Single Nucleotide Mismatch Discrimination Research Articles

A programmable CRISPR/Cas12a-mediated hyperbranched hybridization chain reaction for ultrasensitive sensing of Mycobacterium bovis

Mycobacterium bovis results in a serious threat to domestic animal species, wildlife, and humans. Therefore, a novel colorimetric sensor with high sensitivity and specificity was developed for diagnosing Mycobacterium bovis by combining CRISPR/Cas12a with hyperbranched hybridization chain reaction (HB-HCR). In the presence of target DNA, the active Cas12a protein endowed with trans-cleavage activity showed its role in scgRNA. Thus, an auto-catalytic circuit was formed after the released gRNA molecules were converted to form additional active Cas12a effectors. Simultaneously, the released RNA initiator was used to trigger a cascade of HB-HCR events integrated G-quadruplex. Further, the rather low abundance of target could be observed with naked eyes when Hemin, H2O2 and ABTS were added. The results revealed that the limit of detecting Mycobacterium bovis target was 8.9 aM with single-nucleotide mismatch discrimination. Furthermore, the practical application of our designed colorimetric sensor for detecting Mycobacterium bovis performed by the standard addition method, the recovery ranged from 95.7% and 108.9% and the relative standard deviations (RSD) ranged from 1.2% and 2.8%, opened up a new avenue for highly sensitive and specific diagnosis of Mycobacterium bovis in real biological samples.

Super-Resolution Geometric Barcoding for Multiplexed miRNA Profiling

MicroRNA (miRNA) expression profiles hold promise as biomarkers for diagnostics and prognosis of complex diseases. Herein, we report a super-resolution fluorescence imaging-based digital profiling method for specific, sensitive, and multiplexed detection of miRNAs. In particular, we applied the DNA-PAINT (point accumulation for imaging in nanoscale topography) method to implement a super-resolution geometric barcoding scheme for multiplexed single-molecule miRNA capture and digital counting. Using synthetic DNA nanostructures as a programmable miRNA capture nano-array, we demonstrated high-specificity (single nucleotide mismatch discrimination), multiplexed (8-plex, 2 panels), and sensitive measurements on synthetic miRNA samples, as well as applied one 8-plex panel to measure endogenous miRNAs levels in total RNA extract from HeLa cells.

Super-resolution Geometric Barcoding for Multiplexed miRNA Profiling.

MicroRNA (miRNA) expression profiles hold promise as biomarkers for diagnostics and prognosis of complex diseases. Herein, we report a super-resolution fluorescence imaging-based digital profiling method for specific, sensitive, and multiplexed detection of miRNAs. In particular, we applied the DNA-PAINT (point accumulation for imaging in nanoscale topography) method to implement a super-resolution geometric barcoding scheme for multiplexed single-molecule miRNA capture and digital counting. Using synthetic DNA nanostructures as a programmable miRNA capture nano-array, we demonstrated high-specificity (single nucleotide mismatch discrimination), multiplexed (8-plex, 2 panels), and sensitive measurements on synthetic miRNA samples, as well as applied one 8-plex panel to measure endogenous miRNAs levels in total RNA extract from HeLa cells.

Super‐resolution Geometric Barcoding for Multiplexed miRNA Profiling

AbstractMicroRNA (miRNA) expression profiles hold promise as biomarkers for diagnostics and prognosis of complex diseases. Herein, we report a super‐resolution fluorescence imaging‐based digital profiling method for specific, sensitive, and multiplexed detection of miRNAs. In particular, we applied the DNA‐PAINT (point accumulation for imaging in nanoscale topography) method to implement a super‐resolution geometric barcoding scheme for multiplexed single‐molecule miRNA capture and digital counting. Using synthetic DNA nanostructures as a programmable miRNA capture nano‐array, we demonstrated high‐specificity (single nucleotide mismatch discrimination), multiplexed (8‐plex, 2 panels), and sensitive measurements on synthetic miRNA samples, as well as applied one 8‐plex panel to measure endogenous miRNAs levels in total RNA extract from HeLa cells.

