Single Nucleotide Discrimination Research Articles

Programmable Clostridium perfringens Argonaute-Based, One-Pot Assay for the Multiplex Detection of miRNAs.

Assays for the molecular detection of miRNAs are typically constrained by the level of multiplexing, especially in a single tube. Here, we report a general and programmable diagnostic platform by combining mesophilic Clostridium perfringens Argonaute (CpAgo) with exponential isothermal amplification (EXPAR), which is a dual-signal amplification strategy, allowing for the rapid and sensitive detection of multiple miRNAs with single-nucleotide discrimination in one pot. The CpAgo-based One-Pot (COP) assay achieved a limit of detection of 1 zM miRNA within 30 min of turnaround time and a wide concentration range. This COP assay was applied to simultaneously detect four miRNAs in a single tube from clinical serum samples, showing superior analytical performance in distinguishing colorectal cancer patients from healthy individuals. This programmable, one-pot, multiplex, rapid, and specific strategy offers great promise in scientific research and clinical applications.

Genetically Encoded Light-Up RNA Amplifier Dissecting MicroRNA Activity in Live Cells.

Live cell dissection of microRNA activities is crucial for basic and translational medicine, but current hybridization-based strategies may fail to dissect surrounding-dependent activities. Here, we develop a genetically encoded miRNA-induced light-up RNA amplifier (iLAMP) that enables fast-activated, signal-amplified, fluorogenic imaging of miRNA activities in live cells. iLAMP responds to miRNA targets in the mode of "activation upon cleavage", in which the light-up RNA aptamer restores its fluorescence rapidly upon cleavage by the RNA-induced silencing complex. We demonstrate that iLAMP affords substantial signal amplification of ∼100-fold and high specificity in single nucleotide discrimination because of the miRNA-mediated cyclic cleavage. Combined with a Mango RNA aptamer reference module and a pseudoknot terminal stabilizer, iLAMP is shown for quantitative ratiometric imaging and dynamic monitoring of miRNA activities under exogenous stimulations. iLAMP is featured by a modular "plug and play" design and can be readily adapted to the detection of other miRNAs, highlighting its potential in tracking cell differentiation and screening miRNA therapeutics.

Rapid quantification of miRNAs using dynamic FRET-FISH

MicroRNAs (miRNAs) are short regulatory RNAs that control gene expression at the post-transcriptional level. Various miRNAs playing important roles in cancer development are emerging as promising diagnostic biomarkers for early cancer detection. Accurate miRNA detection, however, remains challenging because they are small and highly homologous. Recently developed miRNA detection techniques based on single-molecule imaging enabled highly specific miRNA quantification without amplification, but the time required for these techniques to detect a single miRNA was larger than 10 minutes, making rapid profiling of numerous miRNAs impractical. Here we report a rapid miRNA detection technique, dynamic FRET-FISH, in which single-molecule imaging at high probe concentrations and thus high-speed miRNA detection is possible. Dynamic FRET-FISH can detect miRNAs in 10 s at 1.2 μM probe concentration while maintaining the high-specificity of single-nucleotide discrimination. We expect dynamic FRET-FISH will be utilized for early detection of cancers by profiling hundreds of cancer biomarkers in an hour.

A multiplexed electrochemical quantitative polymerase chain reaction platform for single-base mutation analysis

Detection of single-based mutation (SbM), which is of ultra-low abundance against wild-type alleles, are typically constrained by the level of multiplexing, sensitivity for single-base resolution and quantification accuracy. In this work, an electrochemical quantitative polymerase chain reaction (E-PCR) platform was developed for multiplexed and quantitative SbM analysis in limited and precious samples with single-nucleotide discrimination. A locked nucleic acid (LNA)-mediated multiplexed PCR system in a single, closed tube setup was firstly constructed to selectively amplify the SbM genes while suppressing the wild-type alleles. The amplicons were detected simultaneously through hybridization with the sequence-specific hairpin probes anchored on a reduced graphene oxide-gold nanoparticles functionalized electrode surface. With the inclusion of an LNA-mediated PCR step upstream of the electrochemical detection, we improved the limit of detection (LOD) by 2 orders of magnitude, down to an ultralow-level of 5 copies μL−1. The platform achieved an ultra-sensitive and specific detection with 0.05% against a background of 10, 000 copies of wild-type alleles. It is highly adaptive and has the potential to enable expanded multiplexed detection in parallel, thus providing a universal tool for multiplexed SbM identification.

