Single-base Mismatch Research Articles

Detection of target DNA with a visual CRISPR-associated hyperbranched rolling circle amplification technique

This paper presents a novel clustered regularly interspaced short palindromic repeat (CRISPR)-associated HRCA technique (CART). During the entire detection process of CART, the target DNA is first specifically recognized and cleaved by a pair of Cas9/sgRNA complexes; then, the cleaved product is ligated into circular DNA as the template of HRCA, and the circular DNA is efficiently amplified by HRCA. Therefore, CART has the advantages of Cas9/sgRNA (single-base mismatch specificity) and HRCA (isothermal reaction temperature and high sensitivity). This technique has been verified by detecting various human papillomavirus (HPV) genes with numerous subtypes. In summary, this study provides a new and effective method for the detection of nucleic acids.

Signal “on-off-off” strategy for improving the sensitivity of BRCA1 electrochemical detection by combining gold substrate amplification, DNA conformational transformation and DSN enzymatic hydrolysis dual reduction

In this article we proposed a new signal amplification strategy based on signal “on-off-off” for improving the detection sensitivity of breast cancer 1 (BRCA1) electrochemical sensor. First, the stem-loop structured captured DNA (C-DNA) modified by 5′ sulfhydryl group and 3′ methylene blue (MB), was self-assembled on electrodeposited nanoAu surface through Au–S reaction. Electrodeposition of nanoAu increased electrode area and strengthened MB electrochemical signal, which indicated “signal on”. Then, BRCA1 hybridization with C-DNA made MB away from nanoAu surface, and reduced the MB signal due to the formation of stiff C-DNA/BRCA1 hybridized structure, which indicated “signal off”. Finally, the double strand specific nuclease (DSN) was used to cleave C-DNA/BRCA1, and led to the further decrease of MB signal, which also indicated “signal off”. Results showed that the designed sensor based on signal “on-off-off” had excellent detection sensitivity for BRCA1. The relative percentage change of MB reduction peak current was linear with BRCA1 concentration from 5 nM to 70 nM, and a low detection limit of 52 pM was achieved. The proposed sensor could distinguish single base mismatch BRCA1, two base mismatch BRCA1, and non-complementary DNA well. In addition, the sensor showed good reproducibility and stability.

Hydrazone chemistry-mediated CRISPR/Cas12a system for bacterial analysis.

In this study, a hydrazone chemistry-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system has been proposed for the fist time and constructed. In our system, hydrazone chemistry is designed and employed to accelerate the formation of a whole activation strand by taking advantage of the proximity effect induced by complementary base pairing, thus activating the CRISPR/Cas12a system quickly and efficiently. Moreover, the introduction of hydrazone chemistry can improve the specificity of the CRISPR/Cas12a system, allowing it to effectively distinguish single-base mismatches. The established system has been further applied to analyze Pseudomonas aeruginosa by specific recognition of the probe strand with a characteristic fragment in 16S rDNA to release the hydrazine group-modified activation strand. The method shows a wide linear range from 3.8 × 102 colony-forming units (CFU)/ml to 3.8 × 106 CFU/ml, with the lowest detection limit of 24 CFU/ml. Therefore, the introduction of hydrazone chemistry may also broaden the application of the CRISPR/Cas12a system.

Electrochemical Detection of Femtomolar DNA via Boronate Affinity-Mediated Decoration of Polysaccharides with Electroactive Tags.

