Site-Specific Fluorophore Labeling of Guanosines in RNA G-Quadruplexes

Abstract

RNA G-quadruplexes are RNA secondary structures that are implicated in many cellular processes. Although conventional biophysical techniques are widely used for their in vitro characterization, more advanced methods are needed to study complex equilibria and the kinetics of their folding. We have developed a new Förster resonance energy-transfer-based method to detect the folding of RNA G-quadruplexes, which is enabled by labeling the 2′-positions of participating guanosines with fluorophores. Importantly, this does not interfere with the required anti conformation of the nucleobase in a quadruplex with parallel topology. Sequential click reactions on the solid phase and in solution using a stop-and-go strategy circumvented the issue of unselective cross-labeling. We exemplified the method on a series of sequences under different assay conditions. In contrast to the commonly used end-labeling approach, our internal labeling strategy would also allow the study of G-quadruplex formation in long functional RNAs.

Inosine Substitutions in RNA Activate Latent G-Quadruplexes

“…To study the properties of putative GI-quadruplexes, we used a well-characterized model sequence comprising three G-quartets (G3 wt ) . We synthesized derivatives of G3 wt with adenosine or inosine at three positions in the third G-tract (Figure a).…”

“…To study the properties of putative GI-quadruplexes, we used a well-characterized model sequence comprising three G-quartets (G3 wt ). 27 We synthesized derivatives of G3 wt with adenosine or inosine at three positions in the third G-tract (Figure 1a). We then measured UVmelting curves and calculated melting temperatures (T m ) to assess the thermal stability of putative quadruplex structures.…”

Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules

“…For example, G-quadruplexes (G4) can appear at the end or in the middle of a nucleic acid strand. Internal base labeling of guanosines [ 62 ] is compatible with the two types of G4 folding but the design should be optimized and verified to not impede the natural secondary structure [ 63 ].…”

Single-molecule fluorescence detection of a tricyclic nucleoside analogue

“…[1][2][3] Most of these studies have used extrinsic fluorophores, which can potentially interfere with the native biomolecular behavior and obscure local structural details. [4][5][6][7] An ideal approach in this regard would be the use of intrinsically fluorescent biomolecules, prepared by the synthetic introduction of only minimal changes. 8 It remains, however, a major challenge to attain adequate brightness and photostability in this approach.…”