A Simple Molecular Rotor for Defining Nucleoside Environment within a DNA Aptamer–Protein Complex

Abstract

The simple 5-furyl-2'-deoxyuridine ((Fur)dU) nucleobase exhibits dual probing characteristics displaying emissive sensitivity to changes in microenvironment polarity and to changes in solvent rigidity due to its molecular rotor character. Here, we demonstrate its ability to define the microenvironment of the various thymidine (T) loop residues within the thrombin binding aptamer (TBA) upon antiparallel G-quadruplex (GQ) folding and thrombin binding. The emissive sensitivity of the (Fur)dU probe to microenvironment polarity provides a diagnostic handle to distinguish T bases that are solvent-exposed within the GQ structure compared with probe location in the apolar duplex. Its molecular rotor properties then provide a turn-on fluorescent switch to identify which T residues within the GQ bind specifically to the protein target (thrombin). The fluorescence sensing characteristics of (Fur)dU make it an attractive tool for mapping aptamer-protein interactions at the nucleoside level for further development of modified aptamers for a wide range of diagnostic and therapeutic applications.

In What Ways Do Synthetic Nucleotides and Natural Base Lesions Alter the Structural Stability of G-Quadruplex Nucleic Acids?

“…The TBA DNA's six loop thymidines were replaced by a fluorescent furyl derivative, the 5-furyl-2′-deoxyuridine ( Figure 8 , 10 ), to demonstrate the analog's ability to determine the positional impact of each thymine on the stability and thrombin-binding activity of the TBA GQ [ 131 ]. Earlier NMR studies suggested that T4 and T13 stack strongly with the neighboring G-tetrad and T9 of GGT T GGTG T GGT T GG interacts with G8 of the TGT loop and with the G-tetrad.…”

Fluorescence Turn-On Sensing of DNA Duplex Formation by a Tricyclic Cytidine Analogue

“…1–16 Recent highlights include Tor’s isomorphic RNA alphabet, 17,18 Wilhelmsson’s internucleobase FRET pair, 12,19 Sasaki’s fluorescent sensors for nucleobase damage, 20 and a number of examples of nucleobase surrogates that can report on events in the major groove, such as protein binding. 21,22 Still, significant unmet needs persist. For example, most fluorescent nucleobase analogues are quenched when base stacked, emit only at wavelengths <525 nm, and many perturb duplex structure.…”

Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes

“…The differential binding affinity exhibited by PDS is likely to be due to differences in the physicochemical environment of the G‐tetrad near the diagonal, lateral, and propeller loops 50. In the absence of a change in emission maximum, fluorescence quenching upon ligand binding could be due to derigidification of the fluorophore or a proximal effect, wherein the close vicinity of a polyaromatic ligand with the fluorophore could induce nonradiative dissipation of energy 50, 52…”

“…[50] In the ab- sence of ac hange in emission maximum, fluorescencequenching upon ligand bindingc ould be due to derigidification of the fluorophore or ap roximale ffect, wherein the close vicinity of ap olyaromatic ligand with the fluorophore could induce nonradiative dissipation of energy. [50,52] The applicabilityo fn ucleoside probes 1 and 2 in estimating the binding of ligand to H-Telo DNA and TERRA GQs in confined environments was studied by preparing stock solutions of DNA and RNA ONs 5-8 containing increasing concentrations of PDS. The individual stock solution was then added to AOT RMs, such that w 0 was maintained at 20.…”