Despite recent advances, major hurdles still need to be cleared for widespread application of therapeutic antisense technologies. In particular, pharmacokinetic properties and efficient cellular uptake need to be improved through chemical derivatization or bioconjugation. The 2'-O-thioethylene nucleotide building block affords easy implementation into standard oligonucleotide synthesis protocols and was used to attach oligolysine chains to phosphodiester oligonucleotides by direct reaction with S-sulfonate protected peptides. Efficient gene silencing was induced in a cell culture model after transfection reagent-free application of the conjugates. A facile optimized procedure for generating oligonucleotide-peptide conjugates was established. The attachment of short basic peptides via a labile linker is sufficient to enhance membrane permeability of oligonucleotides and result in successful gene silencing.