title: Silencing c-MYC expression by targeting quadruplex in P1 promoter using locked nucleic acid trap.
abstract: The nuclease hypersensitive element of P1 promoter in c-MYC gene harbors a potential of unusual structure called quadruplex, which is involved in molecular recognition and function. This Hoogsteen bonded structure is in dynamic equilibrium with the usual Watson-Crick duplex structure, and these competing secondary structures undergo interconversion for execution of their respective biological roles. Herein, we investigate the sensitivity of the c-MYC quadruplex-duplex equilibrium by employing a locked nucleic acid (LNA) modified complementary strand as a pharmacological agent. Our biophysical experiments indicate that the c-MYC quadruplex under physiological conditions is stable and dominates the quadruplex-WC duplex equilibrium in both sodium and potassium buffers. This equilibrium is perturbed upon introducing the LNA modified complementary strand, which demonstrates efficient invasion of stable c-MYC quadruplex and duplex formation in contrast to the unmodified complementary strand. Our data indicate that LNA modifications confer increased thermodynamic stability to the duplex and thus favor the predominance of the duplex population over that of the quadruplex. Further, we demonstrate that this perturbation of equilibrium by a pharmacological agent results in altered gene expression. Our in vivo experiment performed using the LNA modified complementary strand suggests the influence of the quadruplex-duplex structural switch in the modulation of gene expression. We believe that this exploratory approach utilizing the selectivity and specificity of Watson-Crick base pairing of LNA bases would allow the modulation of quadruplex regulated gene expression.
authors.affiliation: Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110007, India.
authors.forename: Niti
authors.initials: N
authors.lastname: Kumar
authors.forename: Ashok
authors.initials: A
authors.lastname: Patowary
authors.forename: Sridhar
authors.initials: S
authors.lastname: Sivasubbu
authors.forename: Michael
authors.initials: M
authors.lastname: Petersen
authors.forename: Souvik
authors.initials: S
authors.lastname: Maiti
mesh_terms: D000818:Animals; D020029:Base Pairing / Q000187:drug effects; D054856:G-Quadruplexes / Q000187:drug effects; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D018390:Gene Targeting / Q000379:methods; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D016271:Proto-Oncogene Proteins c-myc / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D015027:Zebrafish
chemical_list: D009841:Oligonucleotides; D016271:Proto-Oncogene Proteins c-myc; C477371:locked nucleic acid