C 3-symmetric opioid scaffolds are pH-responsive DNA condensation agents.

Natasha Mcstay,Alan Coughlan,Nicholas Gathergood,Vickie Mckee,Attilio Cafolla,Zara Molphy,Andrew Kellett

doi:10.1093/nar/gkw1097

Natasha Mcstay, Alan Coughlan + Show 5 more

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https://doi.org/10.1093/nar/gkw1097

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Abstract

Herein we report the synthesis of tripodal C3-symmetric opioid scaffolds as high-affinity condensation agents of duplex DNA. Condensation was achieved on both supercoiled and canonical B-DNA structures and identified by agarose electrophoresis, viscosity, turbidity and atomic force microscopy (AFM) measurements. Structurally, the requirement of a tris-opioid scaffold for condensation is demonstrated as both di- (C2-symmetric) and mono-substituted (C1-symmetric) mesitylene-linked opioid derivatives poorly coordinate dsDNA. Condensation, observed by toroidal and globule AFM aggregation, arises from surface-binding ionic interactions between protonated, cationic, tertiary amine groups on the opioid skeleton and the phosphate nucleic acid backbone. Indeed, by converting the 6-hydroxyl group of C3-morphine (MC3) to methoxy substituents in C3-heterocodeine (HC3) and C3-oripavine (OC3) molecules, dsDNA compaction is retained thus negating the possibility of phosphate—hydroxyl surface-binding. Tripodal opioid condensation was identified as pH dependent and strongly influenced by ionic strength with further evidence of cationic amine-phosphate backbone coordination arising from thermal melting analysis and circular dichroism spectroscopy, with compaction also witnessed on synthetic dsDNA co-polymers poly[d(A-T)2] and poly[d(G-C)2]. On-chip microfluidic analysis of DNA condensed by C3-agents provided concentration-dependent protection (inhibition) to site-selective excision by type II restriction enzymes: BamHI, HindIII, SalI and EcoRI, but not to the endonuclease DNase I.

Highlights

The search for new synthetic DNA recognition agents is an area of considerable research importance
Tripodal opioid condensation was identified as pH dependent and strongly influenced by ionic strength with further evidence of cationic amine-phosphate backbone coordination arising from thermal melting analysis and circular dichroism spectroscopy, with compaction witnessed on synthetic dsDNA co-polymers poly[d(A-T)2] and poly[d(G-C)2]
Since the biophysical evidence gathered on C3 opioid– DNA binding points toward a surface-binding coordination mode, we considered the possibility of the tertiary amine within the morphine D ring as a possible cationic site responsible for H-bonding and/or electrostatic binding to the phosphate backbone

Summary

Introduction

The search for new synthetic DNA recognition agents is an area of considerable research importance. Non-covalent recognition scaffolds may be further classified to the area of DNA where recognition occurs giving rise to groove binders [7,8,9], intercalators [9,10,11,12] (including threading intercalators [13]) and surface-binding compounds [14]. Within this last category, surface interacting compounds have been designed, and compared to naturally occurring protein binding domains containing, for example, cationic arginine (arginine fork [15]) or lysine (poly-L-lysine [16]) amino acid residues that facilitate their complexation to nucleic acids. An important consideration in the development of new DNA condensation agents is their relative inertness toward chemical reactivity (e.g. artificial nuclease) or secondary interactions (e.g. intercalation) that may interrupt or block efficient transport and incorporation of target nucleic acids into mammalian cells

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