S-Alkyl-Phosphorothioate Modifications Reduce Thermal and Structural Stability of DNA Duplexes

While phosphorothioate (PS) oligonucleotides are usually used in therapeutic applications, they also offer the cheapest and synthetically most straightforward route to introduce hydrophobic modifications for applications in structural DNA nanotechnology and biophysics. For this, the sulfur atom is S-alkylated with alkyl iodides, enabling a hydrophobically tunable interface of DNA nanostructures with lipid bilayers. While longer and more alkyls per helical turn should lead to stronger interactions with lipid membranes, we found that excessive S-alkylations strongly inhibit hybridization of oligonucleotides to their complementary strands and decrease their melting temperature, despite a reduction in electrostatic repulsion between the two strands. Moreover, both the type and placement of alkyl modifications influence the melting temperature. Atomistic molecular dynamics simulations reveal two complementary mechanisms that explain the experimental findings. First, S-alkylated oligonucleotides are more compact and less dynamic than unmodified ones, likely inhibiting their ability to hybridize to their complementary strands. Second, S-alkyls in double-stranded DNA promote defect formation due to alkyl modifications having hydrophobic interactions with other alkyl groups and nucleobases, therefore reducing the thermal and structural stability of alkylated DNA duplexes. This study serves as a practical guide for tuning hydrophobicity while maintaining structural stability in membrane-interfacing DNA nanostructures.