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Naphthalene amine support for G-quadruplex isolation

Abstract

G-quadruplex (G4) is involved in many biological processes, such as telomere function, gene expression and DNA replication. The selective isolation of G4 using affinity ligands that bind tightly and selectively is a valuable strategy for discovering new G4 binders for the separation of G4 from duplexes or the discrimination of G4 structures. In this work, one affinity chromatographic support was prepared using a naphthalene amine as G4 binder. The ligand was immobilized on epoxy-activated Sepharose CL-6B using a long spacer arm and was characterized by HR-MAS spectroscopy. The supercoiled (sc) isoform of pVAX1-LacZ and pVAX1-G4 was isolated from a native sample. Also, the recovery and isolation of the plasmid isoforms from Escherichia coli lysate samples were achieved using an ionic gradient with different concentration of NaCl in 10 mM Tris-HCl (pH 7.4). The retention time of different DNA/single strand sequences that can form G4, such as, c-MYC, c-kit1, c-kit2, tetrameric, telomeric (23AG), thrombin aptamer (TBA) and 58S3 in this support were evaluated. Our experimental results suggest that the support exhibits selectivity for parallel c-MYC and c-kit1 G4s. In vitro transcription was performed using the purified sc pVAX1-G4 and pPH600 to induce G4 formation and the circular dichroism (CD) analysis confirmed that both transcripts adopt parallel G4 topology.

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Publication details

The article was received on 18 Apr 2017, accepted on 05 Jul 2017 and first published on 07 Jul 2017


Article type: Paper
DOI: 10.1039/C7AN00648A
Citation: Analyst, 2017, Accepted Manuscript
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    Naphthalene amine support for G-quadruplex isolation

    J. Ferreira, T. Santos, P. Pereira, M. C. Corvo, J. A. Queiroz, F. Sousa and C. Cruz, Analyst, 2017, Accepted Manuscript , DOI: 10.1039/C7AN00648A

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