*3.5. Electrophoretic Mobility Shift Assay*

The investigation of DOCs self-assembly by EMSA also showed results well-consistent with the DLS data. As shown in Figure 4a, the D-17PG sample forms extremely large structures (or aggregates), which cannot move along the gel and remain close to wells' bottoms (Figure 4a, Lanes 2–5). At the same time, micellar D-13PG particles demonstrated higher electrophoretic mobility than D-22PG (Figure 4a, Lanes 7 and 9 correspondingly). Analysis of the electrophoretic mobility of D-17PG at the indicated time of incubation demonstrated that low-mobility structures form in as little as 30 min (Figure 4a, Lane 2). All control oligonucleotides without dodecyl chains were characterized by significantly higher electrophoretic mobilities as compared to the conjugates (Figure 4a, Lanes 1, 6, 8).

**Figure 4.** Comparative electrophoretic mobilities of control oligonucleotides and dodecyl oligonucleotide conjugates. (**a**) 17 (Lane 1), D-17PG\* (Lanes 2–5), 13 (Lane 6), D-13PG (Lane 7), 22 (Lane 8), D-22PG (Lane 9) investigated by electrophoretic mobility shift assay (EMSA) in non-denaturing 6.5% PAAG after 3 h\* incubation in TAM buffer, at 25 ◦C; each sample contained 20 μM of the oligomer. (**b**) 13 (Lane 1), D-13 (Lane 2), D-13PG (Lane 3), 17 (Lane 4), D-17PG (Lane 5) investigated by non-denaturing 8% PAGE after 3 h incubation at 35 ◦C and additional no (−BSA) or adding (+BSA) of 45 μM bovine serum albumin (BSA without oligomers control lane is depicted on the right); each sample contained 25 μM of the oligomer in TAN buffer. Bands were visualized by Stains-All staining. **\*** This conjugate was loaded into the wells after the indicated time of incubation.

Next, we compared the affinity of DOCs and dodecyl-free oligonucleotides to serum albumin. Having confirmed the formation of DOC micelles by D-13, D-13PG, and D-17PG conjugates in conditions similar to physiologic (Figure 4b, (−BSA), Lanes 2,3,5), we added the bovine serum albumin (BSA) and allowed the reaction mixture to equilibrate for 30 min. After incubation with BSA (1.8 molar excess relative to the oligomer), the dodecyl-containing conjugates completely bound to albumin in contrast to the parent control oligonucleotides 13, 17 (Figure 4b, ((+BSA), Lanes 2,3,5).

Albumin possesses seven major binding sites for fatty acids with high and moderate affinity [44]. Interestingly, we have demonstrated that D-13PG DOC is highly associated with BSA even at the 0.1–0.15 molar excess (that is, the lack) of the protein (Figure S19). This corresponds to the interaction of one albumin molecule with approximately ten DOC molecules. These results point that in blood serum with high levels of albumin (about 0.5–0.6 mM), DOCs would be most probably entirely bound by this protein.

As expected, the EMSA (Figure 5) proved the complete association with the proteins either for FAM-17- /D-17PG duplex (Lanes 6,7) or FAM-D-17PG conjugate (Lanes 10,11) in the medium supplemented with BSA, as well as with 10% FBS. The FAM-17- /17 control duplex remained unbound in this assay (Lanes 2–3).

**Figure 5.** Comparative electrophoretic mobilities of 5 μM control duplex FAM-17- /17 (Lanes 1–4), dodecyl-containing duplex FAM-17- /D-17PG (Lanes 5–8) and the FAM-D-17PG conjugate (Lanes 9–12) investigated by EMSA in non-denaturing 8% PAAG after 2 h incubation at 37 ◦C in DMEM medium (depicted in red), DMEM supplemented with 30 μM BSA (depicted in green), DMEM supplemented with 10% fetal bovine serum (FBS) (depicted in blue). All medium conditions without oligomers control lanes are depicted on the right in corresponding colors. Indicated oligomers after incubation in PBS are depicted in black for additional controls. Bands were visualized after electrophoresis by Stains-All staining (**a**) and by recording the image after scanning with laser excitation at 488 nm (**b**). It is interesting to note that during the electrophoretic analysis of micellar assemblies of FAM-labeled DOCs, we observed significant fluorescence quenching of their bands, in contrast with the bands of non-conjugated control oligomers (Figure 5, Lanes 9, 12, Figure S20).
