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

Abstract

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.

Construction of a Programmable Feedback Network with Continuously Activatable Molecular Beacon Fluorescence for One-Step Quantification of Long Noncoding RNAs in Clinical Breast Tissues

“…The linear equation is F = 220423.35 + 12540.48 lg C ( R 2 = 0.9983), and the detection limit is as low as 5.94 aM (3 copies/μL). The sensitivity of this assay is much higher than those of electrochemical assays based on PAMAM/polyhedral nanogold-modified probe (0.22 fM) and Au NCs/MWCNT-NH 2 -decorated SPCE (42.8 fM), and comparable to those of nucleic acid–based amplification methods including DSN-mediated signal amplification (81 aM) and DSN/T7 RNA polymerase-mediated cascade amplification (31.98 aM) . For comparison, we determined the sensitivity of the traditional nonfeedback MB method (Figure S5).…”

In Vivo Imaging MicroRNA with Bright Fluorescent RNA Aptamer Through Target-Mediated Entropy-Driven Toehold Exchange

“…Fluorescent light-up aptamers (FLAPs), named after vegetables, which consist of cell-permeable small-molecule fluorogenic dyes (fluorophore) and their cognate RNA sequence have shown great potential in studying intracellular biomolecules. − The second structure of these FLAPs can specifically bind with nonfluorescent fluorophores and restrict free rotation-mediated energy dissipation pathway, thus greatly activating their fluorescence for thousands of folds. ,, Moreover, FLAPs are significantly smaller than the MS2-GFP system for easier programmability, making them possess excellent specificity and fast kinetics as ultimate transduction. − Researchers have been looking for ways to shorten the base number and improve the fluorescence and photostability of FLAPs. For example, Li et al describe the structure-guided design of BI that binds Broccoli and produces markedly increased brightness and photostability of Broccoli in cells .…”

“…8−10 The second structure of these FLAPs can specifically bind with nonfluorescent fluorophores and restrict free rotation-mediated energy dissipation pathway, thus greatly activating their fluorescence for thousands of folds. 7,11,12 Moreover, FLAPs are significantly smaller than the MS2-GFP system for easier programmability, making them possess excellent specificity and fast kinetics as ultimate transduction. 13−15 Researchers have been looking for ways to shorten the base number and improve the fluorescence and photostability of FLAPs.…”