Highly Sensitive and Robust Linear Probe for Detection of mRNA in Cells

Asanuma, Hiroyuki; Akahane, Mariko; Niwa, Rie; Kashida, Hiromu; Kamiya, Yukiko

doi:10.1002/anie.201411000

Cited by 29 publications

(12 citation statements)

References 54 publications

(16 reference statements)

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“…Therefore, having demonstrated the power of combination probes on DNA substrates, we determined whether the concept could be applied to RNA detection. Doing so would open up a range of biochemical applications, such as cellular imaging ( 45 ). However, the typically short lifetime of DNA probes with unmodified sugar–phosphate backbones in biological media due to enzymatic degradation severely limits their use in such applications.…”

Section: Resultsmentioning

confidence: 99%

Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection

et al. 2016

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A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics.

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Section: Resultsmentioning

confidence: 99%

Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection

et al. 2016

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“…Recently, several intracellular nanoprobes for sensing RNA have been reported; these nanoprobes include fluorescence resonance energy transfer (FRET)-based nanoflares and nanobeacons, self-assembled gold nanoparticles (Au-NPs) and upconversion NPs 13 - 15 , nature-inspired nanosnail nucleic acid sensor 8 , electrostatic DNA nano-assembly-based hybridization chain reaction, and core-shell nanoprobes 16 - 18 . However, these probes have only focused on imaging or quantifiable detection of RNA in live cells and paid little attention to the specificity of the nanosensors 19 - 21 . DNA hybridization loosely follows the Watson-Crick pairing rules 22 .…”

Section: Introductionmentioning

confidence: 99%

High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells

Bai¹,

Xu²,

Guo³

et al. 2018

Theranostics

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While detection of microRNA with or without signal amplification is highly informative, nanosensors with high specificity for cell-specific RNA detection are rare.Methods: In this study, a tetrahedral DNA nanostructure (TDN) with a specific function was combined with gold nanoparticles (Au-NP) possessing fluorescence quenching effects and a large surface area to fabricate a fluorescence resonance energy transfer based nanosensor (Au-TDNN). The presence of miR-21 (target) can separate the fluorescent dye-labeled detection probe on Au-TDNNs from Au-NPs, which separates the donor and acceptor, thus inducing an intensive fluorescence signal. High specificity for discerning point mutation targets was achieved by rationally designing the nucleic acid strand displacement reaction to occur spontaneously with ΔG0 ≈ 0 based on thermodynamic parameters; under this condition, slight thermodynamic changes caused by base mismatch exert significant effects on hybridization yield.Results: Chemically synthesized DNA of three single-base-changed analogues of target, let-7d, and miR-200b were tested. A discrimination factor (DF) of 15.4 was produced by the expected detection probe on Au-NPs for proximal single-base mismatch. As the control group, the DF produced by an ordinary detection probe on Au-NPs only reached 2.4. The feasibility of the proposed strategy was also confirmed using hepatocyte cancer cells (HepG2).Conclusion: This improved nanosensor opens a new avenue for the specific and easy detection of microRNA in live cells.

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“…Furthermore, background might be significantly increased in complex media such as cells and lysates due to unspecific binding and reduced dye–dye interaction. 5 We inferred that a set of singly labelled probes with chromophores in distinct readout channels would be ideal for RNA multiplexing.…”

Section: Introductionmentioning

confidence: 99%