Molecular Beacon Probes Research Articles

Detection of miR-133a-5p Using a Molecular Beacon Probe for Investigating Postmortem Intervals

Background: When a body is discovered at a crime or murder scene, it is crucial to examine the body and estimate its postmortem interval (PMI). Accurate estimation of PMI is vital for identifying suspects and providing clues to resolve the case. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that remain relatively stable in the cell nucleus even after death-related changes occur. Objective: This study developed a molecular beacon probe for mmu-miR-133a-5p and assessed its use in mouse muscle tissue at temperatures of 4 °C and 21 °C to estimate the PMI. Methods: A total of 36 healthy adult male BALB/c mice were divided into 9 PMI time points (0, 2, 6, 8, and 10 days) with 3 mice per time point, and they were exposed to 4 °C and 21 °C. Next, the expression pattern of mmu-miR-133a in the skeletal muscle tissue over a 10-day PMI period was analyzed using the developed molecular beacon probe. Results: The molecular beacon (MB) probe was designed for optimal thermodynamic stability with a hairpin structure that opened in the presence of mmu-miR-133a-5p, thus separating the fluorophore from the quencher and resulting in a strong fluorescence signal at 495 nm. Fluorescence intensity increased with mmu-miR-133a-5p concentration from 1 ng/μL to 1000 ng/μL and exhibited a strong correlation (R2 = 0.9966) and a detection limit of 1 ng/μL. Subsequently, the expression level of mmu-miR-133a-5p was observed to be stable in mouse skeletal muscle tissue at both 4 °C and 21 °C. Conclusions: This user-friendly assay can complete measurements in just 30 min after RNA extraction and is suitable for point-of-care testing, and it possesses the potential to improve existing complex and time-consuming methods for PMI estimation.

Amplification-free detection of Ascochyta blight in chickpea using a simple molecular beacon assay

Ascochyta blight is a major biotic stress that limits chickpea production globally. Fungicide application remains one of the effective control measures for the endemic spread. Due to the serious threat that synthetic fungicides pose to crop quality, early diagnosis of the pathogen is imperative. Whilst there have previously been several conventional lab-based diagnostic methods developed for early detection of Ascochyta rabiei, they require long assay times, specialised equipment and facilities, and trained personnel to process the samples. To overcome this challenge, a rapid amplification-free detection assay using a molecular beacon probe was developed. The method consists of a simple assembly assay that accurately detects pathogen within 30 min. The developed assay is species-specific and has a similar sensitivity level as conventional amplification-based methods. Although it is still a lab-based technique, considering the simplicity of the assay, it has a great potential to be developed further as a reliable in-field diagnostic device for early detection and quantification of fungal pathogen spores.

Sequence-Unconstrained DNA Computing: DSN cycling and PER circuitry for dynamic miRNAs analysis and multifunctional logic operations

DNA strand displacement and enzyme-driven catalysis are pivotal in regulating molecular interactions and constructing molecular logic circuits. However, their application in intricate hierarchical cascading networks has been hindered by sequence crosstalk restrictions on the multiple inputs and outputs. In this study, we present a sequence-unconstrained strategy to implement versatile molecular logic circuits, relying on duplex-specific nuclease (DSN)-assisted cycling and primer exchange reaction (PER) amplification circuitry (DSN-PER). Employing linear capture probe (CP) conjugated with magnetic beads (MBs) significantly minimizes crosstalk reactions compared to the previous molecular beacon probe (MBP) for DNA barcoding. Importantly, the sequence design of “barcodes” is fully independent of target binding sites on MBs, allowing for the sensitive detection of a broad range of microRNAs (miRNAs) using the DSN-PER method. Specifically, this platform supports the programming of common logic computations (YES, NOT, OR, INHIBIT, AND, and NOR) and hierarchical circuit operation (OR-AND) in response to a specific pattern of varying miRNAs. These versatile logic circuits offer unique features with flexible sequence design, potentially broadening the applications of dynamic nucleic acid nanotechnologies in multiplexed bio-computation, intelligent diagnostics, and sophisticated DNA nanodevices.

