In this study, we consider that the decrease in the transfection activity of polycations in vivo, compared with that in vitro, results from their polyion complex formation. Namely, owing to cross-linking between polycations and plasmid DNAs (pDNAs), the disadvantage of in vivo gene delivery mainly stems from the difficulty in controlling the properties of the resulting polyion complex at the nanoscale size. To avoid the cross-linking by polycations, we have establish the concept of "mono-ion complex" formation between pDNA and a monocationic biocompatible polymer. Here we have synthesized alkylimidazolium end-modified poly(ethylene glycol), that is, R-Im-PEG, and have tuned the electrostatic interaction between the resulting alkylimidazolium group and the phosphate group of pDNA by the length of the alkyl chain to achieve "mono-ion complex" formation with pDNA for in vivo gene delivery. Instead of a polyion complex, our original concept of the "mono-ion complex" consisting of the Bu-Im-PEG and pDNA is expected to offer unique tools to break through the barriers of in vivo gene delivery. As well as the field of gene delivery, this study is considered to have exploded the common sense that it is impossible to form not a polyion complex but a "mono-ion complex" under aqueous conditions for all fields of the modification of biomacromolecules.