Gene Regulation System with an Artificial RNA Switch Operating in Human Cells

Endoh, Tamaki; Sugimoto, Naoki

doi:10.1002/cbic.201100093

Cited by 10 publications

(17 citation statements)

References 37 publications

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“…11,18 The fluorescence increase in TMR-Tat signal as a function of the concentration of RNAs was fitted assuming an equilibrium of a simple 1:1 binding reaction as follows:…”

Section: Assay For Interaction Between Tat Peptide and Aptamer-tars mentioning

confidence: 99%

Rational Design and Tuning of Functional RNA Switch to Control an Allosteric Intermolecular Interaction

Endoh

Sugimoto

2015

Anal. Chem.

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Conformational transitions of biomolecules in response to specific stimuli control many biological processes. In natural functional RNA switches, often called riboswitches, a particular RNA structure that has a suppressive or facilitative effect on gene expression transitions to an alternative structure with the opposite effect upon binding of a specific metabolite to the aptamer region. Stability of RNA secondary structure (-ΔG°) can be predicted based on thermodynamic parameters and is easily tuned by changes in nucleobases. We envisioned that tuning of a functional RNA switch that causes an allosteric interaction between an RNA and a peptide would be possible based on a predicted switching energy (ΔΔG°) that corresponds to the energy difference between the RNA secondary structure before (-ΔG°before) and after (-ΔG°after) the RNA conformational transition. We first selected functional RNA switches responsive to neomycin with predicted ΔΔG° values ranging from 5.6 to 12.2 kcal mol(-1). We then demonstrated a simple strategy to rationally convert the functional RNA switch to switches responsive to natural metabolites thiamine pyrophosphate, S-adenosyl methionine, and adenine based on the predicted ΔΔG° values. The ΔΔG° values of the designed RNA switches proportionally correlated with interaction energy (ΔG°interaction) between the RNA and peptide, and we were able to tune the sensitivity of the RNA switches for the trigger molecule. The strategy demonstrated here will be generally applicable for construction of functional RNA switches and biosensors in which mechanisms are based on conformational transition of nucleic acids.

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“…11,18 The fluorescence increase in TMR-Tat signal as a function of the concentration of RNAs was fitted assuming an equilibrium of a simple 1:1 binding reaction as follows:…”

Section: Assay For Interaction Between Tat Peptide and Aptamer-tars mentioning

confidence: 99%

Rational Design and Tuning of Functional RNA Switch to Control an Allosteric Intermolecular Interaction

Endoh

Sugimoto

2015

Anal. Chem.

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“…[10,21] The fluorescence increase or decrease of TMR-Tat as a function of the concentration of aptamer-TARs, assuming equilibrium of a simple 1:1 binding reaction, was fitted by using Equation (1), where F is the fluorescence signal of TMR-Tat at each concentration of aptamer-TARs, F initial is the initial fluorescence signal of TMR-Tat without aptamer-TAR, F final is the fluorescence signal when all TMR-Tat bound to aptamer-TAR, [Tat] is the total TMR-Tat concentration, and [apTAR] is the concentration of aptamer-TAR.…”

Section: Evaluation Of Association Constantsmentioning

confidence: 97%

“…The allosteric suppression was also confirmed in vitro using a Tat-derived peptide labeled with a fluorophore. [10] This suggested that theophylline binding could be detected by a fluorometric signal resulting from allosteric control of an RNA-peptide interaction. Further, as the molecular recognition and signaling domains are separated, the strategy should enable construction of a universal sensing system.…”

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confidence: 98%

“…[10] In that system, the theophylline aptamer [11] was combined with transactivation-responsive (TAR) RNA derived from bovine immunodeficiency virus (BIV). Allosteric binding by theophylline to the aptamer domain suppressed interaction between the TAR-RNA and the transactivator of transcription (Tat), also derived from BIV, and resulted in repression of transcription in human cells.…”

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confidence: 99%

“…The fluorescence signal of TMR-Tat in the aptamer-TAR bound state is expected to be different depending on each structure of the aptamer-TAR construct. [10] Transfer RNA (tRNA) is usually added to suppress nonspecific interactions between nucleic acids and positively charged peptides or proteins. We observed fluorescence decrease of TMR-Tat with increasing tRNA concentrations (see Figure S2 in the Supporting Information) that is possibly due to weak interaction of the competitor tRNA.…”

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confidence: 99%

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Aptamer‐Based Universal Fluorometric Sensors Based on Allosteric Modulation of RNA–Peptide Interactions

Endoh

Sugimoto

2014

ChemMedChem

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Aptamers are single-stranded DNA or RNA oligonucleotides that serve as molecular recognition units. Aptamers targeting a range of biologically relevant molecules have been developed using selection methods and functional aptamer domains, called riboswitches, are found in natural genomic sequences. Aptamers can be used as starting points for the design of biosensors for diagnostic applications. In this study, we demonstrate a simple strategy to detect binding of an apamer to its target molecule via a fluorometric signal resulting from allosteric suppression of an RNA-peptide interaction. The broad applicability was demonstrated by detection of seven different target molecules-one drug, two antibiotics, and four natural metabolites. This strategy will enable the construction of universal biosensors for various target molecules.

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Noncanonical Structures and Their Thermodynamics of DNA and RNA Under Molecular Crowding

Sugimoto

2014

International Review of Cell and Molecular Biology

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Gene Regulation System with an Artificial RNA Switch Operating in Human Cells

Cited by 10 publications

References 37 publications

Rational Design and Tuning of Functional RNA Switch to Control an Allosteric Intermolecular Interaction

Rational Design and Tuning of Functional RNA Switch to Control an Allosteric Intermolecular Interaction

Aptamer‐Based Universal Fluorometric Sensors Based on Allosteric Modulation of RNA–Peptide Interactions

Noncanonical Structures and Their Thermodynamics of DNA and RNA Under Molecular Crowding

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