The Synthesis of Modified and Locked G Quadruplexes from KRAS 32R as a Novel Therapeutic Device

Abstract

KRAS, an oncogene strongly implicated in pancreatic cancer, possesses G quadruplexes in its promoter region. They are secondary structures formed by G-rich regions in the genome, and are critical cis-regulatory elements of KRAS as they serve as sites for transcription factor binding and gene activation. This process is hijacked in the decoy strategy, where synthetic G4s mimicking those of KRAS bind to the TFs, and reduce their free concentration, thus silencing the oncogene. In this report, modified G4s mimicking the ones in the KRAS promoter are synthesized to facilitate the formation of a covalent bond within the oligonucleotide. This therapeutic approach could ensure that the G4s do not switch in configuration and can stably be recognized by the TFs. The electrophilic phosphorothioester-amine conjugation strategy was used to form the link, where a phosphorothioate group is linked to a primary amine moiety, using Sanger’s reagent (DNFB) as an activator. As a proof-of-concept, the EPT-amine strategy was used to conjugate dodecylamine to a PTO-containing control oligonucleotide, followed by detection using HPLC. Preliminarily, an oligonucleotide fragment was designed to have the amine group at the 3’ end and a PTO moiety in the middle. When such a fragment was treated with DNFB, it was observed that the EPT-conjugation reaction was occurring at biologically relevant pH and temperature, as verified by denaturing PAGE. It was also noted that this method of intra-conjugation was not disrupting the secondary structure of the G4-forming oligonucleotides. Thus, a link could be built between two segments of the modified oligonucleotide.