Structure and functional reselection of the Mango-III fluorogenic RNA aptamer

Abstract

Several turn-on RNA aptamers that activate small molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans-Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to

Nanomechanics and co-transcriptional folding of Spinach and Mango

“…The first step due to unfolding of the base triple occurred at ~ 2.7 pN, with a x u ‡ of ~ 6.8±1.8 nm ( Figure S7). f unfold and x u ‡ values are similar with and without TO1, consistent with lack of direct interaction between TO1 and the base triple in the crystal structure ( Figure S7) 48 . For the second step of unfolding, average f unfold and f refold were ~ 15 and 3.5 pN, respectively (Figure 3l), close to that observed in the absence of TO1 but x u ‡ decreased from 3.8 nm and 2.8 nm upon TO1 addition, suggesting TO1 binding imparts structural changes.…”

“…However, because of the smaller size and simpler geometry compared to Spinach, they were suggested to fold more robustly than Spinach 17 . In view of the superior fluorogenic properties of iMangoIII 48 48 . This is similar to the Mg 2+ -induced stabilization of base triple at the junction between GQ and a flanking stem observed in Spinach2.…”

“…T1 and T2 denote the G-quartets in iMangoIII. TO1 is indicated by the brown arrowhead48 . (b) Schematic of probable sequential folding of iMangoIII GQ and the flanking base triple, in the absence of force.…”

“…(b) Schematic of probable sequential folding of iMangoIII GQ and the flanking base triple, in the absence of force. The structures are drawn with reference to the recently solved crystal structure 48. The Cy5 and Cy3 dyes adjacent to C 28 and G 1 are shown as red and green asterisks, respectively.…”

Rapid, Low-Cost Detection of Water Contaminants Using RegulatedIn VitroTranscription

“…We therefore reasoned that utilizing RNA-level outputs would allow biosensing reactions to be greatly simplified, leading to faster signal generation. To demonstrate this, we focused on fluorescence-activating RNA aptamers that bind and activate the fluorescence of otherwise non-fluorescent dyes [17,18]. These aptamers have been engineered into allosteric biosensors for the in vivo detection of metabolites [19,20] and as real-time reporters for monitoring IVT [21,22], but have yet to be configured within transcriptional biosensing circuits as fast readouts of sensing events.…”