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Naphthalene amine support for G-quadruplex isolation.

João Ferreira¹, Tiago Santos, Patrícia Pereira

¹CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. carlacruz@fcsaude.ubi.pt.

|July 27, 2017

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This study developed a novel affinity chromatography support for isolating G-quadruplex (G4) DNA structures. The new method selectively binds and separates parallel G4s, aiding in the discovery of new G4 binders and applications.

Area of Science:

Biochemistry
Molecular Biology
Chromatography

Background:

G-quadruplex (G4) structures play crucial roles in biological processes like telomere maintenance, gene regulation, and DNA replication.
Selective isolation of G4s is vital for identifying new binders and distinguishing them from duplex DNA.
Affinity chromatography offers a powerful tool for the selective separation and purification of nucleic acid structures.

Purpose of the Study:

To develop and characterize a novel affinity chromatographic support for the selective isolation of G-quadruplex (G4) DNA.
To evaluate the support's ability to separate G4 structures from other DNA forms and identify specific G4 binding preferences.
To demonstrate the utility of the support in isolating G4-forming DNA from complex biological samples.

Main Methods:

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Preparation of an affinity chromatographic support by immobilizing a naphthalene amine G4 binder onto epoxy-activated Sepharose CL-6B using a spacer arm.
Characterization of the support using HR-MAS spectroscopy.
Isolation of supercoiled plasmid isoforms (pVAX1-LacZ and pVAX1-G4) from native samples and bacterial lysates using an ionic gradient (NaCl).
Evaluation of retention times for various G4-forming DNA sequences (c-MYC, c-kit1, c-kit2, telomeric, thrombin aptamer, etc.).
Confirmation of parallel G4 topology in transcripts using circular dichroism (CD) analysis.

Main Results:

The developed affinity support successfully isolated supercoiled plasmid isoforms from native and bacterial lysate samples.
The support demonstrated selectivity, particularly for parallel c-MYC and c-kit1 G-quadruplex structures.
Retention times of various G4-forming sequences were evaluated, indicating differential binding affinities.
In vitro transcription and CD analysis confirmed the parallel G4 topology of specific RNA molecules.

Conclusions:

The novel affinity chromatography support is effective for the selective isolation and purification of G-quadruplex DNA.
The support exhibits selectivity for parallel G4 structures, offering a valuable tool for G4 research.
This method facilitates the discovery of new G4 binders and aids in the separation of G4s from complex biological mixtures.