Purification of supercoiled G-quadruplex pDNA for in vitro transcription

Abstract

Abstract The formation of G-quadruplex in G-rich regions of DNA can be induced by transcription and is formed within plasmid transcribed in Escherichia coli. The G-loops are more evidenced on supercoiled (sc) topology than on relaxed (oc) or linearized (ln) plasmid isoforms. Thus, the present work reports different purification strategies to efficiently purify the pPH600 sc isoform from other plasmid topologies and host contaminants present in a clarified E. coli lysate. To accomplish this purpose, two affinity supports, l -tyrosine-Sepharose and l -tryptophan-Sepharose were prepared by linking l -tyrosine and l -tryptophan onto epoxy-activated Sepharose CL-6B and were further characterized. The commercial support l -arginine Sepharose was also used to purify sc pPH600 since it has already been efficiently applied to separate sc isoforms of other plasmids using mild binding and elution conditions. A first screen was performed to select the best support that allows to obtain highly pure sc pPH600 from a native sample (sc + oc). By comparing the binding/elution conditions of the three supports, l -tyrosine support showed the preeminent result in separation of two isoforms, allowing the total recovery of sc pPH600, using a decreasing (NH4)2SO4 gradient in HEPES 100 mM at 10 °C. The purification of sc pPH600 directly from clarified E. coli lysate was achieved with the support l -tyrosine-Sepharose and the quality control analysis revealed that the level of E. coli impurities (other pDNA topologies, proteins, endotoxins, gDNA and RNA) present in the final sc pPH600 sample was in accordance with the guidelines of regulatory agencies. In vitro transcription was performed using the purified sc pDNA to induce G-quadruplex formation and it was confirmed by circular dichroism (CD) that the transcript adopted parallel G-quadruplex topology. Overall, this work showed that sc pPH600 can be purified using l -tyrosine support and the transcript adopted parallel G-quadruplex topology.