Streamlined and quantitative detection of chimerism using digital PCR

Animal chimeras are widely used for biomedical discoveries, from developmental biology to cancer research. However, the accurate quantitation of mixed cell types in chimeric and mosaic tissues is complicated by sample preparation bias, transgenic silencing, phenotypic similarity, and low-throughput analytical pipelines. Here, we have developed and characterized a droplet digital PCR single-nucleotide discrimination assay to detect chimerism among common albino and non-albino mouse strains. In addition, we validated that this assay is compatible with crude lysate from all solid organs, drastically streamlining sample preparation. This chimerism detection assay has many additional advantages over existing methods including its robust nature, minimal technical bias, and ability to report the total number of cells in a prepared sample. Moreover, the concepts discussed here are readily adapted to other genomic loci to accurately measure mixed cell populations in any tissue.

Electron-Transfer Study and Single Nucleotide Discrimination of a DNA Sequence on a Polymer Gold Electrode (PGE) by Differential Pulse Voltammetry (DPV)

Although the development of DNA sensors, including gold-based electrodes, has made rapid progress, it is essential to characterize DNA-gold surfaces to better understand their operation and improve performance. Recently, a novel polymer gold electrode (PGE) that shows promising performance as DNA biosensor has been designed. Herein, superior electrochemical characteristics of PGE were further studied and reported. For example, a striking difference of potential between Ru(NH3)6 3+ adsorbed onto DNA and in solution was observed, indicating the presence of distinct electron transport pathways of the two states of Ru(NH3)6 3+. Because of the superior electrochemical properties of the PGE, discrimination of single nucleotide mismatch was achieved without signal amplification or sensitive labeling.

Endonuclease IV-mediated substrate structure allosteric for universal and sensitive mutant allele enrichment and discrimination

In this work, the structure allosteric induced by Endonuclease IV (Endo IV) preference to the specific substrate was ingeniously combined with the asymmetric PCR technique. As a result, it could construct a universal and straightforward strategy for mutant allele enrichment (IVME) to improve the sensitivity of downstream mutation detection methods. At the initial time, both the mutant-type target (MT) and the wild-type target (WT) would hybridize with the probe (FP) containing apurinic/apyrimidinic site to form a flap structure, which could not be used to perform asymmetric PCR. Once Endo IV was added, the substrate formed by FP and MT, not the FP-WT, could be recognized and cleaved due to the high single nucleotide discrimination ability of Endo IV. Subsequently, the flap structure at the 3′ terminal was destroyed and exposed an active 3′-OH, which could act as a primer in the asymmetric PCR. Thus, both the absolute amount and the abundance of MT were enhanced. 1% and 0.01% MT could be directly distinguished by coupling with Sanger sequencing and Endo IV-assisted nucleic probe hybridization-based DNA mutation detection method, respectively. EGFR T790M mutation in the peripheral blood of a non-small cell lung cancer patient was detected by IVME coupled with Sanger sequence. The results were also verified by next-generation sequencing, which demonstrated the potential clinical application of our proposed strategy.

1029. Torus Synestia Nucleic Acid Analysis Platform for Fast, High Multiplex Analysis of Nucleic Acids With Single-Nucleotide Discrimination Level

BackgroundNucleic acid amplification testing (NAAT) is an essential tool both for biomedical research and for clinical molecular diagnostics. Currently, there are multiple NAAT platforms available, each offering certain performance and utility advantages and disadvantages as compared to each other. Next generation NAAT platforms aim to deliver increased target detection sensitivity and specificity, low limits of target detection, quantitative high multiplex target capacity, rapid time to results, and simple sample-to-answer workflow.MethodsHere we describe the Torus Synestia System, a NAAT platform capable of rapid, highly multiplexed amplification and detection of both DNA and RNA targets. The platform comprises a small, portable (~ 2kg) amplification and detection device and a disposable single-use cartridge housing a PCR amplification chamber with an integrated label-free microarray for real-time data acquisition and interpretation. The platform offers a 30-min turnaround time with a detection limit of 10 DNA/RNA molecules per assay and single nucleotide discrimination. ResultsWe demonstrate the Synestia System performance and utility with three distinct molecular applications: 1) detection of 20 genetic loci and 30 single nucleotide polymorphisms in human genomic DNA; 2) detection and genotyping of 43 unique bacterial species associated with human urinary tract infections; and 3) detection and profiling human respiratory viral pathogens including SARS-CoV-1/2, seasonal coronaviruses, Influenza A/B, and human respiratory syncytial viruses. In addition, the single-nucleotide specificity of our label-free microarray probes allowed for robust identification and discrimination of newly emerging SARS-CoV-2 lineages, such as B.1.1.7 (a.k.a. UK), B.1.351 (a.k.a. South African), P.1 (a.k.a. Brazilian), and B.1.617 (a.k.a. Indian).ConclusionThe Torus Synestia System has broad applicability in both clinical and research environments. We are confident that the Torus Synestia System will revolutionize syndromic diagnostics at the point of care (PoC) and lead to improved response times during future epidemic and pandemic pathogen outbreaks.Disclosures All Authors: No reported disclosures

