Solution equilibria of the i-motif-forming region upstream of the B-cell lymphoma-2 P1 promoter

The 5′-end of the P1 promoter of the B-cell lymphoma-2 (bcl-2) gene contains a highly guaninecytosine-rich region, which has a role in the regulation of bcl-2 transcription. Whereas the guanine-rich region has been the focus of recent studies, little attention has been paid to the cytosine-rich strand. Here we examine the structural transitions of the cytosine-rich sequence by means of acid–base, mole-ratio and melting experiments monitored by molecular absorption, circular dichroism, and NMR spectroscopies. Two intramolecular i-motif structures have been detected in the pH range 2–7, with maximal formation at pH 4 and 6, respectively. At pH 7.6 the majority species has been associated with a hairpin involving Watson–Crick base pairs. Upon addition of the quadruplex-interacting ligand TmPyP4, bcl-2c structures at pH 6.1 and 7.6 yield identical interaction species with stoichiometries 1:2 (DNA:ligand) and logarithms of formation constant 12.4 ± 0.2 and 11.7 ± 0.1, respectively. The initial i-motif structure at pH 6.1 is lost upon interaction with TmPyP4.