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

Abstract

Overexpression of the c-Myc proto-oncogene is associated with a broad spectrum of human cancers. The nuclease hypersensitivity element III 1 (NHE III 1 ) 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 the c-Myc NHE III 1 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 K + solution. The four loopisomers all form parallel-stranded G-quadruplexes with short loop lengths. While the parallelstranded 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 mM K + , the average difference of the T m values between the most stable loopisomer 14/23 and the least stable loop-isomer 11/20 is greater than 10 degrees. In addition, the capping structures formed by the extended flanking segments are shown to contribute to a stabilization of 2-3°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 help understand their biological roles and drug targeting. Keywordsc-Myc promoter G-quadruplex; loop-isomers; parallel-stranded G-quadruplex; thermodynamic stability; folding kinetics * To whom correspondence should be addressed Telephone: (520) 626-5969 Fax: (520) 626-6988 yang@pharmacy.arizona.edu.. Supporting Information Available.ΔG 25 values for the c-Myc G-quadruplex loop-isomers, hysteresis between CD melting and annealing curves of 11/20 with a temperature gradient of 0.5 °C/min, the imino regions of 1D 1 H NMR spectra of the completely truncated sequences for the four loop-isomers, hysteresis between CD melting and annealing curves of 11/20 and 11/23 loop-isomers with temperature gradients of 2 °C/min and 4 °C/min. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 November 2. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptOverexpression of the c-Myc proto-oncogene is associated with a broad spectrum of human cancers, including colon, breast, prostate, cervical, and lung carcinomas, osteosarcomas, lymphomas, and leukemias (1-9). In addition, elevated levels of c-Myc expression are often associated with poor therapeutic prognosis. c-Myc overexpression can be caused by different mechanisms, including gene amplification (10,11), translocation (12-14), and simple upregulation of transcription (1,15). The transcriptional regulation of c-Myc expression is complex and involves multiple promoters...

Aptamer-Based K+ Sensor: Process of Aptamer Transforming into G-Quadruplex

“…36 For the case of 100 mM K + , T m is too high to be measured. 36 T m is summarized (Table S1 in the Supporting Information), and corresponding thermodynamic parameter ∆G is calculated through van't Hoff analysis. 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 K + sensors have been developed, but it is very difficult to selectively determine extracellular K + concentration due to the large excess of Na + (up to 140 mM) present in physiological conditions.…”

“…36 T m is summarized (Table S1 in the Supporting Information), and corresponding thermodynamic parameter ∆G is calculated through van't Hoff analysis. 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 K + sensors have been developed, but it is very difficult to selectively determine extracellular K + concentration due to the large excess of Na + (up to 140 mM) present in physiological conditions. In order to avoid the interferences from Na + , a complementary strand is selected to totally hybridize with PW17 forming double stranded DNA, which works as a probe for detecting K + instead of PW17.…”

Human Telomeric RNA G-Quadruplex Response to Point Mutation in the G-Quartets

“…Thus, kinetic analysis of the thermal curves was performed. 36 Using two-state model for the folding−unfolding process, the equilibrium between folded (F) and unfolded (UF) fraction of the oligonucleotides is represented as…”

Human Telomeric G-Quadruplex Structures and G-Quadruplex-Interactive Compounds

“…280–295 can indicate hybrid folding topology. For example, the hybrid type BCL2 Mid G-quadruplex shows a small peak at 295 [59] whereas the parallel c-Myc has no peak between 280 and 300 [60]. …”