Turn-on RNA Mango Beacons for trans-acting fluorogenic nucleic acid detection.

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have trans-acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand. Our first design is bimolecular in nature and uses a DNA inhibition strand to prevent folding of the Mango aptamer core until binding to a target RNA. Our second design is unimolecular in nature, and features hybridization arms flanking the core that inhibit G-quadruplex folding until refolding is triggered by binding to a target RNA strand. Our third design builds upon this structure, and incorporates a self-inhibiting domain into one of the flanking arms that deliberately binds to, and precludes folding of, the aptamer core until a target is bound. This design separates G-quadruplex folding inhibition and RNA target hybridization into separate modules, enabling a more universal unimolecular beacon design. All three Mango Beacons feature high contrasts and low costs when compared to conventional molecular beacons, with excellent potential for in vitro and in vivo applications.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immunostaining.

Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.

A molecular beacon design for a colorimetric loop-mediated isothermal amplification assay.

In this study, a molecular beacon (MB) was designed for colorimetric loop-mediated isothermal amplification (cLAMP). The length of complementary bases on the MB, guanine and cytosine content (GC content), and hybridization sites of complementary bases were investigated as key factors affecting the design of the MB. We designed MBs consisting of 10, 15, and 20 complementary bases located at both ends of the HRPzyme. In the case of the long dumbbell DNA structure amplified from the hlyA gene of Listeria monocytogenes, possessing a flat region (F1c-B1) of 61 base pairs (bp), an MB was designed to intercalate into the flat region between the F1c and B1 regions of the LAMP amplicons. In the case of the short dumbbell DNA structure amplified from the bcfD gene of Salmonella species possessing a flat region (F1c-B1) length of 6 bp, another MB was designed to intercalate into the LoopF or LoopB regions of the LAMP amplicons. The results revealed that the hybridization site of the MB on the LAMP amplicons was not crucial in designing the MB, but the GC content was an important factor. The highest hybridization efficiencies for LAMP amplicons were obtained from hlyA gene-specific and bcfD gene-specific MBs containing 20- and 15-base complementary sequences, respectively, which exhibited the highest GC content. Therefore, designing MBs with a high GC content is an effective solution to overcome the low hybridization efficiency of cLAMP assays. The results obtained can be used as primary data for designing MBs to improve cLAMP accessibility.

Droplet digital molecular beacon-LAMP assay via pico-injection for ultrasensitive detection of pathogens.

A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for the ultrasensitive and quantitative identification of pathogens, leveraging the sequence-specific detection capabilities of MBs. The microfluidic device contained three distinct functional units including droplet generation, pico-injection, and droplet counting. Utilizing a pico-injector, MBs are introduced into each droplet to specifically identify LAMP amplification products, thereby overcoming issues related to temperature incompatibility. Our methodology has been validated through the quantitative detection of Escherichia coli, achieving a detection limit as low as 9 copies/µL in a model plasmid containing the malB gene and 3 CFU/µL in a spiked milk sample. The total analysis time was less than 1.5 h. The sensitivity and robustness of this platform further demonstrated the potential for rapid pathogen detection and diagnosis, particularly when integrated with cutting-edge microfluidic technologies.

Enzyme and MicroRNA Dual-Regulated Photodynamic Molecular Beacons for Cell-Selective Amplification of Antitumor Efficacy.

Photodynamic molecular beacons (PMBs) are highly appealing for activatable photodynamic therapy (PDT), but their applications are hindered by limited therapeutic efficacy. Here, by molecular engineering of enzyme-responsive units in the loop region of DNA-based PMBs, we present for the first time the modular design of an enzyme/microRNA dual-regulated PMB (D-PMB) to achieve cancer-cell-selective amplification of PDT efficacy. In the design, the "inert" photosensitizers in D-PMB could be repeatedly activated in the presence of both tumor-specific enzyme and miRNA, leading to amplified generation of cytotoxic singlet oxygen species and therefore enhanced PDT efficacy in vitro and in vivo. By contrast, low photodynamic activity could be observed in healthy cells, as D-PMB activation has been largely avoided by the dual-regulatable design. This work presents a cooperatively activated PDT strategy, which enables enhanced therapeutic efficacy with improved tumor-specificity and thus conceptualizes an approach to expand the repertoire of designing smart tumor treatment modality.

