| Assay Method Information | |
| | In-Vitro Binding to the c-Myc Promoter G-Quadruplex (MycG4) |
| Description: | MycG4 Stabilization Measured by FRET Melting. A F rster Resonance Energy Transfer (FRET)-melting assay was conducted in order to determine whether the 7-aza-8,9-methylenedioxyindenoisoquinoline derivatives can bind and stabilize the c-Myc promoter G-quadruplex (MycG4, FIG. 1 ). MycG4 DNA was labeled with a FRET donor (6-FAM) at the 5′-end, and a FRET acceptor (TAMRA) at the 3′-end. Close proximity of the donor and acceptor fluorophores in the G-quadruplex secondary structure quenches the 6-FAM fluorescence due to FRET transfer to TAMRA. Melting of the secondary structure into single-strand DNA decreases the proximity of the FRET-pair, restoring 6-FAM-based fluorescence emission. Monitoring 6-FAM emission upon G-quadruplex thermal denaturation provides a melting curve from which a melting temperature (Tm), the temperature at which folded and unfolded DNA are equally populated, was derived. The Tm values of MycG4 were measured in the presence of the compounds in 10 mM K+ by FRET-melting experiments. All eight of the 7-aza-8,9-methylenedioxyindenoisoquinolines tested showed clear thermal stabilization (ΔTm) of the MycG4. |
| Affinity data for this assay | |
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