Structural Basis For Fluorophore Binding By Pepper Aptamer Or R8 Aptamer

RNA molecules play vital roles in different biological processes.Spatial-temporal monitoring RNA molecules will facilitate our understanding of the complicated dynamics of RNA,such as expression,translocation and so on.Recently,the fluorogenic RNA aptamers have been developed as simple and effective RNA fluorescent labeling tools in live cell imaging.HBC(4-((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenyl-acetonitrile)is a new fluorophore ligand developed from the GFP fluorophore HBI(4-hydroxybenzylidene imidazolinone).Pepper aptamer that was selected in vitro by SELEX(Systematic Evolution of Ligands by Exponential Enrichment)technology can specifically bind and activate the fluorescence of HBC.Here,we solved the crystal structure of the Pepper-HBC complex at 1.64 (?) resolution using X-ray crystallography method.The overall structure adopts a tuning-fork like architecture,which contains three helices P1,P2 and P3,an internal loop L1,a bulge loop L2 and a hairpin loop L3.The bulge loop L2 forms long-range interactions with L1 and P2,which contribute to the stability of the whole structure.The fluorophore HBC folded in a near-planar conformation is sandwiched between a base quadruple in L1 and a base pair in P2,and further locked by G9-C33 base pair.Based on the structure of the Pepper-HBC complex and in vitro fluorescence experiments,we identified residues of Pepper aptamer that are crucial for the binding of the fluorophore HBC and detected the effects of different metal ions on HBC fluorescence activation by Pepper aptamer.Additionally,we solved the structures of Pepper aptamer in complex with HBC analogs.The overall structures and ligand binding pockets of these complexes are similar to the Pepper-HBC complex,which further verify the binding mechanism of Pepper aptamer and the fluorophore ligands.R8 aptamer is a fluorogenic RNA aptamer,which was selected in vitro based on another new fluorophore ligand RFP.We performed co-crystallization of R8 aptamer with the fluorophore ligand RFP,and determined the structure of the R8-RFP complex at 1.60(?) resolution.The overall structure of R8 aptamer folds in a spindle like architecture and consists of two stems P1 and P2,the junctional region J12 and the hairpin loop L2.Stem P2 co-axially stacks on stem P1.J12 forms long-range interactions with P1,which is important to maintain the stability of the overall structure.The ligand binding pocket resides in the middle of the whole structure.The fluorophore RFP whose three aromatic rings are co-planarity is sandwiched by a base triple in P1 and a base pair in P2.Based on the structure of the R8-RFP complex and in vitro fluorescence experiments,we performed the engineering of tandem R8 array and solved the structure of III?R8(triple-tandem R8)in complex with RFP.We further identified a 24 nt RNA module that can bind and activate the fluorescence of RFP.In addition,we modified the fluorophore ligand according to the conformation of RFP in the structure of the R8-RFP complex.We developed a variety of RFP analogs with different maximum excitation wavelengths and maximum emission wavelengths.We also determined the structures of R8 aptamer in complex with RFP analogs to further understand the mechanism of R8 aptamer to bind the fluorophore ligands.Notably,both the Pepper-HBC and R8-RFP complexes lack a G-quadruplex motif that is observed in most fluorogenic RNA aptamers.The absence of G-quadruplex is favorable for the application of Pepper aptamer and R8 aptamer in live cells.This study reveals the mechanisms of Pepper aptamer and R8 aptamer to bind and activate the fluorescence of fluorophore ligands,and provides a thorough structural basis to engineer the RNA sequence and the fluorophore ligands with improved photophysical properties.