**1. Introduction**

Synthetic oligonucleotide conjugates with lipophilic groups are in the focus of considerable attention as nucleic acid (NA)-based biological tools in a wide range of fields of biotechnology and biomedicine [1,2]. Lipid-oligonucleotide conjugates (LOCs) are particularly interesting as delivery vehicles, which bring therapeutic oligonucleotides (e.g., antisense oligomers or siRNA) to their intracellular targets [3–6]. Negatively charged native oligonucleotides show poor cell penetration [5,6]. Their conjugation with lipophilic moieties can improve cellular uptake by providing an additional anchor for membrane binding [7–11].

Due to their amphipathic nature, oligonucleotide conjugates with lipophilic groups can self-assemble in aqueous solutions and form micelles [10,12–18] and their aggregates [10,11,14], vesicular assemblies [19,20], and more complex self-aggregation structures [2,11,21]. Generally, these structures are nearly spherical in shape [10,15,16,20,22,23]. The size and the shape of self-assembling structures may greatly depend on

experimental conditions: ionic strength, pH, and temperature; the type of lipophilic group; the length and nucleotide sequence of an oligomer in the LOC [2,17].

Cargo systems can be based as well on the attachment of lipophilic groups directly to a specific antisense oligonucleotide [10,24,25], to one of the siRNA strands [11,21,26,27] or more complex delivery formulations [13,23,28,29]. Furthermore, LOCs suit for the construction of oligonucleotide delivery system based on the liposomal-type spherical nucleic acids (SNA) [30–32]. The hydrophobic micellar core of LOC particles also serves as a carrier for extremely poor soluble pharmaceuticals [14,23,29] and fluorescent dyes [13,19].

Substantial characteristics of lipid-oligonucleotide conjugates include good biocompatibility and low toxicity [10,11,33]. LOCs improve pharmacokinetics and biodistribution of antisense oligomers (ASO) compared with native unconjugated oligonucleotides [4]. This advance is generally attributed to enhanced LOC binding to serum proteins. The association of the LOCs with one or more serum proteins (albumin, etc.) is also believed to participate in their cellular uptake mechanisms [4]. Albumin is the major blood protein that binds and transports numerous endogenous and exogenous substances, including fatty acids and other poor water-soluble compounds [34]. Lipid-oligonucleotide conjugates can easily bind as well to hydrophobic sites of the albumin [5,22]. It appears to affect their circulation half-life and bioavailability, e.g., for intravenous administration of LOC-based pharmaceuticals. Modifications of the oligonucleotide backbone (phosphorothioate (PS), morpholino, peptide nucleic acids, 2- -*O*-methyl, 2- -fluoro, and others) combined with lipophilic group enhance nuclease resistance of oligonucleotides, thus increasing their in vivo circulation lifetime [4–6].

So far, cholesterol is one of the most investigated lipophilic groups conjugated with oligonucleotides to improve cellular uptake [4,5,8]. Other promising modifications of this kind are fatty acids and lipid chains [6]. For example, Imetelstat (GRN163L), a palmitoyl-tethered thio-phosphoramidate oligomer, provides telomerase inhibition in the treatment of myeloproliferative disorders or neoplasms [6,24]. An interesting type of conjugates is represented by LOCs with "like-a-brush" lipid moieties attached to the oligonucleotide backbone [10,14,20,35]. Conjugation in such a manner gives the rise of hydrophobicity of the LOC lipophilic segment, in contrast to the consecutive linear combination. Earlier, we designed a non-nucleoside monomer to incorporate dodecyl groups into the oligonucleotides and synthesized dodecyl-containing conjugates with hydrophobicity comparable to that of corresponding cholesterol derivatives [36]. It was recently demonstrated that three dodecyl oligonucleotide conjugates (DOCs) are efficient and non-toxic transport molecules for ASO delivery into the A549 and HEK293 cells, which provide the transfection efficacy comparable to that of Lipofectamine 2000 [33].

In the present work, we report on self-assembly features of amphiphilic "like-a-brush" oligonucleotide conjugates functionalized at 5 and 3- -ends with three dodecyl groups [36]. These DOCs were studied for their abilities to form micellar structures and to penetrate the HepG2 tumor cells. In vitro investigation of self-assembling features of DOCs by dynamic light scattering (DLS), electrophoretic mobility shift assay (EMSA), fluorescence spectroscopy, transmission electron (TEM) and atomic force (AFM) microscopies, flow cytometry, and confocal microscopy substantially extended current knowledge on micelle-like structures of "like-a-brush" lipophilic oligonucleotide conjugates. The obtained results establish the potential of DOCs as nucleic acid delivery tools.