Engineering two-in-one DNA nanohybrids to evaluate anti-cancer effects through activatable RNA imaging

The development of DNA-based imaging techniques provides a promising way for theranostic applications. However, the dramatic chemical difference between DNA probes and therapeutics significantly hinders the construction of an ideal theranostic system for evaluation of therapeutic effects via in situ molecular imaging. In this work, we report a simple approach for the construction of a two-in-one DNA nanohybrid via one-step assembly of DNA probes and small molecular drugs, enabling evaluation of therapeutic effects via sensitive imaging of apoptosis-related mRNA. The nanohybrid was self-assembled from a rationally designed molecular beacon (MB) probe, doxorubicin (DOX, a chemotherapeutic drug) and Fe (II) ions through coordination interactions, which possesses anti-cancer effects from chemotherapeutics and the capability of efficient co-delivery of DNA probes without transfection agents. By tracking pro-apoptotic Bax mRNA expression with the MB, this system allows real-time monitoring of cell apoptotic process in response to drug treatment, enabling assessment of therapeutic effects of small molecular drugs. In addition, the approach is extended to the imaging of target microRNA in the drug treatment based on flexible DNA probe design, demonstrating the universality of this strategy. Moreover, the modular design of this DNA nanohybrid allows the introduction of photosensitizers into this system for efficient photodynamic therapy and simultaneous mRNA monitoring. This strategy expands the theranostic toolbox for the in situ evaluation of treatment response and screening anticancer drugs.

Accurate and affordable detection of rifampicin and isoniazid resistance in Tuberculosis sputum specimens by multiplex PCR-multiple probes melting analysis

BackgroundEscalating cases of multidrug-resistant tuberculosis (MDR-TB) pose a major challenge to global TB control efforts, necessitating innovative diagnostics to empower decentralized detection of gene mutations associated with resistance to rifampicin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis (M. tuberculosis) in resource-constrained settings.MethodsCombining multiplex fluorescent PCR and Multiple Probes Melting Analysis, we identified mutations in the rpoB, katG, ahpC and inhA genes from sputum specimens. We first constructed a reference plasmid library comprising 40 prevalent mutations in the target genes’ resistance determining regions and promoters, serving as positive controls. Our assay utilizes a four-tube asymmetric PCR method with specifically designed molecular beacon probes, enabling simultaneous detection of all 40 mutations. We evaluated the assay’s effectiveness using DNA isolated from 50 clinically confirmed M. tuberculosis sputum specimens, comparing our results with those obtained from Sanger sequencing and retrospective validation involving bacteriological culture and phenotypic drug susceptibility testing (pDST). We also included the commercial Xpert MTB/RIF assay for accuracy comparison.ResultsOur data demonstrated remarkable sensitivity in detecting resistance to RIF and INH, achieving values of 93.33% and 95.24%, respectively, with a specificity of 100%. The concordance between our assay and pDST was 98.00%. Furthermore, the accuracy of our assay was comparable to both Sanger sequencing and the Xpert assay. Importantly, our assay boasts a 4.2-h turnaround time and costs only $10 per test, making it an optimal choice for peripheral healthcare settings.ConclusionThese findings highlight our assay’s potential as a promising tool for rapidly, accurately, and affordably detecting MDR-TB.

Custom-Designed Probes for the Accurate Determination of Epidermal Growth Factor Receptor Mutations and Their Allelic Configuration.

The identification of single nucleotide polymorphisms (SNPs) is of paramount importance for disease diagnosis and clinical prognostication. In the context of nonsmall cell lung cancer (NSCLC), the emergence of resistance mutations, exemplified by the epidermal growth factor receptor (EGFR) T790 M and C797S, is intricately linked to the therapeutic efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Herein, a highly efficient and specific SNP detection platform for T790 M and C797S mutations has been engineered through the integration of an asymmetric polymerase chain reaction (PCR) and an ingeniously tailored four-way junction (4WJ) probe. Notably, a molecular beacon (MB) probe was judiciously designed to discern the allelic configuration of these mutations. The administration of first- and third-generation EGFR-TKIs demonstrates therapeutic efficacy solely when the mutations are in the trans configuration, characterized by a low fluorescence signal. In contrast, significant fluorescence by the MB probe is indicative of the C797S mutation being in a cis arrangement with T790M, thereby rendering the cells refractory to the therapeutic interventions of both first- and third-generation EGFR-TKIs. The assay is capable of concurrently detecting two point-mutations and ascertaining their allelic positions in a single test within 1.5 h, enhancing both efficiency and simplicity. It also exhibits high accuracy in the identification of clinical samples, offering promising implications for therapeutic guidelines. By enabling tailored treatment plans based on specific genetic profiles, our approach not only advances the precision of NSCLC treatment strategies but also marks a significant contribution to personalized medicine.

