Probing the propeller-like loops of DNA G-quadruplexes with looped-out 2-aminopurine for label-free switchable molecular sensing

Abstract

We report a new signal readout mechanism for DNA molecular sensing devices using ligand-free fluorogenic G-quadruplexes in which the propeller-like loops are distinguished from the diagonal and lateral loops with incorporated 2-aminopurine (2-AP, a fluorescent analogue of adenine). We study the fluorescence behavior of looped-out 2-AP in duplexes and G-quadruplexes and demonstrate that it shows better fluorescence properties in shorter loops. In particular, 2-AP in the propeller-like loops of parallel or hybrid G-quadruplexes displays a perfect fluorescence emission whereas that in the diagonal and lateral loops does not. This loop-environment-sensitive feature allows 2-AP to probe the propeller-like loops of G-quadruplexes, illustrated by an ion-tuned allosteric G-quadruplex FG9A and a (3 + 1) hybrid human telomeric DNA. In the presence of K⁺, FG9A folds into a parallel structure where 2-AP is in the propeller-like loops and shows a high fluorescence signal, which can probe K⁺ concentrations down to 25 μM. Upon addition of Pb²⁺, the folded FG9A converts into an antiparallel structure which is revealed by a sharp decrease in 2-AP fluorescence, which can easily be reset with EDTA. This process is utilized to reversibly sense Pb²⁺ with a detection limit of 100 nM. Furthermore, its ability to probe the propeller-like loops may allow 2-AP to identify the folding topologies of unknown G-quadruplexes in human gene regions.