title: Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers.
abstract: The effective tracking and purification of biological RNAs and RNA protein complexes is currently challenging. One promising strategy to simultaneously address both of these problems is to develop high-affinity RNA aptamers against taggable small molecule fluorophores. RNA Mango is a 39-nucleotide, parallel-stranded G-quadruplex RNA aptamer motif that binds with nanomolar affinity to a set of thiazole orange (TO1) derivatives while simultaneously inducing a 103-fold increase in fluorescence. We find that RNA Mango has a large increase in its thermal stability upon the addition of its TO1-Biotin ligand. Consistent with this thermal stabilization, RNA Mango can effectively discriminate TO1-Biotin from a broad range of small molecule fluorophores. In contrast, RNA Spinach, which is known to have a substantially more rigid G-quadruplex structure, was found to bind to this set of fluorophores, often with higher affinity than to its native ligand, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), and did not exhibit thermal stabilization in the presence of the TO1-Biotin fluorophore. Our data suggest that RNA Mango is likely to use a concerted ligand-binding mechanism that allows it to simultaneously bind and recognize its TO1-Biotin ligand, whereas RNA Spinach appears to lack such a mechanism. The high binding affinity and fluorescent efficiency of RNA Mango provides a compelling alternative to RNA Spinach as an RNA reporter system and paves the way for the future development of small fluorophore RNA reporter systems.
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors: Sunny C Y
authors: SC
authors: Jeng
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors: Hedy H Y
authors: HH
authors: Chan
authors: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
authors: Evan P
authors: EP
authors: Booy
authors: Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
authors: Sean A
authors: SA
authors: McKenna
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors: Peter J
authors: PJ
authors: Unrau
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D008024:Ligands; D031022:Mangifera / Q000737:chemistry*; D018749:RNA, Plant / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D018724:Spinacia oleracea / Q000737:chemistry*; D013696:Temperature
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D018749:RNA, Plant
keywords: G-quadruplex; RNA Mango; RNA Spinach; aptamer; concerted binding; fluorophore