Development of an optimized RPA-PfAgo detection system for MTHFR C677T polymorphism genotyping

This study introduces an efficient RPA-PfAgo detection system for the MTHFR C677T polymorphism, proposing a potential strategy to simplify the genotyping process. By optimizing recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute (PfAgo) nucleases, we achieved DNA amplification at a constant temperature. The assay was fine-tuned through meticulous primer and guide DNA selection, with optimal conditions established at 2.0 µL of MgAc, a reaction temperature of 42 °C, and a 10-minute reaction time for RPA. Further optimization of the PfAgo cleavage assay revealed the ideal concentrations of MnCl2, guide DNA, molecular beacon probes, the PfAgo enzyme, and the RPA product to maximize sensitivity and specificity. Clinical validation of 20 samples showed 100% concordance with Sanger sequencing, confirming the method’s precision. The RPA-PfAgo system is a promising tool for on-site genotyping, with broad applications in personalized medicine and disease prevention.

Rapid identification of SARS-CoV-2 variants using stable high-frequency mutation sites.

Respiratory infectious viruses, including SARS-CoV-2, undergo rapid genetic evolution, resulting in diverse subtypes with complex mutations. Detecting and differentiating these subtypes pose significant challenges in respiratory virus surveillance. To address these challenges, we integrated ARMS-PCR with molecular beacon probes, allowing selective amplification and discrimination of subtypes based on adjacent mutation sites. The method exhibited high specificity and sensitivity, detecting as low as 104 copies/mL via direct fluorescence analysis and ~106 copies/mL using real-time PCR. Our robust detection approach offers a reliable and efficient solution for monitoring evolving respiratory infections, aiding early diagnosis and control measures. Further research could extend its application to other respiratory viruses and optimize its implementation in clinical settings.

Microfluidic measurement of intracellular mRNA with a molecular beacon probe towards point-of-care radiation triage.

In large-scale radiation exposure events, the ability to triage potential victims by the received radiation dosage is crucial. This can be evaluated by radiation-induced biological changes. Radiation-responsive mRNA is a class of biomarkers that has been explored for dose-dependency with methods such as RT-qPCR. However, these methods are challenging to implement for point-of-care devices. We have designed and used molecular beacons as probes for the measurement of radiation-induced changes of intracellular mRNA in a microfluidic device towards determining radiation dosage. Our experiments, in which fixed TK6 cells labeled with a molecular beacon specific to BAX mRNA exhibited dose-dependent fluorescence in a manner consistent with RT-qPCR analysis, demonstrate that such intracellular molecular probes can potentially be used in point-of-care radiation biodosimetry. This proof of concept could readily be extended to any RNA-based test to provide direct measurements at the bedside.

Repair-driven DNA tetrahedral nanomachine combined with DNAzyme for 8-oxo guanine DNA glycosylase activity assay, drug screening and intracellular imaging.

8-oxo guanine DNA glycosylase (8-oxoG DNA glycosylase), a crucial DNA repair enzyme, is essential for maintaining genome integrity and preventing diseases caused by DNA oxidative damage. Imaging 8-oxoG DNA glycosylase in living cells requires a dependable technique. In this study, we designed a DNAzyme-modified DNA tetrahedral nanomachine (DTDN) powered by 8-oxoG restoration. Incorporating a molecular beacon probe (MB), the constructed platform was used for amplified in situ monitoring of 8-oxoG DNA glycosylase. Under normal conditions, duplexing with a complementary strand modified with two 8-oxoG sites inhibited the activity of DNAzyme. The restoration of DNAzyme activity by the repair of intracellular 8-oxoG DNA glycosylase on 8-oxoG bases can initiate a signal amplification reaction. This detection system can detect 8-oxoG DNA glycosylase activity linearly between 0 and 20 U mL-1, with a detection limit as low as 0.52 U mL-1. Using this method, we were able to screen 14 natural compounds and identify 6 of them as 8-oxoG DNA glycosylase inhibitors. In addition, a novel approach was utilized to assess the activity of 8-oxoG DNA glycosylase in living cells. In conclusion, this method provides a universal tool for monitoring the activity of 8-oxoG DNA glycosylase in vitro and in living cells, which holds great promise for elucidating the enzyme's functionality and facilitating drug screening endeavors.

