Single-base Mismatch Discrimination Research Articles

Fabrication of on-bead functional nucleic acid nanowires based on cyclic ligation reaction-driven rolling circle amplification for label-free detection of long noncoding RNAs in breast and lung tissues

Long noncoding RNAs (lncRNAs) act as the essential regulators in various biological processes, and are becoming potential diagnostic and prognostic biomarkers. Herein, we demonstrate the fabrication of on-bead functional nucleic acid nanowires based on cyclic ligation reaction-driven rolling circle amplification for label-free detection of long noncoding RNAs in breast and lung tissues. The presence of lncRNA activates the cyclic ligation reaction, converting low-abundance target to numerous biotinylated ligation products through repetitive denaturation-hybridization-ligation. The resulting ligation products immobilize on the magnetic bead to initiate the downstream rolling circle amplification (RCA), facilitating the synthesis of long G-quadruplex nanowires. The resultant nanowires can light up Thioflavin T (ThT) to generate a high fluorescence signal. This method displays ultrahigh sensitivity with a limit of detection of 9.97 aM, and it possesses excellent single-base mismatch discrimination capability. Moreover, it can accurately quantify cellular Homeobox (HOX) gene antisense intergenic RNA (HOTAIR) at single-cell level and distinguish the HOTAIR expression level in healthy person and lung/breast cancer patient tissues, holding great promise in clinical diagnosis.

Sensitive microRNA detection based on bimetallic label photothermal lateral flow locked nucleic acid biosensor with smartphone readout

Fast, convenient, and highly sensitive detection of microRNA (miRNA) is vital for the early diagnosis of diseases. Lateral flow biosensor (LFB) as microdevice has been used in rapid detection, however, the sensitivity is not satisfied for low-abundant miRNA detection, and short length of the miRNA requires high affinity detection probe. To address these issues, herein, we synthesized palladium-gold (Pd-Au) bimetallic nanoplates with high photothermal effect combined locked nucleic acid (LNA) detection probe to develop a photothermal lateral flow locked nucleic acid biosensor (P-LFLNAB) for miRNA detection. The P-LFLNAB enables sensitive and selective miRNA-21 detection with detection limit down to 0.094 pM using a smartphone with external thermal imager, which is 639.5 times lower than that of the conventional gold nanoparticles based colorimetric LFB and the most sensitive enzyme-free LFB for miRNA detection. Besides, the P-LFLNAB achieved single-base mismatch discrimination and can quantify signals in various cell lysates. This highly sensitive, selective and portable P-LFLNAB with smartphone readout has great potential in point-of-care test of miRNAs-related biomedical application.

High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination.

The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9’s REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9’s DNA- and guide RNA- interacting residues to optimize its editing fidelity.

Ferrocene-labeled and purification-free electrochemical biosensor based on ligase chain reaction for ultrasensitive single nucleotide polymorphism detection

Single nucleotide polymorphisms (SNPs) are crucial during the early diagnosis of a given disease as well as in evaluating their response to certain drugs. Thus, this study sought the development of ferrocene (Fc)-labeled electrochemical biosensor for SNP detection. This proposed system involves the ligation of four short probes (e.g., A, B, A′, and B′, where B′ is labeled with an Fc-tag) in the presence of target DNA via ligase chain reaction (LCR), resulting in the formation of Fc-tagged duplex AB-A′B′ in 2n. Subsequently, immobilization of the Fc-tagged duplex AB-A′B′ on a single-stranded DNA capture probe (SC-DNA)-carboxyl multi-wall carbon nanotube (MWCNT-COOH) modified glassy carbon electrode (GCE) was accomplished through hybridization. Owing to the specificity of hybridization, and the use of Fc as electrochemical probe for detection of duplex AB-A′B′, such strategy realized directly analysis of LCR products without the need for purification. By taking advantage of the thermal stability and high-discrimination ability of HiFi Taq DNA ligase for single-base differences, the specificity of hybridization, the EGFR T790 M mutant DNA (MT-DNA) biosensor was developed to offer a low limit of detection (0.75 aM), a high discrimination of single-base mismatches [as low as 0.01% (molar fraction)], a wide linear range of more than 7 orders of magnitude (1 aM–10 pM), and the recovery rates (95.3%–107.8%) from human serum samples. Thus, the biosensor under development was found to be economical, highly-sensitive, and exceptionally selective for detection of SNPs, and as well as extending the versatile applications of LCR to offer great potential for diagnosis and individual clinical regimens.

