Nucleic acids containing guanine-rich sequences have the potential to form G-quadruplexes. These higher-order structures have emerged as attractive targets primarily due to their overrepresentation in genomic regions directly participating in regulatory roles. G-quadruplex structures are composed of stacked G-quartets, each consisting of four guanines arranged in a cyclic planar Hoogsteen hydrogen-bonded array stabilized by monovalent cations, held together through negatively charged loops. G-quadruplex structures vary greatly in topology. Traditionally, the design of G-quadruplex binding ligands focused primarily on nucleic acid sequence specificity, our strategy shifts towards structure specificity, which we look to achieve by generating water-soluble molecularly imprinted polymer (MIP) biomimetic structures. Unlike conventional methods used for MIP preparation, we propose the introduction of G-quadruplex binding domains within synthetic polymers by use of quadruplex-binding ligands as cross-linkers for nucleic acid binding polymers. Polyethyleneimine (PEI) is a cationic polymer, known for its high affinity towards nucleic acids, that forms polyplexes via electrostatic interactions, is cross-linkable, water-soluble and as a result has been selected as the imprinting polymer. Our previous investigations demonstrate that the high charge density of PEI induces conformational change to G-quadruplex structures upon binding. To maintain template topology, the charge density of PEI has been reduced. PEI (10 kDa) was acetylated with increasing amounts of acetic anhydride. The degree of acetylation of primary and secondary amines was determined by a copper (II) UV spectrophotometric assay and NMR spectroscopy. Native PEI and PEI partially acetylated at 18, 26 and 30% was subsequently cross-linked using meso-Tetra(N-carboxymethyl-4-pyridyl)porphine tetrabromide (mTCMePyP) via EDC/sulfo-NHS carbodiimide coupling. Cationic porphyrins are well-known, non-specific, G-quadruplex binding ligands that contain large planar aromatic surfaces capable of π-stacking with G-quartets and cationic charges that interact electrostatically with polyanionic nucleic acids. meso-Tetra(4-pyridyl)porphine (mTPyP) was synthesized using a combination of Lindsey and Adler-Longo methods. mTCMePyP was subsequently prepared via N-alkylation, yielding the quaternized tetracarboxyl-functionalized cationic pyridinium porphyrin cross-linker.

Structure-based design of molecularly imprinted polymers for G-quadruplex nucleic acids

255th National Meeting (2018)

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