Published online 29 November 2016 Nucleic Acids Research, 2017, Vol. 45, No. 2 527–540 doi: 10.1093/nar/gkw1097
C 3-symmetric opioid scaffolds are pH-responsive DNA condensation agents Natasha McStay1,†, Zara Molphy1,†, Alan Coughlan1, Attilio Cafolla2, Vickie McKee1, Nicholas Gathergood3,* and Andrew Kellett1,*
1School of Chemical Sciences and National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland, 2School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland and 3Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia
Received August 23, 2016; Revised October 11, 2016; Editorial Decision October 23, 2016; Accepted October 27, 2016
ABSTRACT INTRODUCTION
Herein we report the synthesis of tripodal C3- The search for new synthetic DNA recognition agents is an symmetric opioid scaffolds as high-affinity conden- area of considerable research importance. These agents can sation agents of duplex DNA. Condensation was be categorized, broadly, into two main areas: (i) those that achieved on both supercoiled and canonical B- covalently bind nucleic acids at electron rich sites on the DNA structures and identified by agarose elec- nucleobase (1–3) and (ii) those that bind in non-covalent fashion through hydrogen (H)-bonding, ionic or -stacking trophoresis, viscosity, turbidity and atomic force interactions (4–6). Accordingly, non-covalent recognition microscopy (AFM) measurements. Structurally, the scaffolds may be further classified to the area of DNA where requirement of a tris-opioid scaffold for conden- recognition occurs thus giving rise to groove binders (7–9), sation is demonstrated as both di- (C2-symmetric) intercalators (9–12) (including threading intercalators (13)) and mono-substituted (C1-symmetric) mesitylene- and surface-binding compounds (14). Within this last cat- linked opioid derivatives poorly coordinate dsDNA. egory, surface interacting compounds have been designed, Condensation, observed by toroidal and globule and compared to naturally occurring protein binding do- AFM aggregation, arises from surface-binding ionic mains containing, for example, cationic arginine (arginine interactions between protonated, cationic, tertiary fork (15)) or lysine (poly-L-lysine (16)) amino acid residues amine groups on the opioid skeleton and the phos- that facilitate their complexation to nucleic acids. Indeed, phate nucleic acid backbone. Indeed, by convert- a commonality between synthetic and naturally occurring surface binders is their ability to efficiently bind electron- ing the 6-hydroxyl group of C -morphine (MC3) 3 rich phosphate oxygen atoms located at the nucleic acid to methoxy substituents in C3-heterocodeine (HC3) backbone, principally through H-bonding and electrostatic and C3-oripavine (OC3) molecules, dsDNA com- interactions. Furthermore, electrostatic binding by polyca- paction is retained thus negating the possibility of tionic molecules can directly lead to a collapse in the B- phosphate––hydroxyl surface-binding. Tripodal opi- DNA conformation with resultant condensation or aggre- oid condensation was identified as pH dependent gation of tertiary DNA helical structures (17). Condensa- and strongly influenced by ionic strength with further tion agents have, in turn, found widespread utility in molec- evidence of cationic amine-phosphate backbone co- ular biology as transfection agents capable of mediating cel- ordination arising from thermal melting analysis and lular delivery of nucleic acids for ubiquitous therapeutic and circular dichroism spectroscopy, with compaction bioprocessing applications (18). Thus, an important con- also witnessed on synthetic dsDNA co-polymers sideration in the development of new DNA condensation agents is their relative inertness toward chemical reactivity poly[d(A-T) ] and poly[d(G-C) ]. On-chip microfluidic 2 2 (e.g. artificial nuclease) or secondary interactions (e.g. inter- analysis of DNA condensed by C3-agents provided calation) that may interrupt or block efficient transport and concentration-dependent protection (inhibition) to incorporation of target nucleic acids into mammalian cells. site-selective excision by type II restriction enzymes: While nucleic acid condensation can be achieved through BamHI, HindIII, SalI and EcoRI, but not to the endonu- mechanisms of neutral crowding, antisolvent precipitation clease DNase I. and liposomal packaging (amongst others), significant at-
*To whom correspondence should be addressed. Tel: +353 1 7005461; Email: [email protected] Correspondence may also be addressed to Nicholas Gathergood. Tel: +372 0 6204381; Email: [email protected] †These authors contributed equally to this work as the first authors.