Naphthalene amine support for G-quadruplex isolation

Tiago Santos, Patrícia Pereira, Marta C. Corvo, João António Queiroz, Fani Sousa, Carla Cruz, Joâo Ferreira

Research output: Contribution to journalArticle

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 a 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 iso-forms from Escherichia coli lysate samples were achieved using an ionic gradient with different concentrations of NaCl in 10 mM Tris-HCl (pH 7.4). The retention times 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 gamma 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 purified sc pVAX1-G4 and pPH600 to induce G4 formation and circular dichroism (CD) analysis confirmed that both transcripts adopt a parallel G4 topology.
Original languageEnglish
Pages (from-to)2982-2994
JournalAnalyst
Volume142
Issue number16
DOIs
Publication statusPublished - 2017

Fingerprint

G-Quadruplexes
Naphthalene
naphthalene
Amines
Binders
DNA
Ligands
Biological Phenomena
ligand
Telomere
Dichroism
Transcription
Circular Dichroism
DNA Replication
Gene expression
Escherichia coli
Spectrum Analysis
Protein Isoforms
MAS
Plasmids

Keywords

  • DYNAMIC BINDING-CAPACITY
  • PLASMID DNA PURIFICATION
  • AFFINITY-CHROMATOGRAPHY
  • IN-VITRO
  • TRANSCRIPTION
  • RESIN
  • NMR

Cite this

Santos, T., Pereira, P., Corvo, M. C., Queiroz, J. A., Sousa, F., Cruz, C., & Ferreira, J. (2017). Naphthalene amine support for G-quadruplex isolation. Analyst, 142(16), 2982-2994. https://doi.org/10.1039/C7AN00648A
Santos, Tiago ; Pereira, Patrícia ; Corvo, Marta C. ; Queiroz, João António ; Sousa, Fani ; Cruz, Carla ; Ferreira, Joâo. / Naphthalene amine support for G-quadruplex isolation. In: Analyst. 2017 ; Vol. 142, No. 16. pp. 2982-2994.
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Santos, T, Pereira, P, Corvo, MC, Queiroz, JA, Sousa, F, Cruz, C & Ferreira, J 2017, 'Naphthalene amine support for G-quadruplex isolation', Analyst, vol. 142, no. 16, pp. 2982-2994. https://doi.org/10.1039/C7AN00648A

Naphthalene amine support for G-quadruplex isolation. / Santos, Tiago; Pereira, Patrícia; Corvo, Marta C.; Queiroz, João António; Sousa, Fani; Cruz, Carla; Ferreira, Joâo.

In: Analyst, Vol. 142, No. 16, 2017, p. 2982-2994.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Naphthalene amine support for G-quadruplex isolation

AU - Santos, Tiago

AU - Pereira, Patrícia

AU - Corvo, Marta C.

AU - Queiroz, João António

AU - Sousa, Fani

AU - Cruz, Carla

AU - Ferreira, Joâo

N1 - sem pdf conforme despacho. Fundo Social Europeu e ao Programa Operacional Potencial Humano - IF/00959/2015 ; Foundation for Science and Technology - (FCOMP-01-0124-FEDER-041068; EXPL/QEQ-MED/1068/2013) ; FEDER funds through the POCI - COMPETE - POCI-01-0145-FEDER-007491 ; FCT - UID/Multi /00709/2013 ; Santander/Totta-UBI - BID/ICI-FCS/CICS/Santander Universidades-UBI/2016

PY - 2017

Y1 - 2017

N2 - 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 a 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 iso-forms from Escherichia coli lysate samples were achieved using an ionic gradient with different concentrations of NaCl in 10 mM Tris-HCl (pH 7.4). The retention times 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 gamma 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 purified sc pVAX1-G4 and pPH600 to induce G4 formation and circular dichroism (CD) analysis confirmed that both transcripts adopt a parallel G4 topology.

AB - 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 a 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 iso-forms from Escherichia coli lysate samples were achieved using an ionic gradient with different concentrations of NaCl in 10 mM Tris-HCl (pH 7.4). The retention times 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 gamma 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 purified sc pVAX1-G4 and pPH600 to induce G4 formation and circular dichroism (CD) analysis confirmed that both transcripts adopt a parallel G4 topology.

KW - DYNAMIC BINDING-CAPACITY

KW - PLASMID DNA PURIFICATION

KW - AFFINITY-CHROMATOGRAPHY

KW - IN-VITRO

KW - TRANSCRIPTION

KW - RESIN

KW - NMR

U2 - 10.1039/C7AN00648A

DO - 10.1039/C7AN00648A

M3 - Article

VL - 142

SP - 2982

EP - 2994

JO - Analyst

JF - Analyst

SN - 0003-2654

IS - 16

ER -

Santos T, Pereira P, Corvo MC, Queiroz JA, Sousa F, Cruz C et al. Naphthalene amine support for G-quadruplex isolation. Analyst. 2017;142(16):2982-2994. https://doi.org/10.1039/C7AN00648A