Thermodynamic Stability and Folding Kinetics of the Major G-Quadruplex and Its Loop Isomers Formed in the Nuclease Hypersensitive Element in the Human c-Myc Promoter: Effect of Loops and Flanking Segments on the Stability of Parallel-Stranded Intramolecular G-Quadruplexes

Overexpression of the c-Myc proto-oncogene is associated with a broad spectrum of human cancers. Nuclease hypersensitivity element III1 (NHE III1) of the c-Myc promoter can form transcriptionally active and silenced forms, and the formation of DNA G-quadruplex structures has been shown to be critical for c-Myc transcriptional silencing. The major G-quadruplex formed in c-Myc NHE III1 is a mixture of four loop isomers, which have all been shown to be biologically relevant to c-Myc transcriptional control. In this study, we performed a thorough thermodynamic and kinetic study of the four c-Myc loop isomers in a K+ solution. The four loop isomers all form parallel-stranded G-quadruplexes with short loop lengths. While the parallel-stranded G-quadruplex has been known to favor short loop lengths, our results show that the difference in thermodynamic and kinetic properties of the four loop isomers, and hence between the parallel G-quadruplexes with similar loop lengths, is more significant than previously recognized. At 20 m M K+, the average difference in the T-m values between the most stable loop isomer 14/23 and the least stable loop isomer 11/20 is more than 10 degrees C. In addition, the capping structures formed by the extended flanking segments are shown to contribute to a stabilization of 2-3 degrees C in T-m for the c-Myc promoter G-quadruplex. Understanding the intrinsic thermodynamic stability and kinetic properties of the c-Myc G-quadruplex loop isomers can aid in our understanding of their biological roles and drug targeting.