title: A Spinach molecular beacon triggered by strand displacement.
abstract: We have re-engineered the fluorescent RNA aptamer Spinach to be activated in a sequence-dependent manner. The original Spinach aptamer was extended at its 5'- and 3'-ends to create Spinach.ST, which is predicted to fold into an inactive conformation and thus prevent association with the small molecule fluorophore DFHBI. Hybridization of a specific trigger oligonucleotide to a designed toehold leads to toehold-initiated strand displacement and refolds Spinach into the active, fluorophore-binding conformation. Spinach.ST not only specifically detects its target oligonucleotide but can discriminate readily against single-nucleotide mismatches. RNA amplicons produced during nucleic acid sequence-based amplification (NASBA) of DNA or RNA targets could be specifically detected and reported in real-time by conformational activation of Spinach.ST generated by in vitro transcription. In order to adapt any target sequence to detection by a Spinach reporter we used a primer design technique that brings together otherwise distal toehold sequences via hairpin formation. The same techniques could potentially be used to adapt common Spinach reporters to non-nucleic acid analytes, rather than by making fusions between aptamers and Spinach.
authors.affiliation: Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: Sanchita
authors.initials: S
authors.lastname: Bhadra
authors.affiliation: Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
mesh_terms: D000483:Alleles; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D009690:Nucleic Acid Conformation; D015347:RNA Probes / Q000737:chemistry*; D018724:Spinacia oleracea / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D052157:Aptamers, Nucleotide; D015347:RNA Probes
keywords: aptamer; aptamer beacon; fluorescent RNA; molecular beacon; nucleic acid diagnostics; nucleic acid engineering; strand displacement