A Multiple Targeting Nanoprobe for Identifying Cancer Metastatic Sites Based on Detection of Various mRNAs in Circulating Tumor Cells.

Identification of cancer metastatic sites is of importance for adjusting therapeutic interventions and treatment choice. However, identifying the location of metastatic lesions with easy accessibility and high safety is challenging. Here we demonstrate that cancer metastatic sites can be accurately detected by a triple targeting nanoprobe. Through coencapsulating molecular beacons probing a cancer biomarker (CXCR4 mRNA), a lung metastatic biomarker (CTSC mRNA), and a bone metastatic biomarker (JAG1 mRNA), the nanoprobe decorated by SYL3C conjugated hyaluronic acid and ICAM-1 specific aptamer conjugated hyaluronic acid can target diverse phenotyped circulating tumor cells (CTCs) during epithelial-mesenchymal and mesenchymal-epithelial transitions in whole blood for sensitive probing. The detection of CTCs from cancer patients shows that the nanoprobe can provide accurate information to distinguish different cancer metastasis statuses including nonmetastasis, lung metastasis, and bone metastasis. This study proposes an efficient screening tool for identifying the location of distant metastatic lesions via facile blood biopsy.

Detecting mir-155-3p through a Molecular Beacon Bead-Based Assay.

Lung cancer (LC) is recognized as one of the most prevalent and lethal cancers worldwide, underscoring an urgent need for innovative diagnostic and therapeutic approaches. MicroRNAs (miRNAs) have emerged as promising biomarkers for several diseases and their progression, such as LC. However, traditional methods for detecting and quantifying miRNAs, such as PCR, are time-consuming and expensive. Herein, we used a molecular beacon (MB) bead-based assay immobilized in a microfluidic device to detect miR-155-3p, which is frequently overexpressed in LC. The assay relies on the fluorescence enhancement of the MB upon binding to the target miRNA via Watson and Crick complementarity, resulting in a conformational change from a stem-loop to a linear structure, thereby bringing apart the fluorophores at each end. This assay was performed on a microfluidic platform enabling rapid and straightforward target detection. We successfully detected miR-155-3p in a saline solution, obtaining a limit of detection (LOD) of 42 nM. Furthermore, we evaluated the method's performance in more complex biological samples, including A549 cells' total RNA and peripheral blood mononuclear cells (PBMCs) spiked with the target miRNA. We achieved satisfactory recovery rates, especially in A549 cells' total RNA.

Detection of a 7SL RNA-derived small non-coding RNA using Molecular Beacons in vitro and in cells.

Small non-coding RNAs (sncRNA) are involved in many steps of the gene expression cascade and regulate processing and expression of mRNAs by the formation of ribonucleoprotein complexes (RNP) such as the RNA-induced silencing complex (RISC). By analyzing small RNA Seq data sets, we identified a sncRNA annotated as piR-hsa-1254, which is likely derived from the 3'-end of 7SL RNA2 (RN7SL2), herein referred to as snc7SL RNA. The 7SL RNA is an abundant long non-coding RNA polymerase III transcript and serves as structural component of the cytoplasmic signal recognition particle (SRP). To evaluate a potential functional role of snc7SL RNA, we aimed to define its cellular localization by live cell imaging. Therefore, a Molecular Beacon (MB)-based method was established to compare the subcellular localization of snc7SL RNA with its precursor 7SL RNA. We designed and characterized several MBs in vitro and tested those by live cell fluorescence microscopy. Using a multiplex approach, we show that 7SL RNA localizes mainly to the endoplasmic reticulum (ER), as expected for the SRP, whereas snc7SL RNA predominately localizes to the nucleus. This finding suggests a fundamentally different function of 7SL RNA and its derivate snc7SL RNA.

