title: Quadruplex-Flanking Stem Structures Modulate the Stability and Metal Ion Preferences of RNA Mimics of GFP.
abstract: The spinach family of RNA aptamers are RNA mimics of green fluorescent protein (GFP) that have previously been designed to address the challenges of imaging RNA inside living cells. However, relatively low levels of free intracellular magnesium limited the practical use of these aptamers. Recent cell-based selections identified the broccoli RNA aptamer, which requires less magnesium for fluorescence, but the basis for magnesium preference remained unclear. Here, we find that the broccoli RNA structure is very similar to that of baby spinach, a truncated version of the spinach aptamer. Differences in stability and metal ion preferences between these two aptamers, and among broccoli mutants, are primarily associated with the sequence and structure of predicted quadruplex-flanking stem structures. Mutation of purine-purine pairs in broccoli at the terminal stem-quadruplex transition caused reversion of broccoli to a higher magnesium dependence. Unique duplex-to-quadruplex transitions in GFP-mimic RNAs likely explain their sensitivity to magnesium for stability and fluorescence. Thus, optimizations designed to improve aptamers should take into consideration the role of metal ions in stabilizing the transitions and interactions between independently folding RNA structural motifs.
authors.affiliation: Department of Chemistry and Biochemistry, Southern Illinois University , Carbondale, Illinois 62901, United States.
authors.forename: Eman A
authors.initials: EA
authors.lastname: Ageely
authors.affiliation: Department of Chemistry and Biochemistry, Southern Illinois University , Carbondale, Illinois 62901, United States.
authors.forename: Zachary J
authors.initials: ZJ
authors.lastname: Kartje
authors.affiliation: Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine , Carbondale, Illinois 62901, United States.
authors.forename: Kushal J
authors.initials: KJ
authors.lastname: Rohilla
authors.affiliation: Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine , Carbondale, Illinois 62901, United States.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Barkau
authors.affiliation: Department of Chemistry and Biochemistry, Southern Illinois University , Carbondale, Illinois 62901, United States.|Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine , Carbondale, Illinois 62901, United States.
authors.forename: Keith T
authors.initials: KT
authors.lastname: Gagnon
mesh_terms: D001937:Brassica / Q000235:genetics; D054856:G-Quadruplexes; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D008670:Metals / Q000737:chemistry*; D018716:Molecular Mimicry; D009154:Mutation; D009690:Nucleic Acid Conformation; D018749:RNA, Plant / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D018724:Spinacia oleracea / Q000235:genetics
chemical_list: D008670:Metals; D018749:RNA, Plant; D049452:Green Fluorescent Proteins