Abstract 4757: Design and optimization of linked nucleic acid (LNA) probes to detect the c-MYC G-quadruplex

The c-MYC oncogene is upregulated in almost 80% of cancers, and in some cases is the causative oncogenic factor. It is regulated at the transcriptional level with up to 90% initiating at the P1 and P2 promoters. The NHE III1 element regulates this region, which consists of a unique guanine-rich string of DNA capable of adopting at least three topologies: single stranded, double stranded (ds) and the non-B-DNA G-quadruplex (G4) structure. G4s are formed when two or more tetrads stack, each comprised of four guanines bonded by Hoogsteen hydrogen bonds, stabilized with monovalent cations. Putative G4 forming regions have at least four runs of three or more consecutive guanines separated by varying nucleotides that comprise the loop structures. The c-MYC G4 structure has been elucidated to involve guanine runs 1α4 (G41-4) or 2Δ5 (G42-5) (of six total runs) in a parallel formation. Regulatory proteins have been identified, and small molecule compounds are under investigation to stabilize the G4 element and downregulate c-MYC expression. In parallel to these efforts, we are developing linked nucleic acid (LNA)-biotin bridge probes to be used as diagnostic tools to identify these formations in biopsies in order to inform therapeutic regimens. Molecular models of the two G4 isoforms were used to determine the distance occupied by the secondary structure to be between 16.4α16.6 angstroms; various length nucleic acid sequences flanking a 17.7α30 angstrom linker were screened and optimized. Probes were shown to be structure specific - either for the c-MYC G41-4 (probe 1a) or the G42-5 (probe 1b) - by EMSA analysis. The probes were amenable to internal biotinylation for ongoing pull-down assays, but truncation of the 5′-complementary sequence limited affinity. Interestingly, the LNA probes thermally stabilized the c-MYC G4 in a unique pattern and are also being examined for their potential therapeutic efficacy. Experiments are underway to show that these hybrid probes will bind intracellularly and show the frequency of G4 formation in quiescent versus activated cells, and following treatment with c-MYC G4-interactive compounds. Through these works, we highlight for the first time that a ‘FISH’-type probe can be developed and used to selectively detect the formation of the c-MYC G4 in vitro. Up to 80% cancer patients overexpress c-MYC and are candidates for G4-targeted therapies currently under development. The developed probes will be of great utility as companion diagnostics in selecting patients for G4-therapeutics and truly personalized medicine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4757. doi:1538-7445.AM2012-4757