Sequence-Resolved Imaging of mRNA G-Quadruplexes for Profiling the Translational Suppression in Oncogene Expression

G-quadruplexes (G4s) are nonclassical DNA or RNA secondary structures with significant regulatory roles in diverse biological processes. Developing advanced G4 detection technologies to elucidate sequence-specific G4 conformations represents one of the active research areas. However, existing methods lack the sequence resolution to image G4s within a specific messenger RNA (mRNA) transcript in living cells. Herein, we report a smart and versatile orthogonal recognition strategy (SORS) that enables sequence-resolved imaging of mRNA G4s by simultaneously targeting both the G4-flanking sequence (via a sequence-specific module, Seq-module) and the G4 structure (via a structure-specific module, Str-module). Upon dual recognition, the two modules hybridize with each other to assemble a complete initiator for the hybridization chain reaction, generating amplified fluorescence signals only when the target mRNA G4 is present. Using SORS, we achieved sequence-resolved detection and imaging of the G4 structure within the proto-oncogene (B-cell lymphoma 2, BCL2) mRNA in living cells and provided direct evidence that a high G4-folding percentage correlates with low BCL2 protein expression, validating the translational suppressive role of this G4 structure. This orthogonal recognition design offers a versatile and robust platform for exploring the configuration dynamics of secondary structures within specific mRNAs and bodes well for the development of oligonucleotide-based molecular diagnostics and therapeutics.