Discrimination of single nucleotide mismatches using a scalable, flexible, and transparent three-dimensional nanostructure-based plasmonic miRNA sensor with high sensitivity

Localized surface plasmon resonance (LSPR) biosensors have attracted much interest due to their capacity for multiplexing, miniaturization, and high performance, which offers the potential for their integration into lab-on-a-chip platforms for point-of-care (POC) diagnostics. The need for microRNA (miRNA)-sensing platforms is particularly urgent because miRNAs are key regulators and biomarkers in numerous pathological processes and diseases. Unfortunately, however, development of such miRNA-sensing platforms has not yet been achieved. In order to realize the detection of these important biomarkers, there has been an increasing demand for POC-sensing platforms that enable label-free quantification with low sample consumption, good sensitivity, real-time responsiveness, and high throughput. Here, we developed a highly specific, sensitive LSPR miRNA-sensing platform on a flexible, scalable plasmonic nanostructure to enable single-base mismatch discrimination and attomole detection of miRNAs in clinically relevant samples. The hairpin probe contained a locked nucleic acid (LNA) that enabled the discrimination of single base mismatches based on differences in melting temperatures of perfectly matched or single base mismatched miRNAs when they formed base pairs with probes. In addition, through hybridization induced signal amplification based on precipitate formation on the gold surface through the enzyme reaction, we observed a dramatic LSPR peak shift, which enabled attomole detection. Additionally, our LSPR miRNA sensor enabled the detection of miR-200a-3p in total RNA extracts from primary cancer cell lines without purification or labeling of the miRNA. This label-free and highly specific miRNA sensing platform may have applications in POC cancer diagnostics without the need for gene amplification.

Two-dimensional titanium carbide (MXene)-based solid-state electrochemiluminescent sensor for label-free single-nucleotide mismatch discrimination in human urine

As a representative member of two dimensional MXenes, the OH-terminated Ti3C2 (Ti3C2Tx) is metallically conducting, with electrical conductivity as high as 6500 S cm−1. In addition, the terminated groups such as OH make Ti3C2 negatively charged. Based on these superiorities, Ti3C2Tx MXene was herein used to form composite film with Nafion for the solid-state ECL sensor construction for the first attempt. The utilization of Ti3C2Tx not only endow the composite film with much higher conductivity, but also enhance the adsorption amount of Ru(bpy)32+ on the electrode surface. The as-proposed sensor was investigated using tripropylamine as a representative ECL coreactant and low detection limit (S N−1 ≥3) of 5 nM was achieved. It also exhibited much excellent reproducibility (relative standard deviations of (0.69% for 107 continuous cycles) and long-term stability (about 90% of its preliminary ECL intensity can be retained after intermittent use over a 90-day period). In addition, the application potential of the as-prepared sensor was verified by the single-nucleotide mismatch discrimination in human urine. As a result, the as-prepared sensor was proved to be with the superiorities of easy-operation, simplicity, long-term stability, and applicability.

Plasmonics-based SERS nanobiosensor for homogeneous nucleic acid detection

Developing a simple and efficient nucleic acid detection technology is essential for clinical diagnostics. Here, we describe a new conceptually simple and selective “turn on” plasmonics-based nanobiosensor, which integrates non-enzymatic DNA strand-displacement hybridization for specific nucleic acid target identification with surface-enhanced Raman scattering (SERS) detection. This SERS nanobiosensor is a target label-free, and rapid nanoparticle-based biosensing system using a homogeneous assay format that offers a simple and efficient tool for nucleic acid diagnostics. Our results showed that the nanobiosensor provided a limit of detection of ~0.1nM (200amol) in the current bioassay system, and exhibited high specificity for single nucleotide mismatch discrimination. From the Clinical EditorSurface-enhanced Raman scattering (SERS) is a sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. The enhancement means that the technique may even detect single molecules. In this article, the authors describe a simple and efficient nucleic acid detection technology using SERS, with "OFF-to-ON" signal switch upon nucleic acid target identification and capture, which provides high sensitivity and specificity for single nucleotide mismatch discrimination. This new technology will be most welcomed in clinical diagnostics.

Influence of the length of target DNA overhang proximal to the array surface on discrimination of single-base mismatches on a 25-mer oligonucleotide array.

BackgroundThe performance of probes on an oligonucleotide microarray can be characterised in terms of hybridisation signal strength and the ability to discriminate sequence mismatches between the probe and the hybridising target strand, such as those resulting from SNPs. Various properties of the probe affect mismatch discrimination, such as probe length and the position of mismatched bases, and the effects of these factors have been well characterised in a variety of array formats.ResultsA low-density microarray was developed to systematically investigate the effect of a probe’s position within hybridised target PCR products on the tolerance and discrimination of single-nucleotide mismatches between the probe and target. In line with previous reports, hybridisation signals were attenuated by different degrees depending on the identity of the mismatch, the position of the mismatch within the probe, and the length of the PCR product. However, the same mismatch caused different degrees of attenuation depending on the position of the probe within the hybridising product, such that improved mismatch discrimination was observed for PCR products where a greater proportion of the total length was proximal to the array surface.ConclusionsThese results suggest that the degree of mismatch discrimination can be influenced by the choice of PCR primers, providing a means by which array performance could be fine-tuned in addition to manipulation of the properties of the probes themselves.

