Strategies for the efficient synthesis of stimuli-responsive ss-DNA bioconjugates for capture and transfection of plasmid DNA

Abstract

The paper describes the preparation of a stimuli-responsive single stranded DNA-bioconjugate and its use for the capture and transient transfection of plasmid DNA. The two segments of the bioconjugate consisted of a thermo-responsive poly-( N-isopropylacrylamide) oligomer (mass average <4000 g/mol, polydispersity <1.3, critical solution temperature in pure water approximately 33 °C) and a homo-pyrimidine oligonucleotide sequence (ss 5′–(CTT) n –3′). The oligomer was produced by chain transfer polymerization in the presence of a suitable chain transfer agent and carried an O-alkylhydroxylamine, respectively, a hydrazide end group. Direct chemical ligation was possible with the oxidized form of the DNA oligonucleotide bearing an additional riboU at the 3′-end. With this single step reaction similar DNA coupling rates (up to 80%) could be achieved as by an established protocol based on the use of a vinyl sulphone-activated oligomer with a thiol-modified oligonucleotide. The bioconjugate was able to selectively extract double stranded plasmid DNA bearing a complementary base sequence from bacterial lysate (recovery yields between 70% and 80%, purity >99%) by triple helix affinity interaction/precipitation. The complex could be directly transfected into mammalian cells (transient transfection, Lipofectamine protocol) with success rates similar or superior to those obtained with pure plasmid DNA obtained by conventional plasmid preparation kits.