Cis-regulatory hairpin-shaped mRNA encoding a reporter protein: catalytic sensing of nucleic acid sequence at single nucleotide resolution

Narita, Atsushi; Ogawa, Kazumasa; Sando, Shinsuke; Aoyama, Yasuhiro

doi:10.1038/nprot.2007.140

Cited by 7 publications

(4 citation statements)

References 27 publications

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“…Historically, a great deal of emphasis has been placed on discriminating SNPs, leading to diverse probe technologies that produce nonquantitative yields for complementary targets. − ,− ,,,, For quantitative expression profiling studies focused on discriminating genes within a genome, where 1-nt resolution is not necessarily needed, we believe it is highly significant that SC probes enable robust, near-quantitative capture of complementary targets and near-quantitative rejection of 2-nt mismatches, providing a powerful framework for genome-wide analysis. Studies with large pools of DNA or RNA mismatched targets demonstrate that SC probes efficiently capture complementary targets and reject mismatched targets when the complementary targets are greatly outnumbered.…”

Section: Discussionmentioning

confidence: 99%

“…In principle, this can be accomplished by using a short unstructured probe (7–15 nt), but this approach has the fundamental flaw that the recognition sequence is then too short to uniquely address genes in the context of a genome. ,, One approach to increasing the length of the recognition sequence while maintaining selectivity is to use the target to template reactions between two unstructured probes . Alternatively, structured probes (of which molecular beacons are the most familiar example) exploit internal base-pairing to compete with probe/complement hybridization, making it possible to adjust the trade-off between affinity and selectivity at a temperature of choice while maintaining a large recognition sequence (Figure b,c). − Structured probes can be designed to achieve selective detection of single-nucleotide mismatches at room or physiological temperatures ,,, and are suitable for in vivo applications. , However, because structured probes achieve selectivity by operating near the melting temperature of the probe/complement duplex, they are unable to stably capture their targets, precluding the use of washes which are critical to applications in vitro and in situ (e.g., removing unbound targets on a microarray or unbound probes within a fixed embryo).…”

Section: Introductionmentioning

confidence: 99%

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Selective Nucleic Acid Capture with Shielded Covalent Probes

et al. 2013

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Nucleic acid probes are used for diverse applications in vitro, in situ, and in vivo. In any setting, their power is limited by imperfect selectivity (binding of undesired targets) and incomplete affinity (binding is reversible, and not all desired targets bound). These difficulties are fundamental, stemming from reliance on base pairing to provide both selectivity and affinity. Shielded covalent (SC) probes eliminate the longstanding trade-off between selectivity and durable target capture, achieving selectivity via programmable base pairing and molecular conformation change, and durable target capture via activatable covalent cross-linking. In pure and mixed samples, SC probes covalently capture complementary DNA or RNA oligo targets and reject two-nucleotide mismatched targets with near-quantitative yields at room temperature, achieving discrimination ratios of 2–3 orders of magnitude. Semiquantitative studies with full-length mRNA targets demonstrate selective covalent capture comparable to that for RNA oligo targets. Single-nucleotide DNA or RNA mismatches, including nearly isoenergetic RNA wobble pairs, can be efficiently rejected with discrimination ratios of 1–2 orders of magnitude. Covalent capture yields appear consistent with the thermodynamics of probe/target hybridization, facilitating rational probe design. If desired, cross-links can be reversed to release the target after capture. In contrast to existing probe chemistries, SC probes achieve the high sequence selectivity of a structured probe, yet durably retain their targets even under denaturing conditions. This previously incompatible combination of properties suggests diverse applications based on selective and stable binding of nucleic acid targets under conditions where base-pairing is disrupted (e.g., by stringent washes in vitro or in situ, or by enzymes in vivo).

