The amount of bolasomes for each compound required to fully bind 25-mer phosphorothioate gapmer oligonucleotide was determined using an unsymmetrical cyanine dye-exclusion assay.50 (link) The fluorescent single-stranded nucleic acid dye, Oligreen (Thermo Fisher Scientific, Wilmington, DE, USA), does not effectively bind to single-stranded phosphorothioate bases following cationic complexation, leading to a large decrease in fluorescence emission intensity compared with free oligonucleotide. Binding was measured by exclusion of the dye from oligonucleotide binding through a stepwise mixing of bolasomes into triplicate MES-buffered 2 µg/mL gapmer ASO solutions in black 96-well optical bottom microplates (Nunc; Thermo Fisher Scientific, Wilmington, DE, USA). Following a 20 minute incubation to stabilize the fluorescent signals, readings (485 nm excitation, 520 nm emission; 20 nm bandwidth) were taken and complexation curves for each experiment created.