Abstract 2756: Custom G4 DNA microarray can determine large-scale binding selectivity of small molecules and proteins to oncogene G-quadruplexes
Authors: Wu Guanhui , Chen Luying , Huseman Kristen , Tillo Desiree , Ray Sreejana , Chang Ta-Chau , Schneekloth John S. , Vinson Charles , Yang Danzhou 
Published: April 2023

G-quadruplexes are novel DNA secondary structures that are formed in guanine-rich genomic regions with functional importance in cancers, such as human telomeres and oncogene promoters. G-quadruplexes adopt globular structures distinct from duplex B-DNA and their structural diversity suggests specific recognition. G-quadruplexes have become attractive drug targets because they provide a means of addressing otherwise undruggable targets. As one example, MYC is an important oncogene that is difficult to target by traditional drug design; however, its regulation involves a promoter G-quadruplex (MycG4) that is a potential drug target. 3,6-bis(1-Methyl-4-vinylpyridinium) carbazole diiodide (BMVC) is the first fluorescent probe developed for detecting G-quadruplex structures in cells and has been tested for cancer cell diagnosis. Although it was designed to detect the G-quadruplexes formed in human telomeres, we found BMVC binds to the MYC promoter G-quadruplex (MycG4) with higher affinity and specificity. We determined the high-resolution NMR K+ solution structure of the 2:1 BMVC-MycG4 complex showing a novel conformational adjustment of BMVC for an optimal binding to G-quadruplexes. Intriguingly, BMVC binds the 5’-end of MycG4 with higher affinity, but it binds the 3’-end with greater sequence selectivity. To determine ligand-binding selectivity on a large-scale, we designed a new custom G-quadruplex microarray platform with more than 25,000 potential oncogene G4 sequences and mutants. The new microarray was able to systematically assess binding affinity and selectivity of drugs targeted to G4s like the G-quadruplex involved in MYC regulation. The microarray results show that BMVC preferentially binds to the parallel type of G-quadruplexes, especially MycG4. Importantly, a strong sequence selectivity of BMVC for a 3’ flanking T was observed, consistent with the NMR data. The new microarray was also able to systematically assess binding of proteins and antibodies in a high-throughput and unbiased manner, and we tested the G-quadruplex binding proteins FANCJ, PIF1, BLM, DHX36, WRN, IGF2, CNBP, nucleolin, as well as the G-quadruplex-specific antibody BG4. In conclusion, we establish the custom G4 DNA microarray which can be used to determine binding selectivity of small molecules and proteins to oncogene G-quadruplexes in large-scale and high-throughput.

Citation Format: Guanhui Wu, Luying Chen, Kristen Huseman, Desiree Tillo, Sreejana Ray, Ta-Chau Chang, John S. Schneekloth, Charles Vinson, Danzhou Yang. Custom G4 DNA microarray can determine large-scale binding selectivity of small molecules and proteins to oncogene G-quadruplexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2756.