*3.3. DLS Experiments*

The size of DOC micellar structures was characterized by DLS measurements. After preincubation in TAM buffer, the average hydrodynamic diameter (Dh) of D-13 particles was found to be 45.77 ± 13.03 nm (Table 1, Figure 2b), as compared to 5.43 ± 1.60 nm for 13-mer control oligonucleotide without dodecyl groups (Figure 2a). As shown in Figure 2, the assemblies of the DOCs have one peak of populations, which is shifted relative to the control oligonucleotide, and fairly low values of polydispersity indexes (PDI) (Table 1). The intensity distribution also showed one peak of populations with an increase in particle diameter compared to the number mean, which is usually typical for DLS results (Figure S9). Similar results were obtained for longer 17-mer DOC (D-17, Table 1, Figure 2d,e), with the addition of the fact that the Dh value of D-17 particles was less than that of D-13 (Table 1).


**Table 1.** The average hydrodynamic diameter (Dh) of the DOC micellar particles.

<sup>1</sup> as calculated by dynamic light scattering (DLS) measurements after 3 h incubation of 5 μM DOC in TAM buffer.

**Figure 2.** Size distributions of the control oligonucleotides 13 (**a**), 17 (**d**) compared with D-13 (**b**), D-13PG (**c**), D-17PG (**e**) and D-17PG (**f**) conjugates (after 3 h incubation at 5 μM in TAM buffer) as measured by DLS. The D-13PG dodecyl oligonucleotide conjugate with two uncharged PG groups assembled into slightly enlarged micellar particles compared to its DNA analog D-13 (Table 1, Figure 2c). Interestingly, we observed a considerable increase in the size of D-17PG self-assemblies (Table 1, Figure 2f). We attributed these results to the multiple micellar complexes that appeared in a short time in this case, or to more complex micellar structures. Such micellar aggregates of 129.96 ± 73.36 (PDI 0.216) nm in diameter were detected after 24 h incubation for D-17 conjugate.

The average hydrodynamic diameter of D-22PG particles was similar to that of D-13PG assemblies (Table 1). DOC particles formed in solution appear to be much larger than typical NA micelles (up to 10 nm), and their size lies in the range characteristic for vesicle or lamellar structures (up to 500 nm). The non-polar, uncharged PG modification in D-13PG and D-17PG may contribute to forming a more densely packed corona of the oligonucleotide chains around the larger micelle core compared to their deoxy counterparts D-13 and D-17. The results demonstrate no direct correlation between the size of micellar particles and the oligomer length of the DOCs due to the additional impact of the nucleotide sequence, which well correlates with our preliminary DLS studies. We measured hydrodynamic diameters of two additional 22-mer dodecyl conjugates with different heteronucleotide sequences. Micellar particles formed by 5- D-CTTGACTTTGGGGATTGTAG\*G\*G 3- (here D is a three-dodecyl unit and \* marks a phosphoryl guanidine modification) and 5- D-AATACTGCCATTTGTACTG\*C\*T 3 conjugates were of 12.15 <sup>±</sup> 0.10 and 11.82 <sup>±</sup> 1.66 nm correspondingly, while D-22PG formed the particles of increased diameter. The reason for these rather contradictory results remains unclear. We can hypothesize that two additionally studied conjugates can form secondary structures: the sequence of 5- D-CTTGACTTTGGGGATTGTAG\*G\*G 3 contains two G-tracts of 3 and 4 consecutive guanines, which suggests the ability of quadruplex formation, and the sequence of 5- D-AATACTGCCATTTGTACTG\*C\*T 3 may form hairpin and partial self-dimer (Figure S10a). Interestingly, the nucleotide sequence of D-17PG and D-17 also enables a formation of self-dimer of eight nucleotide bases (Figure S10b), which may contribute to the formation of larger particles and/or to accelerate their aggregation. Here, we only briefly discussed the effects of the oligonucleotide length and sequence of the LOC on the micelle size. This issue requires further studies on a wider series of oligonucleotides.
