Manipulation of GQ-Based RNA Aptamers at the Single Molecule Level using Integrated Force-Fluorescence Spectroscopy

Abstract

Recent advances in fluorogen-binding RNA aptamers provide an exciting avenue for protein-free detection of RNA in cells. Crystallographic studies have established a G-Quadruplex (GQ) structure at the core of fluorogen binding properties of aptamers such as Spinach and Mango. However, detailed biophysical characterization of folding of such aptamers is still lacking. Additionally, a mechanistic understanding of stability of such GQ-based RNA aptamers is key to their applications as visualization and diagnostic tools in vivo. In this study, we demonstrated GQ folding in RNA aptamers at the single molecule level for the first time. Integrated force-fluorescence spectroscopy revealed step-wise unfolding of the Spinach aptamer applied forces of < 8 pN. Upon force relaxation, refolding was observed in reciprocal steps. Binding of the Spinach-specific fluorogen DFHBI-1T preserved the step-wise unfolding behavior although at slightly higher forces. In contrast, Mango unfolded in one discrete step and refolded via hysteresis at approximately half the unfolding force value. However, upon binding of a functionalized Thiazole Orange fluorogen, no significant changes could be observed in the mechanical unfolding landscape. As cellular processes like replication, transcription etc. exert pico-Newton levels of force, a comprehensive understanding of dynamics of GQ-based RNA aptamers can aid engineering of state-of-the-art fluorogenic modules for cellular applications.