Gene delivery has seen limited clinical success due to poor transfection efficiency or risk of carrier toxicity. Little understanding exists about the dynamic mechanical properties of DNA:​carrier complexes, which we hypothesize are critical for protection and release of DNA. Using optical tweezers, we investigated the DNA condensation behaviors of 19-mer poly-L-lysine (PLL), a histidine-lysine peptide, 25 kDa branched polyethylenimine (PEI), G2-triethylenetetramine conjugated gold nanoparticles (G2- TETA), and two triblock copolymers to identify the optimal force signature for efficient transfection.

Force-extension profiles indicate that PLL and HK peptides condense DNA, showing force plateaus. When free peptide is removed, the force plateau of HK complexes decreased, but hysteresis persisted, indicating that some HK remains bound. Upon changing the pH from 7.4 to 5, HK complexes recovered plateau forces, due to protonation of bound HK. This charge-regulated mechanical behavior is enhanced when the DNA:​HK complex is exposed to Zn²⁺, resulting in the formation of a mechanically stiff complex.

DNA:​PEI complexes showed transient force plateaus with a maximum of 35 pN. Shortening of contour length was observed for condensation with 5 nM PEI. 1 M NaCl destabilized DNA:​PEI complexes suggesting electrostatic interactions as the major force driving complexation. When 50 nM G2-TETA binds DNA, ~10 pN force plateaus appeared, disappeared, and contour length decreased despite pulling forces up to 50 pN. Neither 1 M NaCl nor 5 mg/mL heparin disrupted the complex. Contour length increased in 5% sodium dodecyl sulfate solution indicating that hydrophobic interactions play a major role in forming mechanically rigid condensates.

Both guanidinylated and base copolymers show maximal plateau behavior followed by reduction in contour length. Recovery of the extension for the DNA:​base copolymer complex is achieved by a combination of glutathione and either high salt or heparin. Conversely, high salt or heparin conditions alone are sufficient for destabilization of DNA:​guanidinylated copolymer. Thus, guanidinylation of the copolymer enhanced sensitivity to ionic environments.

Condensed DNA force profiles using different agents were unique regarding their condensation behaviors and responses to environmental changes. Regulation of these interaction forces between DNA and carriers during complex preparation and under physiological conditions will improve transfection efficiencies in vivo.