Detection of small, highly structured RNAs using molecular beacons

Li, J.; Xu, Chengzhen; Shimada, Noritomo; Miyoshi, Y.; Watanabe, Kazunori; Wang, Cong; Ohtsuki, Takashi

doi:10.1039/c7ay00341b

Cited by 6 publications

(8 citation statements)

References 32 publications

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“…We previously showed that the fluorescence intensity of conventional MB in the presence of tRNA Lys3 transcript (15-min reaction) was lower than that of ISMBe and ISMBi in the presence of tRNA transcript (2-h reaction) (Figs 1B and S2B) [18]. These observations suggested that the formation of the ISMBe/tRNA transcript complex requires a relatively long time.…”

Section: Resultsmentioning

confidence: 80%

“…The predicted free energies were the same, indicating lack of correlation between the sensitivity of ISMB and the stability of the ISMBe/eMet transcript complex. In a previous study, we showed that conventional MB molecules targeted to the D arm more sensitively detect tRNA Lys3 transcript than those targeted to the anticodon arm and the T arm [18]. Furthermore, according to thermal unfolding studies, the D arm is more readily melted than other regions [28,29].…”

Section: Discussionmentioning

confidence: 99%

“…Previously, we showed that the fluorescence increase of conventional MB targeted to the T arm, excluding the CCA sequence, is the lowest among the analyzed probes targeting various tRNA regions [18]. On the other hand, the fluorescence increases of ISMBe4 and ISMBi4 molecules targeted to the T arm, including the CCA sequence, in the presence of eMet and iMet transcript, respectively, were higher than those of ISMBe2 and ISMBi2 targeted to the anticodon arm, respectively (Figs 1B and S2B).…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, detecting tRNA molecules by using oligonucleotide-based probes, such as MB, might be difficult. To overcome this problem, we have recently attempted to detect tRNA transcripts by using conventional MBs targeting the various regions of a tRNA molecule [18]. We have previously shown that conventional MB that targets the D arm of a tRNA Lys3 transcript can detect the tRNA Lys3 transcript with higher sensitively than MBs targeted to the anticodon arm and the T arm.…”

Section: Introductionmentioning

confidence: 99%

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In-stem molecular beacon targeted to a 5′-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity

et al. 2019

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Cellular functions are regulated by the up- and down-regulation and localization of RNA molecules. Therefore, many RNA detection methods have been developed to analyze RNA levels and localization. Molecular beacon (MB) is one of the major methods for quantitative RNA detection and analysis of RNA localization. Most oligonucleotide-based probes, including MB, are designed to target a long flexible region on the target RNA molecule, e.g., a single-stranded region. Recently, analyses of tRNA localization and levels became important, as it has been shown that environmental stresses and chemical reagents induce nuclear accumulation of tRNA and tRNA degradation in mammalian cells. However, tRNA is highly structured and does not harbor any long flexible regions. Hence, only a few methods are currently available for detecting tRNA. In the present study, we attempted to detect elongator tRNAMet (eMet) and initiator tRNAMet (iMet) by using an in-stem molecular beacon (ISMB), characterized by more effective quenching and significantly higher sensitivity than those of conventional MB. We found that ISMB1 targeted a 5′- region that includes the D arm of tRNA and that it detected eMet and iMet transcripts as well as mature eMet with high sensitivity. Moreover, the analysis revealed that the formation of the ISMB/tRNA transcript complex required more time than the formation of an ISMB/unstructured short RNA complex. These results suggest that ISMB-based tRNA detection can be a useful tool for various biological and medical studies.

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Section: Resultsmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In-stem molecular beacon targeted to a 5′-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity

et al. 2019

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“…However, the inherent ability of RNA molecules to fold into complex three-dimensional shapes because of base-pair interactions makes interrogation of natural RNA challenging and limits molecular analysis to the easily accessible loop and bulge RNA regions (Herschlag et al 2018). Indeed, scrupulous and costly analysis of the target accessibility is generally required to select an optimal design of a conventional hybridization probe (e.g., molecular beacon probe [MBP]) (Rhee et al 2008;Li et al 2017). The intrinsic stem in the MBP structure stabilizes the probe, thus disfavoring its interactions with a stem-loop folded targeted fragment to form a probe-target complex (Fig.…”

Section: Introductionmentioning

confidence: 99%

Interrogation of highly structured RNA with multicomponent deoxyribozyme probes at ambient temperatures

Reed

Sapia

Dowis

et al. 2020

RNA

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Molecular analysis of RNA through hybridization with sequence-specific probes is challenging due to the intrinsic ability of RNA molecules to form stable secondary and tertiary structures. To overcome the energy barrier toward the probe-RNA complex formation, the probes are made of artificial nucleotides, which are more expensive than their natural counterparts and may still be inefficient. Here, we propose the use of a multicomponent probe based on an RNA-cleaving deoxyribozyme for the analysis of highly structured RNA targets. Efficient interrogation of two native RNA from Saccharomyces cerevisiae-a transfer RNA (tRNA) and 18S ribosomal RNA (rRNA)-was achieved at ambient temperature. We achieved detection limits of tRNA down to ∼0.3 nM, which is two orders of magnitude lower than that previously reported for molecular beacon probes. Importantly, no probe annealing to the target was required, with the hybridization assay performed at 37°C. Excess of nonspecific targets did not compromise the performance of the probe, and high interrogation efficiency was maintained by the probes even in complex matrices, such as cell lysate. A linear dynamic range of 0.3-150 nM tRNA was demonstrated. The probe can be adapted for differentiation of a single mismatch in the tRNA-probe complex. Therefore, this study opens a venue toward highly selective, sensitive, robust, and inexpensive assays for the interrogation of biological RNA.

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Multi-labeled electrochemical sensor for cost-efficient detection of single nucleotide substitutions in folded nucleic acids

Sun

Dou

Chuang

et al. 2019

Sensors and Actuators B: Chemical

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Detection of small, highly structured RNAs using molecular beacons

Abstract: Efficient detection of small, highly structured RNA (e.g. tRNA) was achieved by selecting the best target region.

Cited by 6 publications

References 32 publications

In-stem molecular beacon targeted to a 5′-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity

In-stem molecular beacon targeted to a 5′-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity

Interrogation of highly structured RNA with multicomponent deoxyribozyme probes at ambient temperatures

Multi-labeled electrochemical sensor for cost-efficient detection of single nucleotide substitutions in folded nucleic acids

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