Aligner mediated cleavage of nucleic acids for site-specific detection of single base mismatch

Single base mismatch can always connect with various gene-related diseases, whose determination has aroused widespread interest. So far, various methods have been developed to determine the common base mismatch. However most of them are complex, time-consuming. Herein, we report a novel method, which only need one conventional endonuclease (NEase) and achieve site-specific cleavage in a programmable way, to detect single base mismatch, termed aligner-mediated cleavage-based single base mismatch discrimination (AMCMD). The DNA aligner (DA) is in a stem-loop structure, consistent with an incomplete recognition site of NEase on its stem and a 5′-side arm complementary to the target sequence (TS). Once TS contains matched base and hybridizes with DA, the complete recognition site of NEase is formed, and the TS will be cleavaged with fast speed, while converse is not. Based on it, the method can clearly distinguish mismatched and complementary bases. Without sample pre-processing, we were able to obtain and verify all the test result in about 30 min through the polyacrylamide gel electrophoresis analysis. This endows the proposed method with a simpler advantage. Then we combined AMCMD and EXPAR to create a new method for single base mismatch discrimination, the short sequence obtained by AMCMD as a target to trigger EXPAR, with a detection limit at 1pM level. Another process with human serum underlines that AMCMD is compatible with the complex biological sample, thus it has the potentials for practical applications.

Ultrasensitive Electrochemical Biosensor Developed by Probe Lengthening for Detection of Genomic DNA in Human Serum.

As an alternative to most of the reported nucleic acid amplification-based electrochemical DNA biosensors used for detection of trace levels of genomic DNA, we herein present a novel detection concept. The proposed system involves the conversion of two short double-stranded DNAs (dsDNAs), labeled with a thiol-tag or biotin-tag, into a single integrated dsDNA containing thiol and biotin at both terminals in the presence of target DNA through ligase chain reaction (LCR) and followed by the immobilization of these integrated dsDNAs on a bovine serum albumin (BSA)-modified gold electrode surface. Owing to rapid depletion of the two short dsDNAs via LCR, the integrated dsDNAs were generated in an exponential manner so that this sensoring approach offered a limit of detection of 25 yoctomoles (15 copies in 50 μL sample volumes), a high discrimination of single-base mismatch and a wide linear concentration range (across 6 orders of magnitude) for target DNA. Significantly, the proposed sensor, which has simplicity in operation and ease of miniaturization, detected the target of interest in total nucleic acid extracts derived from clinical serum samples with excellent results, thereby demonstrating its considerable diagnostic potential in fields ranging from virus detection to the diagnosis of genetic diseases.

Ultrasensitive detection of lung cancer-associated miRNAs by multiple primer-mediated rolling circle amplification coupled with a graphene oxide fluorescence-based (MPRCA-GO) sensor.

MicroRNAs (miRNAs) play important roles in gene regulation and have been reported as biomarkers in cancer diagnosis. Herein, we develop an isothermal miRNA detection platform based on the highly efficient, multiple primer-mediated rolling circle amplification method coupled with a graphene oxide-based fluorescence (MPRCA-GO) assay, using lung cancer-associated miRNAs (miR-21 and miR-210) and a reference miRNA (miR-16) as model targets. The combination of the designed ssDNA probe and T4 RNA ligase (T4 Rnl2) used in the MPRCA-GO assay allowed for single-base mismatch discrimination. In addition, the superfluorescence quenching ability of GO allowed for rapid fluorescence detection. The developed platform had a limit of detection as low as 0.87 fM and could detect target miRNAs in cancer cell lines and human serums. Therefore, the MPRCA-GO sensor has the potential for single nucleotide polymorphism (SNP) analysis and applications in clinical diagnostics.

Simultaneous Discrimination of Single-Base Mismatch and Full Match Using a Label-Free Single-Molecule Strategy.

Identification of single-base mismatches has found wide applications in disease diagnosis, pharmacogenetics, and population genetics. However, there is still a great challenge in the simultaneous discrimination of single-base mismatch and full match. Combined with a nanopore electrochemical sensor, a shared-stem structure of molecular beacon was designed that did not need the labeling, but achieved an enhanced signal-to-background ratio of ∼104, high thermodynamic stability to bind with target sequences, and a fast hybridization rate. Fully matched and single-base mismatched sequences were simultaneously discriminated at the single-molecule level, which is expected to have potential applications ranging from the quick detection, early clinical diagnostics to point-of-care research.

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.

