A Thermodynamic Perspective on Potential G‐Quadruplex Structures as Silencer Elements in the MYC Promoter

Abstract

Multiple G‐tracts within the promoter region of the c‐myc oncogene may fold into various G‐quadruplexes with the recruitment of different tracts and guanosine residues for the G‐core assembly. Thermodynamic profiles for the folding of wild‐type and representative truncated as well as mutated sequences were extracted by comprehensive DSC experiments. The unique G‐quadruplex involving consecutive G‐tracts II–V with formation of two one‐nucleotide and one central two‐nucleotide propeller loop, previously proposed to be the biologically most relevant species, was found to be the most stable fold in terms of its Gibbs free energy of formation at ambient temperatures. Its stability derives from its short propeller loops but also from the favorable type of loop residues. Whereas quadruplex folds with long propeller loops are significantly disfavored, a snap‐back loop structure formed by incorporating a 3’‐terminal guanosine into the empty position of a tetrad seems highly competitive based on its thermodynamic stability. However, its destabilization by extending the 3’‐terminus questions the significance of such a species under in vivo conditions.

Thermodynamic Stability of G‐Quadruplexes: Impact of Sequence and Environment

“…This was associated with a loss in Δ G ° of about 8 and 12 kJ/mol at 37 °C as determined by a van’ Hoff analysis of UV melting. It should be mentioned, however, that G4 thermal and thermodynamic stabilities may noticeably differ depending on the ionic strength and in particular on the potassium cation concentration [43,44] . (ii) With a further increase in total loop length, G4 stabilities tend to level off and there is also a growing tendency of G‐rich sequences to fold into antiparallel or hybrid‐type topologies [41,45] .…”

Time-resolved NMR-spectroscopic studies of conformational dynamics in DNA G-quadruplexes

“…99,100 Noteworthy, both studies find melting temperatures (T m ) for each of the single isolated G-register mutants that are lower than for the G-register wt. For a thermodynamic comparison of spare-tire isomers cMYC-1245 and cMYC-1234 also calorimetric studies have been published, 112,523 which are in line with reported T m s based on CD melting for all reported cMYC structures. 111,342,460,461 The plethora of sequence modifications, buffer and DNA concentrations make a detailed comprehensive overview and comparison of all thermodynamic parameters nearly impossible.…”

“…109,110 If more than four G-stretches are nearby in a DNA sequence, also different G-tracts can be incorporated into the G4 structure. This feature is known as spare-tire isomerism and also has been reported for many G4 sequences (cMYC [111][112][113] , VEGF 113 ). If the number of G-tracts increases in longer G-rich sequences, multiple, stacked 114 G-quadruplexes can be formed or potentially switch between hairpin-G-quadruplex arrangements.…”

Structural motifs and intramolecular interactions in non-canonical G-quadruplexes

“…36 It should be noted, that the addition of further non-G residues at the 3 0 -terminus may still allow for a fold-back topology but is expected to compromise the thermodynamic stability as suggested by calorimetric studies on mutated 3 0 -T extended c-myc promoter sequences with five guanine tracts. 73 Like the extended PDGFR-b promoter sequence, a G-rich c-kit promoter sequence d(AG 3 AG 3 CGCTG 3 AG 2 AG 3 ) encompassing four G 3 -tracts and thus capable of forming a regular quadruplex with four continuous G-columns folds into a topology with a snapback loop in K + solution (Fig. 12).…”

“… 36 It should be noted, that the addition of further non-G residues at the 3′-terminus may still allow for a fold-back topology but is expected to compromise the thermodynamic stability as suggested by calorimetric studies on mutated 3′-T extended c-myc promoter sequences with five guanine tracts. 73 …”