In view of their high efficiency and cost-effectiveness, polymers are of great promise as carriers for signal tags in amplified detection. Herein, we present a polysaccharide-amplified method for the electrochemical detection of a BRCA1 breast cancer gene-derived DNA target at the femtomolar levels. Briefly, peptide nucleic acid (PNA) with a complementary sequence was tethered as the capture probe for the DNA target, to which carboxyl group-containing polysaccharides were then attached via facile phosphate-Zr(IV)-carboxylate crosslinking, followed by the decoration of polysaccharide chains with electroactive ferrocene (Fc) signal tags via affinity coupling between a cis-diol site and phenylboronic acid (PBA) group. As the polysaccharide chain contains hundreds of cis-diol sites, boronate affinity can enable the site-specific decoration of each polysaccharide chain with hundreds of Fc signal tags, efficiently transducing each target capture event into the decoration of many Fc signal tags. As polysaccharides are cheap, renewable, ubiquitous, and biodegradable natural biopolymers, the use of polysaccharides for signal amplification offers the benefits of high efficiency, cost-effectiveness, excellent biocompatibility, and environmental friendliness. The linear range of the polysaccharide-amplified method for DNA detection was demonstrated to be from 10 fM to 10 nM (R2 = 0.996), with the detection limit as low as 2.9 fM. The results show that this method can also discriminate single base mismatch with satisfactory selectivity and can be applied to DNA detection in serum samples. In view of these merits, the polysaccharide-amplified PNA-based electrochemical method holds great promise in DNA detection with satisfactory sensitivity and selectivity.

Design of Split G-quadruplex-based DNA–Bridged Nucleic Acid Chimera Nanotweezers That Recognize Short Nucleic Acids with a Single-base Mismatch

Design of Split G-quadruplex-based DNA–Bridged Nucleic Acid Chimera Nanotweezers That Recognize Short Nucleic Acids with a Single-base Mismatch

Development of an MSPQC Nucleic Acid Sensor Based on CRISPR/Cas9 for the Detection of Mycobacterium tuberculosis.

Accurate and rapid detection of nucleic acid plays a vital role in the clinical treatment of tuberculosis caused by Mycobacterium tuberculosis (M.TB). However, false-negative and false-positive results caused by base mismatches could affect the detection accuracy. Inspired by the unique property of CRISPR/Cas9, we proposed a new MSPQC M.TB sensor based on the CRISPR/Cas9 system, which can distinguish single-base mismatches in 10 bases from the protospacer adjacent motif (PAM) region. In the proposed sensor, single-stranded DNA on Au interdigital electrodes was used as a capture probe for the target and an initiator for hybridization chain reaction (HCR). HCR was used to generate long double-stranded DNA (dsDNA), which could span the Au interdigital electrodes. CRISPR/Cas9 was used as recognition components to recognize capture/target dsDNA. When the target existed, the capture probe hybridized with the target to form dsDNA, which could be recognized and cut by CRISPR/Cas9. Thus, the DNA connection between electrodes was cut off and resulted in the MSPQC response. When no target existed, the capture probe remained single-stranded and could not be recognized and cut by CRISPR/Cas9. Therefore, DNA connection between electrodes was reserved. Moreover, silver staining technology was utilized to improve the sensitivity of detection. M.TB was detected by the proposed sensor using specific sequence fragments of 16S rRNA of M.TB as the target. The detection time was down to 2.3 h. The limit of detection (LOD) was 30 CFU/mL.

Nonenzymatic Multiamplified Electrochemical Detection of Medulloblastoma-Relevant MicroRNAs from Cerebrospinal Fluid.

The sensitive analysis of microRNAs (miRNAs) in cerebrospinal fluid (CSF) holds promise for the minimally invasive early diagnosis of brain cancers such as pediatric medulloblastoma but remains challenging due partially to a lack of facile yet sensitive sensing methods. Herein, an enzyme-free triple-signal amplification electrochemical assay for miRNA was developed by integrating the target-triggered cyclic strand-displacement reaction (TCSDR), hybridization chain reaction (HCR), and methylene blue (MB) intercalation. In this assay, the presence of target miRNA (miR-9) initiated the TCSDR and produced primers that triggered the subsequent HCR amplification to generate copious double-stranded DNAs (dsDNAs) on the electrode surface. Intercalation of a large number of MB reporters into the long nicked double helixes of dsDNAs yielded a more enhanced signal of differential pulse voltammetry. The enzyme-free multiple-amplification approach allowed for highly sensitive (detection limit: 6.5 fM) and sequence-specific (single-base mismatch resolution) detection of miR-9 from tumor cells and human CSF with minimal sample consumption (10 μL). Moreover, the clinical utilization of this method was documented by accurate discrimination of five medulloblastoma patients from the nontumoral controls. In light of its sensitivity, specificity, and convenience of use, this electrochemical method was expected to facilitate the early detection of malignant brain tumors.

