A small-molecule platform for selective detection and cellular imaging of DNA G-Quadruplexes

Abstract

G-Quadruplex (G4) plays vital roles in regulating gene expression, replication and is also regarded as a therapeutic target in tumor biology. Approaches for sensitive detection of endogenous DNA G4 without perturbing these secondary structures are urgently needed. However, most of the small molecule probes exhibited strong stabilizing effect on G4 structures which makes it very challenging to achieve endogenous and selective labeling of DNA G4. Moreover, current development of new probes heavily relied either on direct ligand/base conjugation or on serendipitous finding, which calls for a general platform for G4 probe design. This thesis describes a systematic investigation on developing a general molecular scaffold for fluorescence imaging of cellular DNA G-quadruplex with excellent selectivity and sensitivity. Eight novel probes have been designed, synthesized, and characterized. Those probes are essentially non-fluorescent due to the rotary non-radiative decay in buffer. Upon binding to DNA G4, their selective interactions with planar G-tetrads can efficiently restrict molecular rotation, and a strong emission could be released. In particular, NapIM is a sensitive, selective, and cell-permeable probe with deep red “turn-on” emission upon interacting with DNA and RNA G4, which could be used to monitor cellular G4 in real time. Additional five probes (NapDa, NapHoe, Nap2A, NapPD, and NaptBu) have been designed based on the rotary molecular scaffold of NapIM. The N-substituents have been found to affect the quadruplex- vs. duplex selectivity, making NapPD and NaptBu more selective than the rest three probes. Because NapIM outperformed these five derivatives, two new probes, Nap2Ph and Nap2Ph, have been designed based on the knowledge of binding mechanism of NapIM. Nap2Ph, as the second-generation probe, is superior to NapIM in terms of selectivity and sensitive, and it could also surprisingly differentiate DNA G4 from RNA G4. Cellular imaging using Nap2Ph has clearly revealed both big nucleolar and small nuclear foci, which could be competed away by G4 ligand pyridostatin in a dose-dependent manner. Moreover, dynamic G4 formation during cell mitosis has been successfully visualized, revealing the tight correlations between DNA secondary structures, nucleoli assembly and nuclei organizations.