title: A conformation-induced fluorescence method for microRNA detection.
abstract: 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.
authors.affiliation: Institute of Molecular and Cell Biology, 61 Biopolis Drive, 138673, Singapore syaw@imcb.a-star.edu.sg.
authors.forename: Sherry S
authors.identifier: 0000-0003-3067-0323
authors.initials: SS
authors.lastname: Aw
authors.affiliation: Institute of Molecular and Cell Biology, 61 Biopolis Drive, 138673, Singapore.
authors.forename: Melissa Xm
authors.initials: MX
authors.lastname: Tang
authors.affiliation: Molecular Engineering Laboratory, Biomolecular Sciences Institutes, A*STAR, 61 Biopolis Drive, 138673, Singapore Division of Chemistry and Biological Chemistry, SPMS, Nanyang Technological University, 637371, Singapore ynteo1@illumina.com.
authors.forename: Yin Nah
authors.initials: YN
authors.lastname: Teo
authors.affiliation: Institute of Molecular and Cell Biology, 61 Biopolis Drive, 138673, Singapore Department of Cellular and Molecular Medicine,University of Copenhagen, Blegdamsvej 3, Copenhagen 2200 N, Denmark.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Cohen
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001593:Benzyl Compounds / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D048288:Imidazolines / Q000378:metabolism; D035683:MicroRNAs / Q000032:analysis* / Q000737:chemistry; D009690:Nucleic Acid Conformation
chemical_list: C560120:3,5-difluoro-4-hydroxybenzylidene imidazolinone; D052157:Aptamers, Nucleotide; D001593:Benzyl Compounds; D005456:Fluorescent Dyes; D048288:Imidazolines; D035683:MicroRNAs