In‐Stem Molecular Beacon Containing a Pseudo Base Pair of Threoninol Nucleotides for the Removal of Background Emission

Kashida, Hiromu; Takatsu, Tomohiko; Fujii, Tomoyuki; Sekiguchi, Koji; Liang, Xingguo; Niwa, Kosuke; Takase, Tomokazu; Yoshida, Yasuko; Asanuma, Hiroyuki

doi:10.1002/ange.200902367

Cited by 22 publications

(5 citation statements)

References 41 publications

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“…Zhang and Winfree have shown that single-nucleotide mutations, insertions, and deletions in the catalyst strand can result in 10-to 300-fold reductions in its efficiency in catalyzing strand exchange [17]. These reductions in signal observed with single-nucleotide mismatches are comparable to other well-developed DNA sensors [26][27][28][31][32][33]. Consistent with these findings, we observed that when a single nucleotide was changed in the toehold region (Figure 3, inset), the mutant Trigger did not yield any peroxidase activity above background even at 150 nM concentration ( Figure 3, sample #10~12).…”

Section: Resultsmentioning

confidence: 86%

Beyond allostery: Catalytic regulation of a deoxyribozyme through an entropy-driven DNA amplifier

et al. 2010

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The programmability and replicability of RNA and DNA have respectively enabled the design and selection of a number of allosteric ribozymes and deoxyribozymes. These catalysts have been adapted to function as signal transducers in biosensors and biochemical reaction networks both in vitro and in vivo. However, allosteric control of nucleic acid catalysts is currently limited by the fact that one molecule of effector (input) generally regulates at most one molecule of ribozyme or deoxyribozyme (output). In consequence, allosteric control is usually inefficient when the concentration of input molecules is low. In contrast, catalytic regulation of protein enzymes, as in protein phosphorylation cascades, generally allows one input molecule (e.g., one kinase molecule) to regulate multiple output molecules (e.g., kinase substrates). Achieving such catalytic signal amplification would also be of great utility for nucleic acid circuits. Here we show that allosteric regulation of nucleic acid enzymes can be coupled to signal amplification in an entropy-driven DNA circuit. In this circuit, kinetically trapped DNA logic gates are triggered by a specific sequence, and upon execution generate a peroxidase deoxyribozyme that converts a colorless substrate (ABTS) into a green product (ABTS •+). This scheme provides a new paradigm for the design of enzyme-free biosensors for point-of-care diagnostics. FindingsA variety of functional nucleic acids have been engineered over the past two decades, including not only simple binding elements (aptamers [1,2]) and catalysts (ribozymes [3] and deoxyribozymes [4]), but also more 'intelligent' molecular parts, such as aptamer beacons and allosteric ribozymes that can sense biomolecules [5,6], process molecular information [7,8], and regulate biochemical systems [9]. However, most regulatory nucleic acid elements are based on allosteric control, which has a fundamental limitation: one input molecule generally yields only one output molecule. Such stoichiometric or sub-stoichiometric regulation is often insufficient for effective metabolic regulation or diagnostic signal transduction, especially when the concentrations of input molecules are low.In contrast, natural catalytic cascades, such as the phosphorylation of proteins by kinases, readily amplify low input signals. Although in principle ribozymes and deoxyribozymes could participate in similar cascades as catalysts [10][11][12], no generalizable method for implementing such cascades has yet been established. On the other hand, DNA and RNA can catalyze chemical reactions not only by forming intricate tertiary structures, but also by simply forming Watson-Crick base pairs. In fact, by serving as a hybridization template, DNA can control and catalyze a wide range of chemical reactions [13], some of which can yield products capable of regulating downstream reactions. More recently, Zhang and coworkers have designed a scheme for highly efficient, enzyme-free, entropy-driven catalytic reactions that relies only on the dynamic hybrid...

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Section: Resultsmentioning

confidence: 86%

Beyond allostery: Catalytic regulation of a deoxyribozyme through an entropy-driven DNA amplifier

et al. 2010

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“…The use of D ‐threoninol as a scaffold facilitated dimerization or clustering of the dyes in the duplex. We have utilized this base surrogate (threoninol nucleotide) to develop a new in‐stem molecular beacon (ISMB) in which both fluorophore (perylene) and quencher (anthraquinone) on D ‐threoninols are incorporated into the stem region as a pseudo base pair (Scheme ) 10. Direct and strong contact of perylene with the quencher in the stem efficiently removed background emission in the closed ISMB and allowed discrimination of fully matched and one‐base deletion mutant pairs.…”

Section: Methodsmentioning

confidence: 99%

Coherent Quenching of a Fluorophore for the Design of a Highly Sensitive In‐Stem Molecular Beacon

et al. 2010

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Excitonische Wechselwirkung wurde beim Design eines hochempfindlichen „molekularen Beacons“ genutzt, der in seiner Stammregion D‐Threoninole mit Fluorophor und Fluoreszenzlöscher als Pseudobasenpaare trägt (ISMB; siehe Schema: optimierte Kombination mit Cy3 und modifiziertem Methylrot). Durch Minimierung der Differenz zwischen λmax von Fluorophor und Fluoreszenzlöscher wurde die Löscheffizienz maximiert.

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“…Probes for use in solution are expected to emit a signal only when they bind to their targets. [6][7][8][9][10][11][12] This is an essential requirement for bioprobes providing a spatiotemporal response.…”

Section: Introductionmentioning

confidence: 99%

Rational Design for Cooperative Recognition of Specific Nucleobases Using β‐Cyclodextrin‐Modified DNAs and Fluorescent Ligands on DNA and RNA Scaffolds

Futamura

Uemura

Imoto

et al. 2013

Chemistry A European J

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We propose a binary fluorimetric method for DNA and RNA analysis by the combined use of two probes rationally designed to work cooperatively. One probe is an oligonucleotide (ODN) conjugate bearing a β-cyclodextrin (β-CyD). The other probe is a small reporter ligand, which comprises linked molecules of a nucleobase-specific heterocycle and an environment-sensitive fluorophore. The heterocycle of the reporter ligand recognizes a single nucleobase displayed in a gap on the target labeled with the conjugate and, at the same time, the fluorophore moiety forms a luminous inclusion complex with nearby β-CyD. Three reporter ligands, MNDS (naphthyridine-dansyl linked ligand), MNDB (naphthyridine-DBD), and DPDB (pyridine-DBD), were used for DNA and RNA probing with 3'-end or 5'-end modified β-CyD-ODN conjugates. For the DNA target, the β-CyD tethered to the 3'-end of the ODN facing into the gap interacted with the fluorophore sticking out into the major groove of the gap site (MNDS and DPDB). Meanwhile the β-CyD on the 5'-end of the ODN interacted with the fluorophore in the minor groove (MNDB and DPDB). The results obtained by this study could be a guideline for the design of binary DNA/RNA probe systems based on controlling the proximity of functional molecules.

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In‐Stem Molecular Beacon Containing a Pseudo Base Pair of Threoninol Nucleotides for the Removal of Background Emission

Cited by 22 publications

References 41 publications

Beyond allostery: Catalytic regulation of a deoxyribozyme through an entropy-driven DNA amplifier

Beyond allostery: Catalytic regulation of a deoxyribozyme through an entropy-driven DNA amplifier

Coherent Quenching of a Fluorophore for the Design of a Highly Sensitive In‐Stem Molecular Beacon

Rational Design for Cooperative Recognition of Specific Nucleobases Using β‐Cyclodextrin‐Modified DNAs and Fluorescent Ligands on DNA and RNA Scaffolds

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