Single nucleotide discrimination with sub-two nanometer monolayer graphene pore

The effective sensing area is the key point of solid-state nanopore single-molecule detection, which is determined by the diameter of the hole and the thickness of the membrane. High spatial resolution can be achieved by using single-atom thickness two-dimensional materials, such as single-layer graphene and molybdenum disulfide. Helium ion beam microscopy provides an effective technique for manufacturing small-sized nanopores. Here, we show that the precise fabrication of sub-2 nanometer nanopores has been successfully achieved on a single-layer graphene film, which provides the best spatial resolution in room-temperature ionic liquid-assisted single-molecule detection. Many graphene nanopores of different sizes have been fabricated, with a minimum pore diameter of 1.4 nm. The application of these graphene nanopores manufactured by HIM shows that single-layer graphene nanopores can distinguish different homopolymer single-stranded DNA lengths and types, and can even identify four single nucleotides.

Highly multiplexed rapid DNA detection with single-nucleotide specificity via convective PCR in a portable device.

Assays for the molecular detection of nucleic acids are typically constrained by the level of multiplexing (this is the case for the quantitative polymerase chain reaction (qPCR) and for isothermal amplification), turnaround times (as with microarrays and next-generation sequencing), quantification accuracy (isothermal amplification, microarrays and nanopore sequencing) or specificity for single-nucleotide differences (microarrays and nanopore sequencing). Here we show that a portable and battery-powered PCR assay performed in a toroidal convection chamber housing a microarray of fluorescently quenched oligonucleotide probes allows for the rapid and sensitive quantification of multiple DNA targets with single-nucleotide discrimination. The assay offers a limit of detection of 10 DNA copies within 30 min of turnaround time and a dynamic range spanning 4 orders of magnitude of DNA concentration, and we show its performance by detecting 20 genomic loci and 30 single-nucleotide polymorphisms in human genomic DNA samples, and 15 bacterial species in clinical isolates. Portable devices for the fast and highly multiplexed detection of nucleic acids may offer advantages in point-of-care diagnostics.

Sensitive Detection of Single-Nucleotide Polymorphisms by Solid Nanopores Integrated With DNA Probed Nanoparticles.

Single-nucleotide polymorphisms (SNPs) are the abundant forms of genetic variations, which are closely associated with serious genetic and inherited diseases, even cancers. Here, a novel SNP detection assay has been developed for single-nucleotide discrimination by nanopore sensing platform with DNA probed Au nanoparticles as transport carriers. The SNP of p53 gene mutation in gastric cancer has been successfully detected in the femtomolar concentration by nanopore sensing. The robust biosensing strategy offers a way for solid nanopore sensors integrated with varied nanoparticles to achieve single-nucleotide distinction with high sensitivity and spatial resolution, which promises tremendous potential applications of nanopore sensing for early diagnosis and disease prevention in the near future.

Discrimination of a single nucleotide polymorphism in the haptoglobin promoter region, rs5472, using a competitive fluorophore-labeled probe hybridization assay following loop-mediated isothermal amplification.

Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n=36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.

High-Fidelity Determination and Tracing of Small Extracellular Vesicle Cargoes.

Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for elucidating the mechanisms involved in intercellular communication. However, high-fidelity determination of sEVs' molecular cargoes in situ has yet to be achieved due to the difficulty in transporting molecular probes into intact sEVs. Herein, a fLuorescent Intracellular-Guided Hairpin-Tetrahedron (fLIGHT) nanoprobe is described for direct visualization of sEV microRNAs in situ. Integrating the advantages of nondestructive sEV penetration via DNA origami and single-nucleotide discrimination as well as wash-free fluorescence readout using a hairpin probe, the proposed approach enables high-fidelity fluorescence visualization of sEVs' microRNA without RNA extraction or leakage, demonstrating the potential of on-site tracing of sEV cargoes. This strategy opens an avenue to establishing universal molecular detection and labeling platforms that can facilitate both sEV-derived fundamental biological studies and molecular diagnostics.

