4-dinitrotoluene in the environmental water, juice, apple peel, and soil. The high performance makes nanowaxberry to be potentially used for pesticides detection, pollutants monitoring, and forbidden explosives sensing in complex samples.
ABSTRACT
Sophisticated and dynamic membrane-anchored DNA nanostructures were developed to mimic a variety of membrane proteins, which play crucial roles in cellular functions. DNA biomimetic constructions bound on membranes are capable of modulating the morphologies, physical properties, and functions of lipid membranes, via mobility on membranes and/or inherent architectural features. This inspired young field of DNA-lipid-based nanobiomimetic systems is on the foundation of DNA nanotechnology. In this review, we highlight key successes in the development of structural DNA nanotechnology and demonstrate some typical static and dynamic complex DNA nanostructures first. Then, we discuss the biophysical properties of lipid membranes. Primary approaches are shown to attach hydrophilic DNA to hydrophobic lipid membranes. With appropriate designs, membrane-floating DNA nanostructures assemble and disassemble on membranes, modulated by external stimuluses. The aggregation of DNA nanostructures could influence the physical properties of lipid membranes. We also summarize artificial nanochannels made of DNA, analogous to transmembrane proteins. Transformations of DNA nanopores might be achieved under certain conditions and realize the transport of small molecules across membranes. Next, we focus on membrane-shaping functions of membrane-anchored DNA nanostructures. Curvature of the membrane is closely related to the rich diversity of cellular functions. Mimicking membrane-sculpting proteins, such as BAR family domains and SNARE proteins etc., DNA biomimetic nanostructures induce the transformations of lipid membranes and modulate membrane adhesion and membrane fusion processes. Although recent studies in DNA nanostructure-lipid membrane biomimetic nanosystems have made great progress, this field is still facing many challenges. In the future, the designs of more elaborated DNA architectures will be explored. Sophisticated dynamic DNA nanostructures inspired by natural membrane machines will be driven by the synergistic effect of multiple interactions, including hydrophobic force, electrostatic force, and ligand-receptor interactions by chemical modifications on bases, to expand their applications in vivo from model membrane to cell membrane to karyotheca.
Subject(s)
Biomimetic Materials/chemistry , DNA/chemistry , Lipid Bilayers/chemistry , Nanostructures/chemistry , Membrane Fusion/drug effects , Membrane Proteins/chemistry , Nanotechnology/methods , Nucleic Acid ConformationABSTRACT
A formaldehyde-assisted metal-ligand crosslinking strategy is used for the synthesis of metal-phenolic coordination spheres based on sol-gel chemistry. A range of mono-metal (Co, Fe, Al, Ni, Cu, Zn, Ce), bi-metal (Fe-Co, Co-Zn) and multi-metal (Fe-Co-Ni-Cu-Zn) species can be incorporated into the frameworks of the colloidal spheres. The formation of coordination spheres involves the pre-crosslinking of plant polyphenol (such as tannic acid) by formaldehyde in alkaline ethanol/water solvents, followed by the aggregation assembly of polyphenol oligomers via metal-ligand crosslinking. The coordination spheres can be used as sensors for the analysis of nucleic acid variants with single-nucleotide discrimination, and a versatile precursor for electrode materials with high electrocatalytic performance.
ABSTRACT
Circulating methylated DNA has been a new kind of cancer biomarker, yet its small fraction of trace total DNA from clinical samples impairs the accurate analysis. Though fluorescence methods based on quantitative methylation specific PCR (qMSP) have been adopted routinely, yet alternative electrochemistry assay of such DNA from clinical samples remains a great challenge. Herein, we report accurate electrochemistry analysis of circulating methylated DNA from clinical plasma samples based on a paired-end tagging and amplifications strategy. Two DNA primers each labeled with digoxigenin (Dig) and biotin are designed for the recognition and amplification of methylated DNA. Paired-end tagging amplicons and avidin-HRP molecules are successively captured on the electrode modified with Anti-Dig. Then HRP executes catalytic reaction to generate amplified signal. The design of paired-end tagging can readily integrate downstream electrochemical amplified reaction, and two heterogeneous amplifications enable high assay sensitivity. As little as 40 pg of methylated genomic DNA (â¼10 genomic equivalents) is well identified, and our strategy can even distinguish as low as 1% methylation level. Tumor-specific methylated DNA is clearly detected in the plasma of 10 of 11 NSCLC patients. The high clinical sensitivity of 91% (10/11) indicates the good consistency with clinical diagnosis. Excellent spatial control of electrochemistry allows simpler detection of more methylation patterns compared to fluorescence methods. The developed electrochemical assay is a promising liquid biopsy tool for the analysis of tumor-specific circulating DNA.
Subject(s)
Biosensing Techniques/methods , DNA Methylation , DNA, Neoplasm/blood , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Avidin/chemistry , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/diagnosis , DNA Primers/chemistry , DNA Primers/metabolism , Digoxigenin/chemistry , Digoxigenin/immunology , Electrochemical Techniques , Electrodes , Horseradish Peroxidase/chemistry , Humans , Lung Neoplasms/blood , Lung Neoplasms/diagnosis , Nucleic Acid Amplification TechniquesABSTRACT
Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.
Subject(s)
Nucleic Acid Amplification Techniques , Nucleic Acids/analysis , Nucleic Acids/biosynthesis , Temperature , Microfluidic Analytical Techniques , Nanotechnology , Nucleic Acids/geneticsABSTRACT
Primer-dimer artifacts resulting from unintended template-independent primer-primer interactions often hinder the specific amplification of nucleic acids. We demonstrate, for the first time, zero-background helicase-dependent amplification (HDA), with low concentrations of both ATP and dNTPs. This strategy achieved the reliable evaluation of telomerase activity in cancer cells by eliminating primer-dimer artifacts, which have plagued many previous methods with reduced specificity. We found that the performance of the telomerase assay by zero-background HDA was negatively affected by highly concentrated cellular proteins. This inhibitory effect is attributed to the binding of DNA templates to proteins, thus making them unavailable for polymerases. However, gold nanoparticles were demonstrated to highly attenuate such inhibition by abundant proteins, and to enhance the assay sensitivity and reliability when the reaction was performed with concentrated cell extracts.