研究目的
Investigating the development of a multicolor large Stokes shift fluorogen-activating RNA aptamer with cationic chromophores for applications in FRET-based analytical RNA systems.
研究成果
The Chili RNA aptamer in complex with DMHBI+ and DMHBO+ mimics green and red LSS fluorescent proteins. The Chili–DMHBO+ complex is the longest wavelength-emitting (592 nm) and tightest binding (12 nM KD) RNA fluoromodule in the growing family of HBI-binding aptamers. Future work will focus on elucidating the three-dimensional structure of the binding site to comprehend the structural basis of the proton relay in the RNA responsible for the large apparent Stokes shift, and to enable engineering of even brighter LSS aptamer variants for applications in vitro and in cells.
研究不足
The study is limited by the absence of a 3D structural model of the Chili RNA aptamer, which leaves unknown which guanines are involved in putative G quartets. Additionally, the identity of the functional group on the RNA near the phenol that acts as a proton shuttle remains to be identified.
1：Experimental Design and Method Selection:
The study capitalized on 3,5-dimethoxy-substituted HBI (DMHBI) derivatives that have a higher pKa and can be excited at the absorption maximum of the phenol form. The 13-2 aptamer was modified to stabilize an apical hairpin and to assist folding of the ligand binding pocket, resulting in the 52-nt RNA aptamer named Chili.
2：Sample Selection and Data Sources:
The ligands DMHBI, DMHBI+, DMHBI-Imi, and DMHBO+ were synthesized and their fluorescence properties investigated in complex with the Chili RNA aptamer.
3：List of Experimental Equipment and Materials:
The study utilized UV-VIS spectra, fluorescence emission spectra, and polyacrylamide gels for visualization.
4：Experimental Procedures and Operational Workflow:
The fluorescence properties of the dyes and their binding and activation by Chili were investigated. The dissociation constants of the new FLAP complexes were determined by titrating each fluorophore with increasing amounts of RNA.
5：Data Analysis Methods:
The data were analyzed to determine the photophysical properties of the Chili aptamer complexes, including excitation and emission maxima, Stokes shifts, quantum yields, and dissociation constants.
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