Electrochemical sensing of pancreatic cancer miR-1290 based on yeast-templated mesoporous TiO2 modified electrodes.

Electrochemical sensing is an effective, low-cost technology for cancer detection. In this study, mesoporous TiO2 was prepared via biomimetic synthesis based on yeast cell templates, and used to prepare a modified electrode for the sensitive detection of pancreatic cancer miR-1290. The structure and the morphology of the TiO2 were characterized by X-ray diffraction (XRD), N2 adsorption-desorption isotherm (NADI), Atomic force microscopy (AFM), and electron probe microanalysis (EPMA). As a sensing active material, the yeast-templated mesoporous TiO2 could detect pancreatic cancer miRNAs with single-nucleotide discrimination. The sample prepared by calcination at 400°C showed the best electrochemical sensing activity. Moreover, compared with the blank electrode, the yeast mesoporous TiO2 sensing electrode could oxidize the pancreatic cancer microRNAs at a lower potential, which minimized the interference from oxygen evolution reaction at high potentials.

Enhanced SNP-sensing using DNA-templated reactions through confined hybridization of minimal substrates (CHOMS).

DNA or RNA templated reactions are attractive for nucleic acid sensing and imaging. As for any hybridization-based sensing, there is a tradeoff between sensitivity (detection threshold) and resolution (single nucleotide discrimination). Longer probes afford better sensitivity but compromise single nucleotide resolution due to the small thermodynamic penalty of a single mismatch. Herein we report a design that overcomes this tradeoff. The reaction is leveraged on the hybridization of a minimal substrate (covering 4 nucleotides) which is confined by two guide DNAs functionalized respectively with a ruthenium photocatalyst. The use of a catalytic reaction is essential to bypass the exchange of guide DNAs while achieving signal amplification through substrate turnover. The guide DNAs restrain the reaction to a unique site and enhance the hybridization of short substrates by providing two π-stacking interactions. The reaction was shown to enable the detection of SNPs and SNVs down to 50 pM with a discrimination factor ranging from 24 to 309 (median 82, 27 examples from 3 oncogenes). The clinical diagnostic potential of the technology was demonstrated with the analysis of RAS amplicons obtained directly from cell culture.

Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss

Since most dominant human mutations are single nucleotide substitutions1,2, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can be difficult to achieve3 because commonly used endonucleases, such as Streptococcus pyogenes Cas9 (SpCas9), can tolerate up to seven mismatches between guide RNA (gRNA) and target DNA. Furthermore, the protospacer-adjacent motif (PAM) in some Cas9 enzymes can tolerate mismatches with the target DNA3,4. To circumvent these limitations, we screened 14 Cas9/gRNA combinations for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, we used Beethoven mice5, which harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner-ear hair cells6. We identified a PAM variant of Staphylococcus aureus Cas9 (SaCas9-KKH) that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1 allele, in Beethoven mice and in a DFNA36 human cell line. Adeno-associated virus (AAV)-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to one year post injection. Analysis of current ClinVar entries revealed that ~21% of dominant human mutations could be targeted using a similar approach.

Sol-Gel Synthesis of Metal-Phenolic Coordination Spheres and Their Derived Carbon Composites.

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.

Single-Molecule Counting of Point Mutations by Transient DNA Binding

High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

Single Nucleotide Discrimination with Electro-Optical Nanopore

Nanopore is an emerging technique, during the past two decades, has been explore to analysis the information about the identity, concentration, structure, and dynamics of the target molecules. However, the readout signal is mainly related on the tiny changes of ionic current as single molecules pass through the nanopore. Here, we develop the ultra-low current analyzer and combine plasmonic resonance scattering spectroscopy with nanopore technique, which could acquire the optical-electrical dual signals as a molecule pass though the nanopore. It provides more unique information of single biomolecule, which have a significant potential in life science. By virtue of this integrated strategy, we have realize single nucleotide discrimination, and even for the detecting the substituent difference in a molecule, such as DNA cytosine methylation.1. Yi-Tao Long, Rui Gao, Chan Cao, Yi-Lun Ying, Da-Wei Li. CN203572764U[P].2. Gang Logan Liu, Yi-Tao Long, Yeonho Choi, Taewook Kang & Luke P Lee, Nat. Methods, 2007, 4(12), 1015-1017.

Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA.

Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within double-stranded DNA generated from real-life human mitochondrial DNA samples. Aside from the potential diagnostic value, the current study represents an additional way to control the strand displacement reaction rate without altering other reaction parameters and provides new insights into the influence of single nucleotide substitutions on 3- and 4-way branch migration efficiency and kinetics.