PCDetection: PolyA-CRISPR/Cas12a-based miRNA detection without PAM restriction

MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The aberrant expression of miRNAs is related to many diseases. MiRNAs can serve as potential biomarkers for the prognosis and diagnosis of cancers and other human diseases. However, the short sequence and high sequence similarity of miRNAs impede detection. Herein, we propose a method to integrate polyA-tailing and CRISPR/Cas12a to amplify and detect all miRNAs with high specificity and sensitivity. PolyA-tailing enables efficient amplification of RNA and introduces a universal PAM sequence for Cas12a to unlock its PAM restriction. The CRISPR-Cas system guarantees the specific recognition of nucleic acid sequences with a single base mismatch. A limit of detection (LOD) as low as 50 fM was achieved. The practical application ability of polyA-CRISPR/Cas12a-based miRNA detection was validated by miRNA analyses in multiple cancer cell samples. With the increasing stability of RNA samples, low cost, excellent specificity, and sensitivity, this method demonstrates great potential to scale up to parallel diagnostic sets for miRNA-related disease.

REVEALR-Based Genotyping of SARS-CoV-2 Variants of Concern in Clinical Samples.

The SARS-CoV-2 virus has evolved into new strains that increase viral transmissibility and reduce vaccine protection. The rapid circulation of these more harmful strains across the globe has created a pressing need for alternative public health screening tools. REVEALR (RNA-encoded viral nucleic acid analytic reporter), a rapid and highly sensitive DNAzyme-based detection system, functions with perfect accuracy against patient-derived clinical samples. Here, we design REVEALR into a novel genotyping assay that detects single-base mismatches corresponding to each of the major SARS-CoV-2 strains found in the United States. Of 34 sequence-verified patient samples collected in early, mid, and late 2021 at the UCI Medical Center in Orange, California, REVEALR identified the correct variant [Wuhan-Hu-1, alpha (B.1.1.7), gamma (P.1), epsilon (B.1.427/9), delta (B.1.617.2), and omicron (B.1.1.529)] with 100% accuracy. The assay, which is programmable and amenable to multiplexing, offers an important new approach to personalized diagnostics.

A clamp-improved universal amplified system for ratiometric fluorescent detection of single-nucleotide polymorphisms coupled with a novel dual-emissive silver nanocluster

Rapid, sensitive and precise detection of single-nucleotide polymorphisms (SNPs) with risk association is of great significance but still remains challenging in resource-limited clinical settings. Herein, a convenient, low-cost and label-free ratiometric fluorescent sensing strategy is developed by coupling peptide nucleic acid (PNA) clamp-improved catalyzed hairpin assembly (CHA) reaction with a novel hairpin DNA-templated dual-emissive silver nanocluster (AgNC). In the presence of targets containing single-base mismatches, the sensing platform initiates the amplification and enlarges the distance between the Ag emitter pair in AgNC, resulting in a decrease of red emission accompanied by a significant optical readout. On the contrary, the PNA clamp is capable of specifically blocking the perfectly homologous counterpart in DNA sequence and thereby impeding unwanted amplification. In this study, the SNP of oncogenic Kirsten ras (KRAS) gene has been accurately and effectively genotyped with a detection limit of 9.6 pM. Moreover, this nanosensor can effectively discriminate a series of SNPs with vivid color change and its universality can be simply achieved by a rational design of the relative sequences. This platform performs well for both target DNA spiked cell lysate samples and PCR amplicons from standard cell lines, implying its great practical application prospect for bioanalysis and biosensors.