Super-Resolution Tension PAINT Imaging with a Molecular Beacon.

DNA-PAINT enabled super-resolution imaging through the transient binding of fluorescently-labelled single-stranded DNA (ssDNA) imagers to target ssDNA. However, its performance is constrained by imager background fluorescence, resulting in relatively long image acquisition and potential artifacts. We designed a molecular beacon (MB) as the PAINT imager. Unbound MB in solution reduces the background fluorescence due to its natively quenched state. They are fluorogenic upon binding to target DNA to create individual fluorescence events. We demonstrate that MB-PAINT provides localization precision similar to traditional linear imager DNA-PAINT. We also show that MB-PAINT is ideally suited for fast super-resolution imaging of molecular tension probes in living cells, eliminating the potential of artifacts from free-diffusing imagers in traditional DNA-PAINT at the cell-substrate interface.

Coding Intrinsic Disorder into DNA Hybridization Probes Enables Discrimination of Single Nucleotide Variants over Wide and Tunable Temperature Ranges.

DNA hybridization probes are commonly used tools to discriminate clinically important single nucleotide variants (SNVs) but often work at elevated temperatures with very narrow temperature intervals (ΔT). Herein, we investigated the thermodynamic basis of the narrow ΔT both in silico and experimentally. Our study revealed that the high entropy penalty of classic hybridization probe designs was the key attributor for the narrow ΔT. Guided by this finding, we further introduced an entropy-compensate probe (Sprobe) design by coding intrinsic disorder into a stem-loop hybridization probe. Sprobe expanded ΔT from less than 10 °C to over 30 °C. Moreover, both ΔT and the optimal reaction temperature can be fine-tuned by simply altering the length of the loop domain. Sprobe was clinically validated by analyzing EGFR L858R mutation in 36 pairs of clinical tumor tissue samples collected from lung cancer patients, which revealed 100 % clinical sensitivity and specificity. We anticipate that our study will serve as a general guide for designing thermal robust hybridization probes for clinical diagnostics.

Dual isothermal amplification all-in-one approach for rapid and highly sensitive quantification of plasma circulating MYCN gene of neuroblastoma.

A dual isothermal amplification assay with dual fluorescence signal detection strategy, named dual isothermal amplification all-in-one approach, was developed for rapid, one-step, highly sensitive quantification of plasma circulating MYCN copy number of neuroblastoma (NB). The developed strategy consisted of rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) on a real-time PCR system using highly specific probe, molecular beacon (MB), as detection probe. The developed strategy possessing a broad linear dynamic range of 10 aM to 1 pM for both target gene (MYCN) and reference gene (NAGK). The ratio of the MYCN copy number to NAGK copy number (M/N ratio) was detected by the developed approach in cell lines, NB tumor tissues, hepatoblastoma tumor tissues and Wilms' tumor tissues, to which the M/N ratios were consistent with previous reports. In particular, the M/N ratio in NB clinical tissue specimens and NB plasma specimens detected with the developed approach were in keeping with the standard RT-PCR approach. More importantly, the M/N ratio in NB tissue samples and corresponding plasma samples of NB patients were consistent with each other with a correlation coefficient of 0.9690, indicating that plasma circulating MYCN is a promising indicator for the risk classification of NB.

Molecular beacon based real-time PCR p1 gene genotyping, macrolide resistance mutation detection and clinical characteristics analysis of Mycoplasma pneumoniae infections in children.