A rapid, ratiometric, enzyme-free, and sensitive single-step miRNA detection using three-way junction based FRET probes

MicroRNAs (miRNAs) are single stranded endogenous molecules composed of only 18–24 nucleotides which are critical for gene expression regulating the translation of messenger RNAs. Conventional methods based on enzyme-assisted nucleic acid amplification techniques have many problems, such as easy contamination, high cost, susceptibility to false amplification, and tendency to have sequence mismatches. Here we report a rapid, ratiometric, enzyme-free, sensitive, and highly selective single-step miRNA detection using three-way junction assembled (or self-assembled) FRET probes. The developed strategy can be operated within the linear range from subnanomolar to hundred nanomolar concentrations of miRNAs. In comparison with the traditional approaches, our method showed high sensitivity for the miRNA detection and extreme selectivity for the efficient discrimination of single-base mismatches. The results reveal that the strategy paved a new avenue for the design of novel highly specific probes applicable in diagnostics and potentially in microscopic imaging of miRNAs in real biological environments.

A Dual-signal Amplification Method for DNA Detection Based on Exonuclease III and Fluorescence Quenching Ability of MoS 2 Nanosheet

Abstract A highly sensitive and selective DNA biosensor was described based on the fluorescence quenching ability of MoS2 nanosheet and exonuclease III (Exo III) assisted dual-signal amplification. In the absence of target DNA, two single oligonucleotides linked fluorophores (HP1 and HP2) resisted the digestion by Exo III due to the 3′-termini protrusion, resulting in low fluorescent signal by the adsorption and the quenching ability of MoS2 nanosheets toward single oligonucleotides and fluorophores. In the presence of target DNA, HP1 and HP2 were digested by Exo III because of the target DNA-induced two-step hybridization, and desorbed from the surface of MoS2 nanosheets, trigging dual-signal amplification. As a result, a large amount of fluorescent fragments and high fluorescent signal were generated. Under the optimal conditions, the proposed strategy could detect target DNA with a detection limit of 0.28 pM. In comparison with single amplification method, this method provided an improvement in the sensitivity and discrimination of single-base mismatch.

Highly sensitive electrochemiluminescent DNA biosensor based on hydrazide-modified graphene quantum dots and hemin/G-quadruplex DNAzyme

Graphene quantum dots (GQDs), an alternative of conventional luminescent reagents, has been widely used in electrochemiluminescent (ECL) analysis because of its low cost, non-toxicity and ease of preparation. To further improve the ECL signal of GQDs, we herein succeeded in synthesizing hydrazide modified graphene quantum dots (HM-SGQDs). In comparison with GQDs, HM-SGQDs possessed abundant luminol like units, thus greatly enhancing the ECL intensity. On the other hand, luminol involved ECL system typically needed the participation of H2O2 as a coreactant, while hemin/G-quadruplex DNAzyme (hGQ) DNAzyme could efficiently catalyze the decomposition of H2O2. Accordingly, taking p53 gene as a model, a novel ECL DNA biosensor was developed based on HM-SGQDs and hGQ DNAzyme. The experimental results indicated target DNA can be quantified in a linear range from 100fM to 100nM with a detection limit of 66fM (S/N=3). Meanwhile, the discrimination of single-base mismatch was also achieved with the proposed analytical approach, suggesting broad potential applications of nucleic acid-related clinical diagnosis.

Accurate and visual discrimination of single-base mismatch by utilization of binary DNA probes in gold nanoparticles-based biosensing strategy

Herein we report a colorimetric biosensing strategy to discriminate single-nucleotide mutation in DNA with high selectivity using unmodified gold nanoparticles (AuNPs) as indicators. In the AuNPs-based colorimetric strategy, binary DNA probes were produced by splitting a long DNA probe in the middle for sensitive differentiation of single-base mismatch. The detection limit of this method toward target DNA was 5nM. The developed system has superior advantages of utilization of inexpensive materials, simplicity and visualization. Moreover, binary DNA probes not only can distinguish single-base mutation in the target DNA very well, as compared to long DNA probe, but also can construct “AND” logic gate using two distinct target DNAs as inputs, which holds great potential for increasing the accuracy of disease diagnosis in clinical applications.

Single-base mismatch discrimination by T7 exonuclease with target cyclic amplification detection.

T7 exonuclease is reported for the first time to have high specificity in discriminating single-base mismatch and utilized for developing a target cyclic amplification biosensor strategy for sensitive SNP detection based on graphene oxide quenching of uncleaved probes.

A restriction enzyme-powered autonomous DNA walking machine: its application for a highly sensitive electrochemiluminescence assay of DNA.