C5-amino acid functionalized LNA: positively poised for antisense applications.

Incorporation of positively charged C5-amino acid functionalized LNA uridines into oligodeoxyribonucleotides (ONs) results in extraordinary RNA affinity, binding specificity and stability towards 3'-exonucleases.

Structure-Function Relationships of Three Kinds of Immobilized DNA Probes in the Discrimination Abilities of Single Nucleotide Variation

We have compared here the nucleic acid hybridization abilities of three kinds of immobilized probes with different structures. Under the conventional structural design, we found that the share-stem hairpin structure probe (SHP) was more easily hybridized with the target than the linear probe and conventional hairpin shaped probe (HP). The HP probe had the lowest hybridization ability. However, it was shown a contrary result in the single-nucleotide mismatch discrimination ratio. Subsequently, we increased the Tm of the two hairpin-shaped probes and found both of the two probes were improved on the hybridization specificities even though the hybridization abilities were decreased. The result of the hybridization temperature optimization experiment showed that at 45 degrees C, the Dr Ratio (mismatch discrimination ratio) of HP-28T and SHP-32T were both as high as 8.5 while the ratios of linear probe were -0.3-0.7. The results suggested that the immobilized shared stem hairpin shaped probes also had the ability to discriminate single-nucleotide variation under proper design.

Nanoresonator chip-based RNA sensor strategy for detection of circulating tumor cells: response using PCA3 as a prostate cancer marker

There is widespread interest in circulating tumor cells (CTCs) in blood. Direct detection of CTCs (often < 1/mL) is complicated by a number of factors, but the presence of ∼103 to 104 copies of target RNA per CTC, coupled with simple enrichments, can greatly increase detection capability. In this study we used resonance frequency shifts induced by mass-amplifying gold nanoparticles to detect a hybridization sandwich bound to functionalized nanowires. We selected PCA3 RNA as a marker for prostate cancer, optimized antisense binding sites, and defined conditions allowing single nucleotide mismatch discrimination, and used a hybrid resonator integration scheme, which combines elements of top-down fabrication with strengths of bottom-up fabrication, with a view to enable multiplexed sensing. Bound mass calculated from frequency shifts matched mass estimated by counting gold nanoparticles. This represents the first demonstration of use of such nanoresonators, which show promise of both excellent specificity and quantitative sensitivity. From the Clinical EditorCancer cell detection from blood is an emerging method for more sensitive screening for malignancies. In this work, RNA detection with nanoresonators is demonstrated to have high specificity and sensitivity, suggesting that such technology may be feasible for laboratory medicine-based cancer detection.

Highly sensitive PNA Array Platform Technology for Single Nucleotide Mismatch Discrimination

Reliable discrimination of single nucleotide mismatch was demonstrated using arrays with peptide nucleic acid (PNA) probes. Newly developed PNA probes immobilization method and hybridization conditions for PNA arrays gave excellent specificity and sensitivity. And we compared the specificity, sensitivity, and stability obtained with the PNA and DNA arrays in discriminating single nucleotide mismatches. PNA arrays had superior perfect match-to-mismatch signal ratios and sensitivities. The relative signal intensities of mismatch PNA probes ranged from 1.6% to 12.1% of the perfect match PNA probes. These results demonstrated that the PNA arrays were 2.0 to 37.3 times more specific and about 10 times more sensitive than DNA arrays. A PNA array showed the same specificity and sensitivity after 12-month storage at room temperature.

Molecular Recognition with DNA Nanoswitches: Effects of Single Base Mutations on Structure

This paper investigates the properties of a simple DNA-based nanodevice capable of detecting single base mutations in unlabeled nucleic acid target sequences. Detection is achieved by a two-stage process combining first complementary-base hybridization of a target and then a conformational change as molecular recognition criteria. A probe molecule is constructed from a single DNA strand designed to adopt a partial cruciform structure with a pair of exposed (unhybridized) strands. Upon target binding, a switchable cruciform construct (similar to a Holliday junction) is formed which can adopt open and closed junction conformations. Switching between these forms occurs by junction folding in the presence of divalent ions. It has been shown from the steady-state fluorescence of judiciously labeled constructs that there are differences between the fluorescence resonance energy transfer (FRET) efficiencies of closed forms, dependent on the target sequence near the branch point, where the arms of the cruciform cross. This difference in FRET efficiency is attributed to structural variations between these folded junctions with their different branch point sequences arising from the single base mutations. This provides a robust means for the discrimination of single nucleotide mismatches in a specific region of the target. In this paper, these structural differences are analyzed by fitting observed time-resolved donor fluorescence decay data to a Gaussian distribution of donor-acceptor separations. This shows the closest mean separation (approximately 40 A) for the perfectly matched case, whereas larger separations (up to 50 A) are found for the single point mutations. These differences therefore indicate a structural basis for the observed FRET differences in the closed configuration which underpins the operation of these devices as biosensors capable of resolving single base mutations.

