Sherry S. Aw1,*, Melissa XM Tang1, Yin Nah Teo2,3,* and Stephen M. Cohen1,4
1 Institute of Molecular and Cell Biology, 61 Biopolis Drive, 138673, Singapore,
2 Molecular Engineering Laboratory, Biomolecular Sciences Institutes, A*STAR, 61 Biopolis Drive, 138673, Singapore and
3 Division of Chemistry and Biological Chemistry, SPMS, Nanyang Technological University, 637371, Singapore
4 Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3, Copenhagen 2200N, Denmark.
*Correspondence: Sherry Aw, email: firstname.lastname@example.org
Teo Yin Nah, email: email@example.com
Published online in Nucleic Acids Research on 7 March 2016. (doi: 10.1093/nar/gkw108)
MicroRNAs play important roles in a large variety of biological systems and processes through their regulation of target mRNA expression, and show promise as clinical biomarkers. However, their small size presents challenges for tagging or direct detection. Innovation in techniques to sense and quantify microRNAs may aid research into novel aspects of microRNA biology and contribute to the development of diagnostics. By introducing an additional stem loop into the fluorescent RNA Spinach and altering its 3′ and 5′ ends, we have generated a new RNA, Pandan, that functions as the basis for a microRNA sensor. Pandan contains two sequence-variable stem loops that encode complementary sequence for a target microRNA of interest. In its sensor form, it requires the binding of a target microRNA in order to reconstitute the RNA scaffold for fluorophore binding and fluorescence. Binding of the target microRNA resulted in large changes in fluorescence intensity. The median fold change in fluorescence observed for the sensors tested was ∼50-fold. Pandan RNA sensors exhibit good signal-to-noise ratios, and can detect their target microRNAs within complex RNA mixtures.
Pandan is a bimolecular miRNA sensor. (A) Absence of the target miRNA prevents duplexes P3 and P4 from forming, hence destabilizing the G-quadruplex and base triplet required for proper folding and binding of DFHBI. (B) Pairing between miRNA and sensor backbone in stem-loops P3 and P4 allows for stable complex folding with DFHBI. Drawing is based on Deigan-Warner et al. (20).
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