*3.2. CAC Determination by Nile Red Encapsulation Assay*

Further testing by Nile Red encapsulation assay to determine the critical concentration of aggregation for D-17PG partially explained our observations. The Nile Red dye fluoresces intensively in the hydrophobic lipid environment but shows negligible fluorescence in an aqueous medium. Due to these properties, it is applied to determine the LOC's critical aggregation concentration (CAC) value [42]. The incubation of a certain amount of the dye with D-17PG at varying concentrations revealed an increase in the fluorescence intensity of Nile Red (Figure S7), which evidenced for its encapsulation in a non-polar microenvironment of micellar structure. Control oligonucleotide without dodecyl chains gave no significant increase in Nile Red fluorescence intensity (Figure S7). The CAC value of the DOC micellar particles was extremely sensitive to the presence of magnesium ions. In our work, the CAC of D-17PG conjugate in 15 mM MgCl2 (in TAM buffer) was above 1.2 μM (Figure S8a), while without magnesium ions, the CAC value (in TA buffer) was above 25 μM (Figure S8b).

We supposed that the self-assembly of the DOCs occurs with the formation of micellar particles composed of hydrophobic dodecyl inner core and hydrophilic oligonucleotide in the exterior shell corona. Considering this, Mg2<sup>+</sup> ions can impact the CAC value of DOC micelles by reducing electrostatic repulsion between the oligonucleotides' charged phosphate groups, stabilizing the micelle structure, and facilitating its formation.