Template-controllable rolling circle amplification for dual protein sensitive analysis.

Conjoint analysis of multiple protein biomarkers can improve the accuracy of disease analysis. Rolling circle amplification (RCA) generates different products by designing circular templates, which can subsequently bind with specific probes to generate various fluorescence signals; thus, it has potential for application in the analysis of various protein biomarkers. Current RCA approaches to detect proteins commonly follow an indirect primer-controlled RCA mode. And the molecular beacon probe combines with RCA products through free collision to generate signals, resulting in lower reaction efficiency. Herein, we propose a direct template-controlled RCA mode using nanosheets as carriers and quenchers for fluorescent probes to simultaneously detect two protein biomarkers. A dual functional magnetic bead was first designed to recognize and capture two proteins while releasing two templates for subsequent RCA. RCA products compete with probes loaded on two-dimensional metal-organic framework nanosheets for hybridization, completing the transition from single-stranded to double-stranded DNA. Double-stranded DNA is far from the nanosheets, and the recovered fluorescence signal can be used to evaluate the concentration of target proteins. This method exhibits excellent analytical performance and can successfully achieve the analysis of Tau and AβO in Alzheimer's disease clinical cerebrospinal fluid samples.

DNA four-way junction-driven dual-rolling circle amplification sandwich-type aptasensor for ultra-sensitive and specific detection of tumor-derived exosomes

There is an urgent need to accurately quantify tumor-derived exosomes, which have emerged as promising non-invasive tumor diagnostic biomarkers. Herein, a bispecific-aptamer sandwich-type gold nanoparticle-modified electrochemical aptasensor was developed based on a four-way junction (4-WJ)-triggered dual rolling circle amplification (RCA)-assisted methylene blue (MB)/G-quadruplex strategy for extremely specific and sensitive exosome detection. This aptamer/exosome/aptamer sandwich-type design contained a CD63-specific aptamer and a cancerous mucin-1 (MUC1) protein-specific aptamer. The CD63 aptamer modified on a gold electrode captured exosomes, and then the sandwich-type aptasensor was formed with the addition of the MUC1 aptamer. The MUC1 aptamer's 3′-end sequence facilitated the formation of 4-WJ, assisted by a molecular beacon probe and a binary DNA probe. Subsequently, a dual-RCA reaction was triggered by binding to two cytosine-rich circle DNA templates at both ends of 4-WJ. Ultimately, dual-RCA products containing multiple G-quadruplex conformations were generated with the assistance of K+ to trap abundant MB indicators and amplify electrochemical signals. The aptasensor exhibited high specificity, sensitivity, repeatability, and stability toward MCF-7-derived exosomes, with a detection limit of 20 particles/mL and a linear range of 1 × 102 to 1 × 107 particles/mL. Moreover, it showed excellent applicability in clinical settings to recover exosomes in normal human serum. Our aptasensor is anticipated to serve as a versatile platform for detecting various specific aptamer-based targets in biomedical and bioanalytical applications.

Lanthanide‐FRET Molecular Beacons for microRNA Biosensing, Logic Operations, and Physical Unclonable Functions