BACKGROUND: Mycoplasma pneumoniae can be divided into different subtypes on the basis of the sequence differences of adhesive protein P1, but the relationship between different subtypes, macrolide resistance and clinical manifestations are still unclear. In the present study, we established a molecular beacon based real-time polymerase chain reaction (real-time PCR) p1 gene genotyping method, analyzed the macrolide resistance gene mutations and the relationship of clinical characteristics with the genotypes. METHODS: A molecular beacon based real-time PCR p1 gene genotyping method was established, the mutation sites of macrolide resistance genes were analyzed by PCR and sequenced, and the relationship of clinical characteristics with the genotypes was analyzed. RESULTS: The detection limit was 1-100 copies/reaction. No cross-reactivity was observed in the two subtypes. In total, samples from 100 patients with positive M. pneumoniae detection results in 2019 and 2021 were genotyped using the beacon based real-time PCR method and P1-1 M. pneumoniae accounted for 69.0%. All the patients had the A2063G mutation in the macrolide resistance related 23S rRNA gene. Novel mutations were also found, which were C2622T, C2150A, C2202G and C2443A mutations. The relationship between p1 gene genotyping and the clinical characteristics were not statistically related. CONCLUSION: A rapid and easy clinical application molecular beacon based real-time PCR genotyping method targeting the p1 gene was established. A shift from type 1 to type 2 was found and 100.0% macrolide resistance was detected. Our study provided an efficient method for genotyping M. pneumoniae, valuable epidemiological monitoring information and clinical treatment guidance to control high macrolide resistance.

Hairpin oligosensor using SiQDs: Förster resonance energy transfer study and application for miRNA-21 detection.

MicroRNAs are known to be tumor suppressors and promoters and can be used as cancer markers. In this work, a novel oligosensor was designed using Si quantum dots (SiQDs) for the detection of miRNAs. Five-nanometer SiQDs were synthesized, with a band gap of 2.8 eV, fluorescence lifetime of 4.56 µs (τ1/2 = 3.26 µs), quantum yield of 25%, fluorescence rate constant of 6.25 × 104, and non-radiative rate constant of 1.60 × 105 s-1. They showed excellent water dispersibility, good stability (with 95% confidence for 6-month storage) without photobleaching, and high biocompatibility, with an IC50 value of 292.2 µg/L. The SiQDs and Black Hole Quencher-1 (BHQ1) were conjugated to the 5' and 3' terminals of an oligomer, respectively. The resulting hairpin molecular beacon showed resonance energy transfer efficiency of 63%. A distance of 0.91 R (Förster distance) between SiQD and BHQ1 was obtained. In the presence of a stoichiometric amount of the complementary oligonucleotide (ΔGhybridization = -35.09 kcal mol-1), 98% of the fluorescence was recovered due to loop opening of the hairpin structure. The probe showed good selectivity toward miRNA-21, with a limit of detection of 14.9 fM. The oligosensor recoveries of miRNA-21 spiked in human serum and urine were 94-98% and 93-108%, respectively.

Signal-on fluorescent sensing strategy for Pb2+ detection based on 8-17 DNAzyme-mediated molecular beacon-type catalytic hairpin assembly circuit.

Based on a Pb2+-specific 8-17 DNAzyme-induced catalytic hairpin assembly (CHA), a simple signal-on fluorescence strategy for lead ion detection was established. 8-17 DNAzyme was used as the recognition element of Pb2+, which catalyzed the cleavage of the RNA base embedded in the DNA substrate strand, while releasing part of the substrate strand (S') as CHA initiator. And two hairpin probes (H1 and H2-FQ) were designed according to the sequence of S' for CHA, in which H2-FQ was labeled with the fluorophore FAM and quencher BHQ-1 as fluorescent "molecular switch" based on fluorescence resonance energy transfer (FRET). In the presence of Pb2+, the CHA reaction was triggered to form a large number of H1-H2 complexes, enabling enzyme-free isothermal amplification and a signal-on fluorescence strategy. In the concentration range of 0.5-1000 nM, the fluorescence signal increases with the increase of Pb2+ concentration. The quantitative detection limit of Pb2+ by this method is 0.5 nM, which has better detection performance compared with the FQ-labeled 8-17 DNAzyme method. The established biosensor exhibits good specificity and can be effectively used for the detection of Pb2+ in real samples of river water and grass carp. Through ingenious nucleic acid sequence design, DNAzyme and CHA reactions are integrated to realize the enzyme-free isothermal amplifications and sensitive detection of Pb2+, which holds potential versatility in food supervision and environmental monitoring.

