Abstract 5225: Novel loop interactions within a parallel-stranded G-quadruplex formed in the human BCL-2 proximal promoter

The human BCL-2 gene contains a 39-bp GC-rich region upstream of its P1 promoter which has been shown to be critical for BCL-2 transcriptional regulation. Therapeutic inhibition of BCL-2 expression can reduce cell proliferation and the apoptotic threshold of tumorigenic cells. Previously, we have reported that the major G-quadruplex (G4) formed in the Pu39 G-rich sequence adopts a stable, parallel-stranded structure with two 1-nt short loops and a unique 13-nt long central loop. Parallel G-quadruplexes have been found to be prevalent in promoter sequences; the 13-nt loop in BCL-2 Pu39G4 is the longest in this prototype parallel G-quadruplexes identified in human gene promoters. The structure of this G4 with such a long loop is important because it can enable specific small molecule recognition of this BCL-2 G-quadruplex over other G-quadruplexes and provide a potential basis for rational drug design targeting BCL-2 transcription. Thus, the objective of our study is to determine the molecular structure of the BCL-2 Pu39 G-quadruplex and the molecular interactions of this long loop in physiologically relevant K+ solution by Nuclear Magnetic Resonance (NMR) spectroscopy. Our NMR study shows that the BCL-2 Pu39G4 adopts a well-defined parallel-stranded G-quadruplex structure which contains three G-tetrads stabilized by Hoogsteen hydrogen bonding and three double-chain reversal loops containing 1:13:1 bases. The two 1-nt double-chain reversal loops on two edges of the tetrad provide overall stability of the BCL-2 Pu39G4. The central 13-nt double-chain reversal long loop is more dynamic in conformation. Specific interactions of the long loop are observed at both the 5’ and 3’ end with external tetrads as well as the flanking residues, which may also contribute the stable formation of this G4. The middle region of the 13-nt central long loop is highly dynamic and doesn’t show much interaction with the G-quadruplex core. The specific molecular interactions of the unique long central loop are specific to the BCL-2 Pu39G4 sequence/structure and significantly differ from those in other parallel-stranded structures, e.g. c-myc and VEGF. The specific interactions are supported by our mutational analyses. The results are also supported by the DMS footprinting results which show that the guanines involved in the tetrad formation in this major G-quadruplex are well-protected against DMS methylation. Therefore, the knowledge about the molecular structure of this non B-DNA conformation of the BCL-2 promoter region is essential for using the G-quadruplex as a target for anticancer drugs, which could be a novel approach to anti-BCL-2 drug discovery in cancer therapy. Citation Format: Guanhui Wu, Clement Lin, Danzhou Yang. Novel loop interactions within a parallel-stranded G-quadruplex formed in the human BCL-2 proximal promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5225. doi:10.1158/1538-7445.AM2017-5225