Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase‐Modified Fluorogenic RNA Aptamers

Abstract

RNA aptamers form compact tertiary structures and bind their ligands in specific binding sites. Fluorescence‐based strategies reveal information on structure and dynamics of RNA aptamers. Herein, we report the incorporation of the universal emissive nucleobase analog 4‐cyanoindole into the fluorogenic RNA aptamer Chili, and its application as a donor for supramolecular FRET to the bound ligands DMHBI+ or DMHBO+. The photophysical properties of the new nucleobase–ligand‐FRET pair revealed structural restraints for the overall RNA aptamer organization and identified nucleotide positions suitable for FRET‐based readout of ligand binding. This strategy is generally suitable for binding‐site mapping and may also be applied for responsive aptamer devices.

Fundamental studies of functional nucleic acids: aptamers, riboswitches, ribozymes and DNAzymes

“…42,53 Initial hints into the organization of the aptamer and the relative location of the ligand were obtained by supramolecular FRET. 54 The fluorescent nucleobase analogue 4-cyanoindol was covalently incorporated into the RNA aptamer and served as donor for energy transfer to the non-covalently bound DMHBI + /DMHBO + ligands that served as acceptor. 54 Additional fundamental studies of ESPT photophysics in combination with structural studies may reveal the identity and function of the proton acceptor.…”

“…54 The fluorescent nucleobase analogue 4-cyanoindol was covalently incorporated into the RNA aptamer and served as donor for energy transfer to the non-covalently bound DMHBI + /DMHBO + ligands that served as acceptor. 54 Additional fundamental studies of ESPT photophysics in combination with structural studies may reveal the identity and function of the proton acceptor. Preliminary data regarding the involvement of a crucial nucleobase nitrogen have been obtained by mutagenesis experiments, but more detailed investigations are needed to surface the mechanism of how Chili mimics the large Stokes shift (LSS) fluorescent proteins, such as LSSmOrange and LSSmKate.…”

Changed reactivity of secondary hydroxy groups in C8-modified adenosine – lessons learned from silylation

“…Accordingly, the field has developed to a stage that allows custom-design of RNA probes and tools for specific application. For example, investigations of RNA structures by NMR, EPR, or fluorescence spectroscopy require labeling of the RNA molecules with specific reporter groups [ 2 , 4 , 7 – 10 ]. Likewise, assays that implement separation steps require RNA molecules conjugated to an affinity tag such as biotin, or any other functionality for functional selection [ 11 – 12 ].…”

Changed reactivity of secondary hydroxyl groups in C8-modified adenosine – lessons learned from silylation

“…Oligoribonucleotides carrying site-specific modifications are highly required as models for structure and function studies, driven by the ongoing discovery of new RNAs and their investigation. [1][2][3][4][5][6] This has put demand also on synthetic chemistry to provide suitable compounds at monomeric and oligomeric level. Accordingly, the field has developed to a stage that allows custom-design of RNA probes and tools for specific application.…”

“…For example, investigation into RNA structures by NMR, EPR, or fluorescence spectroscopy requires labelling of the RNA molecules with specific reporter groups. 2,4,[7][8][9][10] Likewise, assays that implement separation steps require RNA molecules conjugated to an affinity tag such as biotin, or any other functionality for functional selection. [11][12] Very importantly, terminal modification/functionalization is not always suitable to a specific aim.…”