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RNA. 2014 Aug;20(8):1183-94. doi: 10.1261/rna.045047.114. Epub 2014 Jun 18.

A Spinach molecular beacon triggered by strand displacement.

Author information

1
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.

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.

KEYWORDS:

aptamer; aptamer beacon; fluorescent RNA; molecular beacon; nucleic acid diagnostics; nucleic acid engineering; strand displacement

PMID:
24942625
PMCID:
PMC4105745
DOI:
10.1261/rna.045047.114
[Indexed for MEDLINE]
Free PMC Article

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