AbstractTime‐resolved or time‐gated (TG) biosensing and bioimaging with luminescent lanthanide probes and Förster resonance energy transfer (FRET) have significantly advanced bioanalytical chemistry. However, the development of lanthanide‐based molecular beacons (MBs) has been rather limited. Here, we designed DNA stem‐loop MB probes against two different microRNAs (miR‐21 and miR‐27b) using Tb and Eu FRET donors and quenching (BHQ2) and fluorescent (Cy3) FRET acceptors. Limits of detection down to 190 pM and duplexed miR‐21/miR‐27b quantification at low nanomolar concentrations with Tb‐BHQ2 and Eu‐BHQ2 TG‐FRET MBs demonstrated the versatility and high analytical performance of lanthanide‐based MBs. The particular donor‐acceptor distances in the Tb‐Cy3 MB resulted in inverted nucleic acid target concentration‐dependent TG PL intensities in short (e. g., 0 to 40 μs) and long (e. g., 0.1 to 2.1 ms) TG detection windows after pulsed excitation. We showed that this specific feature of our TG‐FRET MBs can be adapted to the design of molecular logic devices (NOR, OR, NAND, AND, XNOR, XOR, IMPLEMENT, and INHIBIT). Moreover, the almost unlimited choice of TG detection windows and the distinct spectral features of Tb and Cy3 over a broad visible spectral range could be exploited to devise biophotonic physical unclonable functions for highly secure authentication and identification. Our study manifests the versatility of lanthanides for advanced biophotonic applications.

Visual and rapid detection of escolar (Lepidocybium flavobrunneum) using loop mediated isothermal amplification in conjunction with a specific molecular beacon probe

Species adulteration has become a main reason for the unexpected exposure to escolar, which is often related with the gastrointestinal disease called keriorrhea. Sensitive and accurate identification of escolar is required to protect consumers from commercial and health frauds. The present study established a visual and rapid method for escolar detection using LAMP (loop-mediated isothermal amplification) in conjunction with a MB (molecular beacon) probe. The visual MB-LAMP assay demonstrated high specificity and superb sensitivity (1 pg DNA) for escolar and low to 0.1 % (w/w) simulated adulteration could be detected within 25 min. Additionally, method validation on commercial products highlighted the umbrella term of white tuna for escolar on Chinese market. All these results indicated that the MB-LAMP method is a useful tool for rapid, sensitive and convenient detection of escolar and can also be used as a point-of-care molecular diagnostic technique since it does not require the expensive equipment.

A contamination-free and visual method using LAMP (loop-mediated isothermal amplification) and molecular beacon for sensitive and specific detection of rainbow trout (Oncorhynchus mykiss)

Fish species adulteration is a global issue, negatively affecting consumer's rights, public health and marine conservation. LAMP (loop-mediated isothermal amplification) has been widely used for fish species identification, while the commonly-used detection techniques are often time-consuming, prone to carry-over contamination, and sequence-independent. This work selected rainbow trout as a case study, and aimed to develop a novel LAMP assay, combing with a sequence-specific MB (molecular beacon) probe and a simple and portable end-point visual detection device, for rapid detection of rainbow trout with great specificity and superb sensitivity.With the results of the present study, a cost-effective and reliable strategy for the selection of MB's stem-loop structure based on the fluorescence analysis was proved. The molecular beacon LBP-4-1 in the final protocol is characterized by the low background signal and high amplification efficiency. The visual MB-LAMP assay, after optimization, has proved its specificity and applicability, and can detect rainbow trout DNA as little as 0.1 pg in 25 min. Moreover, the risk of cross-contamination can be greatly reduced by the omission of lid-opening, and with the use of a novel end-point visual device, the detection system could be used to screen rainbow trout in the field, without the constraints of complex instruments and other factors. Therefore, the present study offers a simple, fast, accurate method for rainbow trout screening, which could support the identification of authentic aquatic products and help control rainbow trout adulteration.

Synergistic effect enhancing the energy transfer efficiency of carbon dots-based molecular beacon probe for ultrasensitive detection of microRNA

An ultrasensitive molecular beacon (MB) probe employing carbon dots (CDs) as energy donor and organic dye and graphene oxide (GO) as synergistic quencher was constructed for microRNA-21 (miRNA-21) determination. The MB probe (CDs-MB-BHQ1) was first fabricated by conjugating CDs on the 5′ end of BHQ1-tagged MB, and the fluorescence of CDs decreased owing to the occurrence of luminescence resonance energy transfer (LRET). GO was introduced as a synergistic quencher, thus effectively enhancing the energy transfer efficiency through the interaction between GO and the loop region of MB. In the presence of miRNA-21, it hybridized with loop of MB, thus bringing CDs-MB-BHQ1 away from the surface of GO and increasing the distance between CDs and BHQ1. Owing to the inhibition of LRET process, the fluorescence of CDs at 513 nm (excitation at 490 nm) was recovered. This probe exhibited excellent specificity and could be applied to miRNA-21 determination (0.5–800 pM). Moreover, this proposed probe could monitor the content of miRNA-21 in real human serums, and accuracy were verified by qRT-PCR method (Er ≤ 15.3%) and recovery experiments (recoveries ranged from 88.0% to 105%).