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Selective Nucleic Acid Capture with Shielded Covalent Probes

et al. 2013

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“…Molecular beacons offer a powerful approach to real-time visualization of specific endogenous mRNAs and simultaneous monitoring of gene expression in cancer cells [ 10 , 11 , 12 , 13 ]. Therefore, a molecular beacon strategy might be suitable for the detection of expression levels of endogenous small molecules in living subjects.…”

Section: Introductionmentioning

confidence: 99%

Quantum Dot-Based Molecular Beacon to Monitor Intracellular MicroRNAs

Lee

Moon

Lee

et al. 2015

Sensors

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Fluorescence monitoring of endogenous microRNA (miRNA or miR) activity related to neuronal development using nano-sized materials provides crucial information on miRNA expression patterns in a noninvasive manner. In this study, we report a new method to monitor intracellular miRNA124a using quantum dot-based molecular beacon (R9-QD-miR124a beacon). The R9-QD-miR124a beacon was constructed using QDs and two probes, miR124a-targeting oligomer and arginine rich cell-penetrating peptide (R9 peptide). The miR124a-targeting oligomer contains a miR124a binging sequence and a black hole quencher 1 (BHQ1). In the absence of target miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon and remained in quenched state because the BHQ1 quenches the fluorescence signal of the R9-QD-miR124a beacon. The binding of miR124a to the miR124a binding sequence of the miR124a-targeting oligomer triggered the separation of the BHQ1 quencher and subsequent signal-on of a red fluorescence signal. Moreover, enhanced cellular uptake was achieved by conjugation with the R9 peptide, which resulted in increased fluorescent signal of the R9-QD-miR124a beacons in P19 cells during neurogenesis due to the endogenous expression of miR124a.

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“…(1) The present mRNA probe with no functionalization with organic dye can be applied also in the form of double-strand DNA, which is in situ transcribed into RNA in an in vitro translation system or in live cells, (2) the present system is signal-amplifying, since the transcription (DNA to RNA), translation (RNA to protein luciferase), and the chemiluminescence-generating, enzymatic sensing reaction of luciferase are all catalytic, and (3) the choice of reporter proteins in terms of wavelength or color of chemiluminescence is arbitrary so that one can assign different colors to different alleles of the target to allow multicolor sensing of SNP (single nucleotide polymorphism). 44 5.2 Light-Up AptamerFluorophore Pair for Transcription Monitoring. The Nobel Prize in Chemistry for 2008 went to the discovery of green fluorescent protein (GFP) and its application.…”

Section: Manipulated Biological Systems Withmentioning

confidence: 99%

Structure and Function of Molecular Assembly. A Personal Reminiscence

Aoyama¹

2009

Bulletin of the Chemical Society of Japan

Self Cite

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In this account is described an outline of research the author has conducted over the last 35 years. Depending on the type of molecular assemblies dealt with, the whole work is divided into four parts, i.e., small hostguest systems in organic media, infinite solid systems, huge but finite nano systems in aqueous media, and manipulated biological systems. The essence of the hostguest systems (Section 2) is molecular recognition based on convergent multiple interactions involving OH£O hydrogen bonds, where sugar binding and detailed thermodynamic analysis of multipoint recognition may be taken as highlights. A couple of new fields arise from the hostguest chemistry by paradigm shift. One is in the direction of multiple interactions, from convergent to divergent. Section 3 thus concerns molecular alignment control in crystals using orthogonal aromatic triad and diad building blocks. Catalysis by microporous metallo-organic frameworks as organic zeolites is also described. The other shift in paradigm is in the media, from organic to aqueous, and also in the significance of sugar binding, from sugars as a guest to sugars as a probe. Section 4 is devoted to the highly adhesive (hydrogen bonding) nature of glycocluster compounds and their micelle-like aggregates, focusing on the formation of glycoviruses and glycoviral gene delivery. With DNA and RNA manipulation techniques in hand, we became interested in in-cell gene sensing and transcription monitoring. The latest activity in this area is shown in Section 5. The entire work is design-based. The author, who is soon retiring from Kyoto University, now has mixed feelings about this apparently natural approach, as briefly referred to in the Closing Remark (Section 6).

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Cis-regulatory hairpin-shaped mRNA encoding a reporter protein: catalytic sensing of nucleic acid sequence at single nucleotide resolution

Cited by 7 publications

References 27 publications

Selective Nucleic Acid Capture with Shielded Covalent Probes

Selective Nucleic Acid Capture with Shielded Covalent Probes

Quantum Dot-Based Molecular Beacon to Monitor Intracellular MicroRNAs

Structure and Function of Molecular Assembly. A Personal Reminiscence

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