A self-assembly amplification strategy for ultra-sensitive detection of microRNA based on phosphorothioated probes

Based on self-assembly amplification, we designed a novel microRNA (miRNA)-detection method with high specificity and sensitivity. Two unique DNA probes named Linker A and Linker B were modified with phosphorothioate (PS) at both ends. In the presence of the target miRNA, these two DNA probes were ligated together by T4 DNA ligase enzyme to form a dumbbell-shaped DNA. Then the dumbbell-shaped structure would be extended with Bst 2.0 DNA polymerase enzyme, triggering the strand displacement activity without using additional primers. These results revealed our method's ultralow detection limit (300 fM), excellent selectivity, simple operation, and capability to discriminate single-base mismatches. It is believed that this proposed approach would have great application potential in clinical diagnosis and other involved fields.

Label-free detection of HPV mRNA with an artificial chaperone-enhanced MNAzyme (ACEzyme)-based electrochemical sensor

Nucleic acid biosensors for point-of-care (POC) diagnostic applications are highly desirable. The ability to detect DNA and RNA in a simple, rapid, affordable and portable format leads to a range of important applications for early screening in the field of disease monitoring and management. Herein, we report the development of an isothermal, label-free electrochemical biosensor that was designed on the basis of target-driven MNAzyme cleavage activity. Hybridization with HPV mRNA, a model nucleic acid target, activated MNAzyme and initiated the cleavage of immobilized hairpin substrates, leading to changes in the electrochemical signal. Under optimal conditions, a detection limit of 2.6 pM was obtained with an incubation time of 60 min. Furthermore, an artificial chaperone-enhanced MNAzyme (ACEzyme) system was integrated to an electrochemical biosensor for the first time. The analytical performance of the biosensor was enhanced, and the detection time was significantly reduced by the addition of PLL-g-Dex, which exhibits nucleic acid chaperone-like activity. A detection limit of 0.88 pM was obtained with a threefold decrease in incubation time without prior amplification. The proposed biosensing platform shows the advantages of simple fabrication and operation, good selectivity in the presence of single-base mismatch, and excellent versatility in a complex mixture of total RNA. We believe that this isothermal, label-free, and protein-free nucleic acid analysis platform could provide foundations for the further development of a universal nucleic acid biosensing platform for clinical application.

An ultrafast ratiometric electrochemical biosensor based on potential-assisted hybridization for nucleic acids detection

The development of rapid nucleic acid detection methods, which are promising the diagnostic standard of the infectious disease, could expand more options for disease traceability and controllability. Nucleic acid hybridization-based biosensing techniques still encounter limitations in meeting the requirements for rapid detection. Therefore, we proposed a potential-assisted ratiometric electrochemical biosensor for rapid and accurate target DNA detection. This dual-signal analysis system actuated an enzyme-free detection process. The application of an external electric field accelerated molecular dynamics, resulting in highly efficient collision chances. This hybridization process was thus improved from hours to minutes compared with passive hybridization approach. The biosensor not only had a high sensitivity with the detection limit of 12 fM, but also features a robust capability in identifying single-base mismatch. Moreover, the biosensor realized sensitive detection of target DNA in complex biological environments. Overall, this sensing strategy exhibits a promising potential for application in point-of-care testing.

Electrochemiluminescent detection of epilepsy biomarker miR-134 using a metal complex light switch

The detection of a key biomarker in epilepsy, miR-134, using an environmentally sensitive electrochemiluminescent luminophore, [Ru(DPPZ)2 PIC]2+, is reported, DPPZ is dipyrido[3,2-a:2′,3′-c]phenazine) and PIC is (2,2′-bipyridyl)-2(4-carboxy phenyl) imidazo [4,5][1,10] phenanthroline. A thiolated capture strand is first labelled with [Ru(DPPZ)2 PIC]2+ and then adsorbed onto a gold electrode. No significant electrochemiluminescence, ECL, is observed for immobilised Ru-labelled capture strands which is consistent with the light-switch dye being exposed to the aqueous solution. In sharp contrast, binding of the target turns on ECL. The ECL intensity, IECL, depends on the number of adenine “spacer” bases between the end of the capture sequence and the dye. The ECL intensity for the optimised system increases linearly with increasing miR-134 concentration from 100 nM to approximately 20 μM. Single and double base mismatches produce IECL that are only approximately 30% and 8% respectively of that observed for the fully complementary target reflecting differences in their association constants. Significantly, the presence of BSA protein causes IECL to increase by less 5% in either the single or duplex circumstances. Finally, the ability of the sensor to quantify miR-134 in unprocessed plasma samples from healthy volunteers and people with epilepsy is reported.