Complementary DNA Significantly Enhancing Signal Response and Sensitivity of a Molecular Beacon Probe to Aflatoxin B1

This paper reported an improved molecular beacon method for the rapid detection of aflatoxin B1 (AFB1), a natural mycotoxin with severe carcinogenicity. With the assistance of a complementary DNA (cDNA) chain, the molecular beacon which consists of a DNA aptamer flanked by FAM and BHQ1 displayed a larger fluorescent response to AFB1, contributing to the sensitive detection of AFB1. Upon optimization of some key experimental factors, rapid detection of AFB1 ranging from 1 nM to 3 μM, within 20 min, was realized by using this method. A limit of detection (LoD) of 1 nM was obtained, which was lower than the LoD (8 nM) obtained without cDNA assistance. This aptamer-based molecular beacon detection method showed advantages in easy operation, rapid analysis and larger signal response. Good specificity and anti-interference ability were demonstrated. This method showed potential in real-sample analysis.

Molecular Beacon Probe (MBP)-Based Real-Time PCR.

In the advancement of molecular biology techniques, several probe-based techniques, like molecular beacon probe (MBP) assay, TaqMan probe, and minor groove binder (MGB) probe assay, have been reported to identify specific sequences through real-time polymerase chain reaction (PCR). All probe-based methods are more sensitive than the conventional PCR for the detection and quantification of target genes. MBP is a hydrolysis probe that emits fluorescence when getting the specific sequences on the gene. Here, we describe the application of MBP for the identification of the motif sequences present in the promoters of differentially expressed genes.

OWL2: a molecular beacon-based nanostructure for highly selective detection of single-nucleotide variations in folded nucleic acids.

Hybridization probes have been used in the detection of specific nucleic acids for the last 50 years. Despite the extensive efforts and the great significance, the challenges of the commonly used probes include (1) low selectivity in detecting single nucleotide variations (SNV) at low (e.g. room or 37 °C) temperatures; (2) low affinity in binding folded nucleic acids, and (3) the cost of fluorescent probes. Here we introduce a multicomponent hybridization probe, called OWL2 sensor, which addresses all three issues. The OWL2 sensor uses two analyte binding arms to tightly bind and unwind folded analytes, and two sequence-specific strands that bind both the analyte and a universal molecular beacon (UMB) probe to form fluorescent 'OWL' structure. The OWL2 sensor was able to differentiate single base mismatches in folded analytes in the temperature range of 5-38 °C. The design is cost-efficient since the same UMB probe can be used for detecting any analyte sequence.

An Advanced Detection System for In Situ Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe.

In situ hybridization (ISH) is a powerful method for detecting specific RNAs at the cellular level. Although conventional ISH using hapten-labeled probes are useful for detecting multiple RNAs, the detection procedures are still complex and required longer time. Therefore, we introduced a new application of fluorescence resonance energy transfer (FRET)-based molecular beacon (MB) probes for ISH. MCF-7 cells and C57BL/6J mouse uterus were used for ISH. MB probes for ERα mRNA and 28S rRNA were labeled with Cy3/BHQ-2 and 6-FAM/DABCYL, and conventional probes were labeled with digoxigenin. Fluorescence measurements revealed that of more-rapid hybridization kinetics compared to conventional probes. In MCF-7 cells, 28S rRNA was detected in nucleolus and cytoplasm of all cells, whereas ERα mRNA was detected in some nucleolus. In the uterus, 28S rRNA was clearly detected using complementary MB probe, but there were no signals in control slides. Moreover, 28S rRNA was detected in all cells, whereas ERα mRNA was detected mainly in the epithelium. Fluorescence intensity of 28S rRNA was decreased significantly in 1 or 2 base-mismatched sequences, that indicates highly specific detection of target RNAs. In conclusion, the FRET-based MB probes are very useful for ISH, providing rapid hybridization, high sensitivity and specificity.