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.

Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine

“…3d, e, g ). To confirm this result, the recently described fluorescent 4-cyanoindol ribonucleoside (r4CI) was incorporated at positions 34 or 46 of the Chili RNA, where it reported on ligand binding by serving as a FRET donor to DMHBO + 28 . No reduction in donor emission due to FRET was observed in c 7 G15 mutants of r4CI-modified Chili RNA, indicating that N7 of G15 is strictly required for ligand binding (Supplementary Fig.…”

“…RNAs containing c 7 G and r4CI were prepared by enzymatic ligation of synthetic oligonucleotides obtained by solid phase synthesis using 2′- O -TOM-protected phosphoramidites under standard conditions 28 , 51 . The c 7 G phosphoramidite was purchased form ChemGenes, the r4CI phosphoramidite, chemical 5′ phosphorylation reagent, and 3′-phosphate CPG support were prepared as described 28 . Sequence information for RNAs are given in Supplementary Table 2 .…”

Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics

“…A unique and attractive feature inherent to FBA labeling is that the fluorophore is firmly located in the oligomer stack, which allows for positioning of the label at a specific site of interest with a level of spatial control that is not attainable with external fluorophores. FBAs have previously been employed as oligomer-incorporated reporters to study DNA duplex- 22 , triplex- 23 , and G-quadruplex 24 formation, as well as in Förster resonance energy transfer (FRET) applications to monitor ligand binding 25 , 26 , duplex form transitions 27 , 28 , and protein-nucleic acid interactions 29 , 30 . These studies and others have provided important novel insights on isolated biological systems, while there is a notable lack of FBA applications in more complex biological matrixes.…”

Concentration‐, Temperature‐ and Solvent‐Dependent Self‐Assembly: Merocyanine Dimerization as a Showcase Example for Obtaining Reliable Thermodynamic Data

“…Thus, when calculating Δ G 0 293 the resulting value is fairly constant, independent of the wavelength at which the fit is performed (Figure 4c). We interpret this in a way that the optimized values for Δ H 0 and Δ S 0 are very sensitive to the shape of the self‐assembly curve, since both parameters can influence the inflection point of the melting curve and the slope at this point [56] . For the non‐linear regression, a change in one parameter can thus to some degree be compensated by a corresponding change in the other and the results are therefore strongly influenced by the small errors due to thermochromism which distort the shape of aggregation curve.…”

“…In the literature also slightly different approaches can be found to evaluate temperature‐dependent self‐assembly data. Often the parameter of the melting temperature T m is introduced which is defined as the temperature at which α agg =0.5 [27,39,56–58] . For examples and more details the reader is referred to the cited literature.…”