Cleavable molecular beacon-based loop-mediated isothermal amplification assay for the detection of adulterated chicken in meat.

A simple, sensitive, specific and fast method based on the loop-mediated isothermal amplification (LAMP) technique and cleavable molecular beacon (CMB) was developed for chicken authentication detection. LAMP and CMB were used for DNA amplification and amplicon analysis, respectively. Targeting the mitochondrial cytochrome b gene of chickens, five primers and one CMB probe were designed, and their specificity was validated against nine other animal species. The structure of CMB and concentrations of dNTPs, MgSO4, betaine, RNase H2, primers and CMB were optimized. The CMB-LAMP assay was completed within 17 min, and its limit of detection for chicken DNA was 1.5 pg µL-1. Chicken adulteration as low as 0.5% was detected in beef, and no cross-reactivity was observed. Finally, this assay was successfully applied to 20 commercial meat products. When combined with our developed DNA extraction method (the extraction time was 1 min: lysis for 10 s, washing for 20 s and elution for 30 s), the entire process (from DNA extraction to results analysis) was able to be completed within 20 min, which is at least 10 min shorter than other LAMP-based methods. Our method showed great potential for the on-site detection of chicken adulteration in meat.

Systematically investigating the fluorescent signal readout of CRISPR-Cas12a for highly sensitive SARS-CoV-2 detection.

The CRISPR/Cas system is widely used for molecular diagnostics after the discovery of trans-cleavage activity, especially now with the COVID-19 outbreak. However, the majority of contemporary trans-cleavage activity-based CRISPR/Cas biosensors exploited standard single-strand DNA (ssDNA) reporters, which were based on the FRET principle from pioneering research. An in-depth comparison and understanding of various fluorescent readout types are essential to facilitate the outstanding analytical performance of CRISPR probes. We investigated various types of fluorescent reporters of Cas12a comprehensively. Results show that trans-cleavage of Cas12a is not limited to ssDNA and dsDNA reporters, but can be extended to molecular beacons (MB). And MB reporters can achieve superior analytical performance compared with ssDNA and ds DNA reporters at the same conditions. Accordingly, we developed a highly-sensitive SARS-CoV-2 detection with the sensitivity as low as 100 fM were successfully achieved without amplification strategy. The model target of ORF1a could robustly identify the current widespread emerging SARS-CoV-2 variants. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. And a minimum of 27 copies/mL was achieved successfully. This inspiration can also be applied to other Cas proteins with trans-cleavage activity, which provides new perspectives for simple, highly-sensitive and universal molecular diagnosis in various applications.

Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes.

BACKGROUND: RNA viruses periodically trigger pandemics of severe human diseases, frequently causing enormous economic losses. Here, a nucleic acid extraction-free and amplification-free RNA virus testing probe was proposed for the sensitive and simple detection of classical swine fever virus (CSFV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on a double-stranded molecular beacon method. This RNA virus probe contains two base sequences-a recognition strand that binds to the specific domain of CSFV N2 or SARS-CoV-2 N, with a fluorophore (FAM) labeled at the 5' end, and a complementary strand (CSFV-Probe B or SARS-CoV-2-Probe B), combined with a quencher (BHQ2) labeled at the 3' end. RESULTS: Using linear molecular beacon probe technology, the detection limit of the RNA virus probe corresponding to CSFV and SARS-CoV-2 were as low as 0.28 nM and 0.24 nM, respectively. After CSFV E2 and SARS-CoV-2 N genes were transfected into corresponding host cells, the monitoring of RNA virus probes showed that fluorescence signals were dramatically enhanced in a concentration- and time-dependent manner. These results were supported by those of quantitative (qRT-PCR) and visualization (confocal microscopy) analyses. Furthermore, CSF-positive swine samples and simulated SARS-CoV-2 infected mouse samples were used to demonstrate their applicability for different distributions of viral nucleic acids in series tissues. CONCLUSIONS: The proposed RNA virus probe could be used as a PCR-free, cost-effective, and rapid point-of-care (POC) diagnostic platform for target RNA virus detection, holding great potential for the convenient monitoring of different RNA viruses for early mass virus screening.

