Engineering Logic-Gated i-Motif/G-Quadruplex (iG4) Hybrid Structures for DNA Nanotweezer-Based Aptasensing

Abstract

Given high levels of both K⁺ and H⁺ in the tumor microenvironment, G-quadruplex (G4) and i-motif can in principle be utilized as two cooperative modules to build logic-gated DNA nanodevices for microenvironment recognition and targeting. Combined use of G4 and i-motif in DNA nanoassemblies, however, usually causes the uncontrolled DNA aggregation. To address this trouble, we well design a novel i-motif/G-quadruplex (iG4) hybrid structure that integrates two four-stranded DNA helices in a folding topology, with a parallel mini-duplex flanking at the 5’ end to provide a binding site for fluorescent ligands. This design enables that the folded and lighting-up DNA structure is favored by H⁺ and K⁺ together, consistent with a two-input AND gate behavior. We then employ the iG4 structure to guide a DNA nanotweezer that is clamped by an altered split ATP aptamer, which brings a proximity effect contributing to the proper folding of iG4 in slightly acidic microenvironments and enables the sensitive detection of endogenous ATP in cancer cell lysates.