Electrochemical Detection of Single-Nucleotide Mismatches Using an Electrode Microarray

Gold electrode arrays with electrode diameters of 10 mum were used for the detection of eight single-nucleotide mismatches in unlabeled and prehybridized DNA by electrochemical impedance spectroscopy (EIS). Because of the differences in the electrical properties of films of duplex DNA (normal duplex DNA in B-form) in the presence and absence of Zn(2+) at pH > or = 8.6, Randles equivalent circuits were employed to evaluate the EIS results. The difference in the charge-transfer resistance (DeltaR(CT)) between B-DNA (absence of Zn2+ at pH > or = 8.6) and M-DNA (presence of Zn2+ at pH > or = 8.6) allows unequivocal detection of all eight single-nucleotide mismatches within a 20-mer DNA sequence. After dehybridization/rehybridization with target DNA, DeltaR(CT) allows the discrimination of single-nucleotide mismatches with concentrations of the target strand as low as 10 fM. Although the presence of protein impurities (bovine serum albumin, 10 microg/mL) interferes with the detection of the target strand (1 pM detection limit), the presence of nontarget DNA (calf thymus DNA, 10(-8) M) does not interfere, and the detection limit for recognition of the target strand remains at 10 fM.

DNA hybridization and discrimination of single-nucleotide mismatches using chip-based microbead arrays.

The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a "plug and play" approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The microcavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microbeads. Collectively, these features allow the identification and quantitation of target DNA analytes to occur in near real time using fluorescence changes that accompany binding of the target sample. The unique three-dimensional microenvironment within the agarose bead and the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of < or = 10(2) different DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this bead array method exceed in many respects the operational characteristics of the commonly utilized planar DNA chip technologies. The power and utility of this microbead array DNA detection methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10000 and limit of detection values of approximately 10(-13) M are obtained readily with this microbead array system.

Amplifying the electrical hybridization signals of DNA array by multilayer assembly of Au nanoparticle probes.

This paper describes a versatile method for amplifying the signals of Au-nanoparticle-based DNA hybridization detecting systems. The Au nanoparticles usually serve as labels to enhance DNA hybridization signal. We further assembled several layers of nanoparticles to selectively increase the number of labelled nanoparticles. Through silver enhancement, the multilayer nanoparticles may produce significantly higher amounts of metal silver on the their surfaces than the monolayer nanoparticles did. This finally accounts for the greatly enhanced DNA hybridization signal. Particularly, the amplification of electrical detection system was demonstrated here. Electrical measuring results indicated that the current values were enhanced by approximately 3 orders of magnitude, and the single nucleotide mismatch discrimination ratio was enlarged to approximately 10(9):1.

Thin Film Biosensor for Rapid Visual Detection of Nucleic Acid Targets

We have developed a silicon-based biosensor that generates a visual signal in response to nucleic acid targets. In this system, capture oligonucleotide probes are immobilized on the surface of the biosensor. Interaction of the capture probes with a complementary target and a biotinylated detector oligonucleotide allows initiation of formation of an organic thin film on the biosensor. Thin film formation is completed by enzymatic activity of peroxidase conjugated to an anti-biotin antibody. Peroxidase catalyzes deposition of an insoluble product onto the silicon surface, generating a uniform thin film. The increased thickness on the surface alters the perceived color of the biosensor through changes in the interference patterns of reflected light from the surface, causing a color change from gold to purple. The biosensor results may be evaluated by direct visual inspection or quantified by ellipsometry. Results are obtained in 25 min with a detection limit of 5 pmol/L (150 amol/sample). Selectivity of the biosensor is demonstrated by discrimination of single nucleotide mismatches. Multitarget arrays are also analyzed with the thin film biosensor, and the system is capable of detecting targets from human serum and urine. The biosensor surface is inexpensive to produce, and the assay format is simple and rapid. The thin film biosensor is adaptable to a wide variety of nucleic acid detection applications, including rapid diagnostic testing for infectious disease panels, antibiotic resistance panels, or allelic discrimination of specific genetic markers.