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Design of a synthetic luminescent probe from a biomolecule binding domain: selective detection of AU-rich mRNA sequences.

ABSTRACT: We report the design of a luminescent sensor based upon the zinc finger (ZF) protein TIS11d, that allows for the selective time-resolved detection of the UUAUUUAUU sequence of the 3'-untranslated region of messenger RNA. This sensor is composed of the tandem ZF RNA binding domain of TIS11d functionalized with a luminescent Tb3+ complex on one of the ZFs and a sensitizing antenna on the other. This work provides the proof of principle that an RNA binding protein can be re-engineered as an RNA sensor and, more generally, that tunable synthetic luminescent probes for biomolecules can be obtained by modifying biomolecule-binding domains.

SUBMITTER: Raibaut L

PROVIDER: S-EPMC5364516 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Design of a synthetic luminescent probe from a biomolecule binding domain: selective detection of AU-rich mRNA sequences.

Raibaut Laurent L Vasseur William W Shimberg Geoffrey D GD Saint-Pierre Christine C Ravanat Jean-Luc JL Michel Sarah L J SLJ Sénèque Olivier O

Chemical science 20161116 2

We report the design of a luminescent sensor based upon the zinc finger (ZF) protein TIS11d, that allows for the selective time-resolved detection of the UUAUUUAUU sequence of the 3'-untranslated region of messenger RNA. This sensor is composed of the tandem ZF RNA binding domain of TIS11d functionalized with a luminescent Tb<sup>3+</sup> complex on one of the ZFs and a sensitizing antenna on the other. This work provides the proof of principle that an RNA binding protein can be re-engineered as ...[more]

PMID: 28451295

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Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. Different probe designs were investigated with respect to sensitivity and selectivity of microarray hybridization. An array with 11 different variants of oligonucleotides for miR-122a and miR-16 was produced. 5 µg of respective total RNA was fluorescently labelled by 3’ ligation. Total RNA was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 5 fmol of each of 816 non redundant miRNAs sequences and miRControl 3 sequences. Local background was subtracted from the signal to obtain the net signal intensity and the mean of the net signal intensities of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was two-fold the mean background value (2bkg dataset).

Dataset Information

Design of a synthetic luminescent probe from a biomolecule binding domain: selective detection of AU-rich mRNA sequences.

Publications

Design of a synthetic luminescent probe from a biomolecule binding domain: selective detection of AU-rich mRNA sequences.

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