Mismatched fluorescent probes with an enhanced strand displacement reaction rate for intracellular long noncoding RNA imaging

Zhang, Yan; Du, Xueke; Su, Xianwei; Zou, Xiaoran; Zhang, Chunyang

doi:10.1039/d1cc05270e

Cited by 7 publications

(4 citation statements)

References 28 publications

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“…A LOD of 1 cell is obtained. The sensitivity of this Corn-based biosensor is superior to that of the mismatched fluorescent probe-based biosensor (10 cells) . These results clearly demonstrate that this Corn-based biosensor can accurately quantify the expression level of cellular lncRNA MALAT1 and even differentiate the lncRNA MALAT1 level between normal cells and tumor cells.…”

Section: Resultsmentioning

confidence: 76%

Corn-Based Fluorescent Light-Up Biosensors with Improved Signal-to-Background Ratio for Label-Free Detection of Long Noncoding RNAs

et al. 2023

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Long noncoding RNAs (lncRNAs) play pivotal roles in multifarious physiological and pathological processes, and their aberrant expression may disturb the normal regulatory network of gene expression to induce diverse human diseases. Herein, we construct a fluorescent light-up biosensor with a low background for label-free detection of lncRNAs by coupling duplex-specific nuclease (DSN)-assisted target recycling amplification with transcription-driven synthesis of fluorogenic RNA aptamer-Corns. We design two linear probes, including a capture probe for initiating a cyclic cleavage reaction and a linear template for transcribing RNA aptamer-Corn. Target lncRNA is recognized by capture probes assembled on magnetic bead (MB) surfaces to trigger a DSN-assisted cyclic cleavage reaction, releasing abundant T7 promoter sequences. After magnetic separation, free T7 promoter hybridizes with a linear template to induce efficient transcription amplification with the assistance of T7 RNA polymerase, producing numerous fluorogenic RNA aptamer-Corns that can light up small-molecule fluorogens 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO). Notably, the introduction of MBs facilitates both the separation of cleaved capture probes and the enrichment/isolation of target lncRNAs from the complex biological matrix. Benefiting from the high efficiency of DSN/T7 RNA polymerase-mediated cascade amplification and high signal-to-background ratio of the Corn–DFHO complex, this biosensor is capable of sensitively quantifying lncRNA with a detection limit of 31.98 aM. Moreover, it can precisely quantify lncRNA at the single-cell level and even in complex biological samples, and it can differentiate tumor cells from normal cells. Importantly, this Corn-based biosensor is readily extended to detect other lncRNAs by altering capture probe sequences, opening a new avenue for molecular diagnosis.

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Section: Resultsmentioning

confidence: 76%

Corn-Based Fluorescent Light-Up Biosensors with Improved Signal-to-Background Ratio for Label-Free Detection of Long Noncoding RNAs

et al. 2023

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“…This assay has outstanding features: (1) it can be performed in a mix-and-read manner within 10 min, which is the rapidest and simplest method reported so far for the lncRNA MALAT1 assay; (2) this assay can be performed in an absolutely isothermal condition (50 °C), eliminating the requirement for a sophisticated thermal cycler and precise temperature control in a conventional PCR assay; (3) high amplification efficiency of multiple cyclic ERA and high cleavage efficiency of APE1-assisted cyclic catalysis endow this assay with improved sensitivity; (4) this assay can be extended to detect any nucleic acids without considering the length of targets. In comparison with the previously reported methods (Table S1), this assay has the advantages of rapidity (within 10 min), lower LOD (0.87 aM), and good performance in clinical samples. ,,− Moreover, the proposed assay can accurately measure cellular lncRNA MALAT1 at the single-cell level and even discriminate the lncRNA expression level between breast cancer tissues and their corresponding healthy adjacent tissues. Importantly, this assay can be easily reconfigured to detect a series of RNAs (e.g., miRNA, circRNA, mRNA, and SARS-CoV-2 RNA) by just changing the recognition sequences, and it can be integrated with many signal amplification approaches (e.g., ELOSA, SDA, and RCA) , to sensitively detect RNAs and other targets (e.g., DNAs, proteins, and small molecules), providing a promising platform for biomedical research, clinical diagnostics, and therapeutics.…”

Section: Discussionmentioning

confidence: 92%

Mix-and-Detection Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Ultrasensitive Detection of Long Noncoding RNAs in Breast Tissues

Zhao

Tian

et al. 2023

Anal. Chem.

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Long noncoding RNAs (lncRNAs) are valuable biomarkers and therapeutic targets, and they play essential roles in various pathological and biological processes. So far, the reported lncRNA assays usually suffer from unsatisfactory sensitivity and time-consuming procedures. Herein, we develop a mix-and-read assay based on multiple cyclic enzymatic repairing amplification (ERA) for sensitive and rapid detection of mammalian metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1). In this assay, we design two three-way junction (3WJ) probes including a 3WJ template and a 3WJ primer to specifically recognize lncRNA MALAT1, and the formation of a stable 3WJ structure induces cyclic ERA to generate triggers. The resulting triggers subsequently hybridize with a free 3WJ template and act as primers to initiate new rounds of cyclic ERA, generating abundant triggers. The hybridization of triggers with signal probes forms stable double-stranded DNA duplexes that can be specifically cleaved by apurinic/apyrimidinic endonuclease 1 to produce a high fluorescence signal. This assay can be carried out in a mix-and-read manner within 10 min under an isothermal condition (50 °C), which is the rapidest and simplest method reported so far for the lncRNA MALAT1 assay. This method can sensitively detect lncRNA MALAT1 with a limit of detection of 0.87 aM, and it can accurately measure endogenous lncRNA MALAT1 at the single-cell level. Moreover, this method can distinguish lncRNA MALAT1 expression in breast cancer patient tissues and their corresponding healthy adjacent tissues. Importantly, the extension of this assay to different RNAs detection can be achieved by simply replacing the corresponding target recognition sequences.

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“…The detection of displacement probes is achieved by comparing the binding ability of the receptor with uorescent indicator and the analyte. [63][64][65] Therefore, there are high requirements for receptors and uorescent indicators. The uorescent indicator needs to bind to the receptors, and the binding ability cannot be too strong.…”

Section: Mechanism Of Uorescent Probementioning

confidence: 99%

Non-destructive real-time monitoring and investigation of the self-assembly process using fluorescent probes

Ji,

Wang,

Wang

et al. 2024

Chem. Sci.

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Mismatched fluorescent probes with an enhanced strand displacement reaction rate for intracellular long noncoding RNA imaging

Abstract: We design a mismatched fluorescent probe to directly monitor the long noncoding RNA (lncRNA) in living cells. The introduction of mismatched bases in the fluorescent probe greatly enhances the strand...

Cited by 7 publications

References 28 publications

Corn-Based Fluorescent Light-Up Biosensors with Improved Signal-to-Background Ratio for Label-Free Detection of Long Noncoding RNAs

Corn-Based Fluorescent Light-Up Biosensors with Improved Signal-to-Background Ratio for Label-Free Detection of Long Noncoding RNAs

Mix-and-Detection Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Ultrasensitive Detection of Long Noncoding RNAs in Breast Tissues

Non-destructive real-time monitoring and investigation of the self-assembly process using fluorescent probes

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