Isomerization Transmission to G-Quadruplex (IT2G4) for Discriminating Natural Isoquinoline Alkaloid Isomers

Exploring efficient strategies to discriminate bioactive natural isomers using nucleic acids is of great importance to advanced biosensing platform development and pharmaceutical discovery. The DNA duplex-based strategy gets trouble in discriminating isomers containing conjugated multicycles like natural protoberberine alkaloids due to their comparable affinities with the universally retentive helix structure. Herein, we developed an isomerization transmission-to-G-quadruplex (IT2G4) strategy to discriminate protoberberine alkaloid isomers, as exemplified using coptisine (COP) and isocoptisine (iCOP) that involve only one adjacent positional isomerization. COP binds in a 2:1 mode with the human telomeric G-quadruplex (htG4) without structural alteration, whereas iCOP transmits its isomerization feature to htG4 by uniquely switching the hybrid folding to an antiparallel chair structure via a 3:1 binding manner. This G4 remodeling, validated by CD, PAGE, and ITC, enables highly selective discrimination of iCOP from COP and even other alkaloid congeners. Based on this distinction, a dual TAMRA-labeled htG4 with self-quenched fluorescence was employed to develop a sensitive discrimination method. The iCOP-stabilized chair structure exhibits pronounced resistance to DNase I digestion, preventing fluorescence recovery in contrast to the facile digestion of htG4 upon binding with COP and other congeners. Finally, the method was successfully applied to quantify iCOP in herbal extracts. This work proves the IT2G4 strategy as a versatile, structure-switch-based platform for high-performance isomer discrimination and advances the potential regulation of htG4 bioactivity by designing iCOP-like anticancer pharmaceuticals in the future.