Folding/unfolding kinetics of G-quadruplexes upstream of the P1 promoter of the human BCL-2 oncogene

Abstract

The G-rich Pu39 region of the P1 promoter of the oncogene BCL-2, an apoptosis regulator, can fold into multiple G-quadruplex (G4) structures. Bcl2-2345 and Bcl2-1245 are two major G4 species forming with high thermal stability and distinct topologies in the Pu39 region, but their folding/unfolding kinetics have not yet been investigated. Here, we used magnetic tweezers to measure the mechanical stability and the folding/unfolding kinetics of the Bcl2-2345 and Bcl2-1245 G4 structures. We report that the hybrid-stranded Bcl2-2345 G4 had a lower mechanical stability than the parallel-stranded Bcl2-1245 G4. We observed that the Bcl2-2345 G4 is a kinetically favored structure, whereas the Bcl2-1245 G4, with a slow unfolding rate, may function as a kinetic barrier for transcription. We also determined that in addition to the Bcl2-2345 and Bcl2-1245 G4s, other stable DNA secondary structures, such as a hybrid-stranded Bcl2-1234 G4, can also form in the Pu39 sequence. The characterization of the folding/unfolding kinetics of specific G4s reported here sheds light on the participation of G4s during gene transcription and provides information for designing G4-targeting small molecules that could modulate BCL-2 gene expression.