Abstract
Modulating the expression or function of the enigmatic MYC protein has demonstrated efficacy in an array of cancer types and a marked potential therapeutic index and safety profile. Despite its high therapeutic value, specific and selective inhibitors or downregulating therapeutics have proven difficult to develop. In the current study, we expanded our work on a MYC promoter G-quadruplex (G4) stabilizing DNA clamp to develop an oligonucleotide interfering DNA (DNAi) therapeutic. We explored six DNAi for G4-stabilization through EMSA, DMS footprinting, and thermal stability studies, focusing on the DNAi 5T as the lead therapeutic. 5T, but not its scramble control 5Tscr, was then shown to enter the nucleus, modulate cell viability, and decrease MYC expression through G4-stabilization. DNAi 5T is thus described to be our lead DNAi, targeting MYC regulation through stabilization of the higher-order DNA G4 structure in the proximal promoter, and it is poised for further preclinical development as an anticancer therapeutic.
Highlights
Despite its high therapeutic value, specific and selective inhibitors or downregulating therapeutics have proven difficult to develop
In order to assess the nature of the DNA structure separated and represented by each band, Pu46 was incubated with each demonstrated selectivity for an individual promoter G4 (DNAi) at a 1:1 ratio, the species were separated by Electromobility shift assays (EMSAs), isolated from the gel, and subjected to Dimethyl sulfate (DMS) footprinting (Figure 2B)
We examined the effects of G4-mediated changes in MYC expression as demonstrated by the dose-dependent decreased expression in CA46 cells of MYC mRNA containing exon 1, but not exon 2, by DNAi 5T (Figure 4B), no changes in either exon were noted with 5Tscr
Summary
Adding DNAi that complements the regions flanking the G4-forming promoter section can shift transient basis due to superhelical stress and is capable of forming a higher-order G4 stru the equilibrium to maintain more G4 formation, enhancing the silencing function within the MYC. EMSA separation of the higher-order species can differentiate between un DNA where the DNAi captures both flanking regions. Linear or G4 DNA, DNA wherein the DNAi complements the G4-forming region on one flan
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