The construction of a restriction enzyme (Nt.AlwI)-powered DNA walking machine and its application for highly sensitive detection of DNA are described. DNA nanostructure tracks containing four overhang sequences with electrochemiluminescence (ECL) labels and complementary to the walker (target DNA) are self-assembled on the sensing electrode. The walker hybridizes with the complementary sequences on the tracks and forms specific recognition sites for Nt.AlwI, which cleaves the overhang sequences, releases the ECL labels and enables directional movement of the walker along the tracks. The formation of the nanostructure tracks and the Nt.AlwI-assisted cleavage of the overhang sequences in the presence of the walker are verified by using polyacrylamide gel electrophoresis analysis and cyclic voltammetry. The successive movement of the walker on the nanostructure tracks leads to continuous removal of massive ECL labels from the sensing electrode, which results in a significantly amplified suppression of the ECL emission for highly sensitive detection of sequence-specific DNA down to 0.19 pM. Results show that this DNA walking machine can also offer single-base mismatch discrimination capability. The successful application of the DNA walking machine for sequence-specific DNA detection can thus offer new opportunities for molecular machines in biosensing applications.

DNA biosensor based on hybridization refractory mutation system approach for single mismatch detection

We report on a simple approach to enhance solid-phase hybridization-based single base mismatch discrimination at high ionic strength based on the deliberate insertion of a natural DNA base mismatch in the surface-tethered probe. A large drop in hybridization signal of single base mismatched alleles using the designed probe as compared with the conventional probe, from 80% to less than 25% of the signal obtained with the fully complementary, non-mutation-containing sequence, when using colorimetric detection was further improved to 20% when using electrochemical detection, attributable to a difference of spacing of immobilized probes. Finally, the designed probe was used for the electrochemical detection of the DQA1*05:05 allele amplified from real human blood samples.

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.

A General Strategy for Label-Free Sensitive DNA Detection Based on Quantum Dot Doping

Development of rapid, sensitive, and cost-effective DNA detection is of great significance to meet the growing demand of disease diagnostics. Herein, we report a new general strategy for label-free sensitive in-solution DNA detection using quantum dot (QD) doping-induced photoluminescence as a fluorogenic reporter system. The dopant mercury (Hg(II)) ions are initially sequestered in the hairpin-structured probe through T-Hg(2+)-T mismatch formation. Upon hybridization with the DNA target, the hairpin is disrupted and Hg(2+) ions are released and incorporated into ZnSe QDs, leading to a dopant-specific emission peak at 560 nm for DNA detection. Unlike the other methods, this method does not require any chemical modification of the DNA probe. It could provide high signal-to-noise ratio, robust single-base mismatch discrimination capability, and 3 orders of magnitude lower limit of detection (LOD) than the traditional molecular beacon (MB)-based fluorescence spectroscopy without any type of amplification. The method could be used for the detection of a variety of clinical significant DNA targets containing single mutations. To the best of our knowledge, this is the first study on applying chemical transformation of inorganic nanostructures to sensitive DNA detection.

Self-assembling protein platform for direct quantification of circulating microRNAs in serum with total internal reflection fluorescence microscopy

MicroRNA (miRNA) has recently emerged as a new and important class of cellular regulators. Strong evidences showed that aberrant expression of miRNA is associated with a broad spectrum of human diseases, such as cancer, diabetes, cardiovascular and psychological disorders. However, the short length and low abundance of miRNA place great challenges for conventional techniques in the miRNA quantification and expression profiling. Here, we report a direct, specific and highly sensitive yet simple detection assay for miRNA without sample amplification. A self-assembled protein nanofibril acted as an online pre-concentrating sensor to detect the target miRNA. Locked nucleic acid (LNA) of complimentary sequence was served as the probe to capture the target miRNA analyte. The quantification was achieved by the fluorescence intensity measured with total internal reflection fluorescence microscopy. A detection limit of 1pM was achieved with trace amount of sample consumption. This assay showed efficient single-base mismatch discrimination. The applicability of quantifying circulating mir-196a in both normal and cancer patient’s serums was also demonstrated.

Massively Parallel and Highly Quantitative Single-Particle Analysis on Interactions between Nanoparticles on Supported Lipid Bilayer

Observation of individual single-nanoparticle reactions provides direct information and insight for many complex chemical, physical, and biological processes, but this is utterly challenging with conventional high-resolution imaging techniques on conventional platforms. Here, we developed a photostable plasmonic nanoparticle-modified supported lipid bilayer (PNP-SLB) platform that allows for massively parallel in situ analysis of the interactions between nanoparticles with single-particle resolution on a two-dimensional (2D) fluidic surface. Each particle-by-particle PNP clustering process was monitored in real time and quantified via analysis of individual particle diffusion trajectories and single-particle-level plasmonic coupling. Importantly, the PNP-SLB-based nanoparticle cluster growth kinetics result was fitted well. As an application example, we performed a DNA detection assay, and the result suggests that our approach has very promising sensitivity and dynamic range (high attomolar to high femtomolar) without optimization, as well as remarkable single-base mismatch discrimination capability. The method shown herein can be readily applied for many different types of intermolecular and interparticle interactions and provide convenient tools and new insights for studying dynamic interactions on a highly controllable and analytical platform.