Signal-On Fluorescence Biosensor for Highly Sensitive Detection of miRNA-21 Based on DNAzyme Assisted Double-Hairpin Molecular Beacon.

Although miRNAs exist in small quantities in the human body, they are closely related to the abnormal expression of genes in diseases such as tumors. Therefore, sensitive detection of miRNAs is very important for the prevention and treatment of various tumors and major diseases. The purpose of this study is to develop a label-free sensing strategy based on the co-action of double-hairpin molecular beacons and deoxyribozymes (DNAzymes) for highly sensitive detection of miRNA-21. The target miRNA-21 promotes the assembly of DNAzyme with a complete catalytic core region. At the presence of Mg2+, DNAzyme cuts a substrate into short chains, which open the double hairpin molecular beacon, and then form G-quadruplexs at both ends, specifically binding more ThT to generate a amplified fluorescent signal. The cut substrate will be replaced by the uncut ones in the next stage, increasing the concentration of reactants, and thus further improving the fluorescence intensity. This DNAzyme assisted double hairpin molecular beacon has a certain degree of discrimination for substances with single base mismatches, and the detection limit of miRNA-21 is 0.13 pM, lower than that of the many other analysis. Further, this detection has good selectivity and sensitivity in serum. Therefore, this strategy provides a simple, fast and low-cost platform for the sensitive detection of miRNA-21, having potential applications in early cancer diagnosis.

Synergistic enhanced rolling circle amplification based on mutS and radical polymerization for single-point mutation DNA detection

The detection of nucleic acids in biofluids is essential for changing the paradigm of disease diagnosis. As there are very few nucleic acids present in human biofluids, a high sensitivity method is required to detect nucleic acids for disease diagnosis. The Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation is associated with non-small cell lung cancer. It is a point mutation and requires a highly selective detection technique. In this study, high sensitivity and selectivity were achieved for the detection of KRAS mutation using rolling circle amplification (RCA), atomic transfer radical polymerization (ATRP), mutS enzyme, and electrochemical sensors. Although RCA can isothermally amplify DNA, it has low selectivity for detecting single-base mismatch DNA, and its sensitivity is not suitable for circulating tumor DNA detection. The selectivity of RCA was improved by using mutS, which can bind specifically to point mutations. In addition, as a method of isothermal radical polymerization, ATRP was used to amplify the weak signal of RCA. Since RCA and ATRP reactions occur simultaneously, detection time was reduced, and the calculated detection limit was 3.09 aM. Computational and experimental analyses were conducted to verify each detection step and the combination of mutS, ATRP, and RCA. The experiment was performed using normal human serum samples for biological application, and the proposed detection method was confirmed to have excellent potential for diagnosing cancer patients.

Adjusting the Structure of a Peptide Nucleic Acid (PNA) Molecular Beacon and Promoting Its DNA Detection by a Hybrid with Quencher-Modified DNA

In this study, we performed an elaborate adjustment of the structure of peptide nucleic acid (PNA) molecular beacons as probes for detecting nucleic acids. We synthesized the PNA beacons with various numbers of Glu, Lys, and dabcyl (Dab) quenchers in them, and we investigated their fluorescence changes (F1/1/F0) with and without full-match DNA. As the numbers of Glu/Lys or Dab increased, the F1/1/F0 tended to decrease. Among the different beacons, the PNA beacon with one Glu and one Lys (P1Q1) showed the largest F1/1/F0. On the other hand, a relatively large F1/1/F0 was obtained when the number of Glu/Lys and the number of Dab were the same, and the balance between the numbers of Glu/Lys and Dab seemed to affect the F1/1/F0. We also investigated the DNA detection by the prehybrid of P1Q1, which consists of the T790M base sequence, [P1Q1(T790M)], with quencher-modified DNA (Q-DNA). We examined the DNA detection with single-base mismatch by P1Q1(T790M), and we clarified that there was difficulty in detecting the sequence with P1Q1 alone, but that the sequence was successfully detected by the prehybrid of P1Q1 with the Q-DNA.

Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection.

Massive DNA testing requires novel technologies to support a sustainable health system. In recent years, DNA superstructures have emerged as alternative probes and transducers. We, herein, report a multiplexed and highly sensitive approach based on an allele-specific hybridization chain reaction (AS-HCR) in the array format to detect single-nucleotide variants. Fast isothermal amplification was developed before activating the HCR process on a chip to work with genomic DNA. The assay principle was demonstrated, and the variables for integrating the AS-HCR process and smartphone-based detection were also studied. The results were compared to a conventional polymerase reaction chain (PCR)-based test. The developed multiplex method enabled higher selectivity against single-base mismatch sequences at concentrations as low as 103 copies with a limit of detection of 0.7% of the mutant DNA percentage and good reproducibility (relative error: 5% for intra-assay and 17% for interassay). As proof of concept, the AS-HCR method was applied to clinical samples, including human cell cultures and biopsied tissues of cancer patients. Accurate identification of single-nucleotide mutations in KRAS and NRAS genes was validated, considering those obtained from the reference sequencing method. To conclude, AS-HCR is a rapid, simple, accurate, and cost-effective isothermal method that detects clinically relevant genetic variants and has a high potential for point-of-care demands.

Design and fabrication of Gr/Ag-coated tilted grating sensor for ultra-sensitive detection of DNA hybridization

A graphene sensitized biosensor based on 6° tilted fiber Bragg grating (TFBG) was proposed. The introduction of the graphene coating boosted the sensitivity and stability of the Ag/TFBG sensor, and improved the probe loading and molecular capture of the sensitized layer. A quantitative analytical model was proposed to calculate the hybridization efficiency and maximum sensor response of DNA single base mismatch. The results showed that the kinetic affinity response of the Gr/Ag/TFBG sensor was stably demonstrated with a dissociation constant (kd=9.392 ± 0.178·10−3 min−1) and the detection limit of DNA was as low as 3 pM. This indicates that the sensor has the ability to distinguish single base mutations quantitatively in real time. The surface modification, probe binding and DNA hybridization were characterized based on atomic force microscopy (AFM) and Raman detection spectrum detection system. A 3D diversity analysis model for TFBG sensors was innovatively proposed based on COMSOL. The multi-mode coupling and polarization response analysis of the inner core, the electric field distribution and polarization transmission mode of the sensitized layer, and the polarization gain brought by graphene are systematically described. Excellent bioanalytical capabilities combined with well-established analytical methods make prepared sensor promising for high-throughput screening of genetic variants and disease markers.

Specific and robust hybridization based on double-stranded nucleic acids with single-base resolution

Nucleic acid hybridization plays a critical role in medical diagnostics and nanotechnology, but its selectivity and robustness remain to be improved. Here, focusing on double-stranded nucleic acid-based hybridization, we present a series of related strategies. Above all, two simple strategies for enriching toehold-included double-stranded nucleic acids have been proposed. On this basis, two universal hybridization methods with higher selectivity than typical toehold exchange reaction and a long target detection method using short probes to extend the detectable length range are realized. We also provide a double-stranded nucleic acids-catalyzed cycle amplification reaction to improve sensitivity, which has superior interference resistance and excellent discrimination for single-base mismatches. Besides, double-stranded nucleic acids with forked toeholds are used as essential elements to construct a series of logic gates that can evaluate different input combinations. Given the unique advantages of double-stranded nucleic acids, we expect the current work to advance the application of double-stranded nucleic acid-based hybridization in medical diagnostics and nanotechnology.