Fluorescent Aptasensor for Highly Specific Detection of ATP Using a Newly Screened Aptamer.

Owing to the significant roles of adenosine triphosphate (ATP) in diverse biological processes, ATP level is used to research and evaluate the physiological processes of organisms. Aptamer-based biosensors have been widely reported to achieve this purpose, which are superior in their flexible biosensing mechanism, with a high sensitivity and good biocompatibility; however, the aptamers currently used for ATP detection have a poor ability to discriminate ATP from adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Herein, an ATP-specific aptamer was screened and applied to construct a fluorescent aptasensor for ATP by using graphene oxide (GO) and strand displacement amplification (SDA). The fluorescence intensity of the sensor is linearly related to the concentration of ATP within 0.1 µM to 25 µM under optimal experimental conditions, and the detection limit is 33.85 nM. The biosensor exhibits a satisfactory specificity for ATP. Moreover, the experimental results indicate that the biosensor can be applied to determine the ATP in human serum. In conclusion, the screened aptamer and the biosensor have promising applications in the determination of the real energy charge level and ATP content in a complex biological system.

Molecular Beacon for Detection miRNA-21 as a Biomarker of Lung Cancer.

Lung cancer (LC) is the leading cause of cancer-related death worldwide. Although the diagnosis and treatment of non-small cell lung cancer (NSCLC), which accounts for approximately 80% of LC cases, have greatly improved in the past decade, there is still an urgent need to find more sensitive and specific screening methods. Recently, new molecular biomarkers are emerging as potential non-invasive diagnostic agents to screen NSCLC, including multiple microRNAs (miRNAs) that show an unusual expression profile. Moreover, peripheral blood mononuclear cells' (PBMCs) miRNA profile could be linked with NSCLC and used for diagnosis. We developed a molecular beacon (MB)-based miRNA detection strategy for NSCLC. Following PBMCs isolation and screening of the expression profile of a panel of miRNA by RT-qPCR, we designed a MB targeting of up-regulated miR-21-5p. This MB 21-5p was characterized by FRET-melting, CD, NMR and native PAGE, allowing the optimization of an in-situ approach involving miR-21-5p detection in PBMCs via MB. Data show the developed MB approach potential for miR-21-5p detection in PBMCs from clinical samples towards NSCLC.

Aptamer Molecular Beacon Sensor for Rapid and Sensitive Detection of Ochratoxin A.

Ochratoxin A (OTA) is a carcinogenic fungal secondary metabolite which causes wide contamination in a variety of food stuffs and environments and has a high risk to human health. Developing a rapid and sensitive method for OTA detection is highly demanded in food safety, environment monitoring, and quality control. Here, we report a simple molecular aptamer beacon (MAB) sensor for rapid OTA detection. The anti-OTA aptamer has a fluorescein (FAM) labeled at the 5' end and a black hole quencher (BHQ1) labeled at the 3' end. The specific binding of OTA induced a conformational transition of the aptamer from a random coil to a duplex-quadruplex structure, which brought FAM and BHQ1 into spatial proximity causing fluorescence quenching. Under the optimized conditions, this aptamer sensor enabled OTA detection in a wide dynamic concentration range from 3.9 nM to 500 nM, and the detection limit was about 3.9 nM OTA. This method was selective for OTA detection and allowed to detect OTA spiked in diluted liquor and corn flour extraction samples, showing the capability for OTA analysis in practical applications.