## 4 Conclusions  
While trying to dissect the process of interaction of DNA duplex structures with carrier surfaces in our study on GNPs, we should address in detail the question of which state of DNA, single- or double-stranded, has prevailing affinity for the surface of the material (Figure 9). Figure 9: Schematic representation of the competitive processes of formation of inter- and/or intramolecular structures by oligonucleotides R and FLn (where n = 0, 1, 2, or 4) and their association with GNPs at different molar ratios (the capacity of GNPs is denoted as k, m, z, l, and i) in a single-stranded, double-stranded, or internally structured conformation. The orange background highlights these processes involving R, the gray background indicates the processes involving FLn, the yellow color highlights the formation of the FLn–R duplex, and the green background shows processes involving the FLn–R duplex and GNPs. Index h denotes an internally structured (hairpin) conformation of oligonucleotides R or FLn. Dashed lines show some possible competitive processes. It would seem that the extension of an oligonucleotide via the introduction of the gold-binding L motif, especially when several such motifs are attached, should noticeably increase the capacity of GNPs for DNA molecules. By contrast, using an analogous series of homothymidylate motif-extended oligonucleotides showing statistically significantly weakened affinity for GNPs, we were able to demonstrate that the increased likelihood of intramolecular structures in ssDNA weakens its expected affinity for GNPs. Moreover, by means of the \({}^{32}\)P label introduced separately into each strand of interacting oligonucleotides, we were able to show for the first time that on the surface of GNPs, the DNA duplex only partially retains its duplex structure. Meanwhile, it is known [51] that \(\Delta\)G of association of the duplex DNA is \(-23.8\) kcal/mol, and \(\Delta\)G of adsorption of the same DNA duplex on GNPs is \(-6.8\) kcal/mol, i.e., energies of these processes are of the same order of magnitude and, hence, competition between them is likely. It is more likely that the affinity of ssDNA, that is, of its individual strands, determines the capacity of the gold surface for dsDNA molecules. In this context, the affinity of each complementary interacting strand is of paramount importance. Our study unambiguously shows that ssDNA has much more pronounced affinity for GNPs in comparison with its duplex or intramolecularly structured state. With all this, while examining the material balance of noncovalent interaction of a pair of complementary oligonucleotides with GNPs, a researcher should take into account a wide range of implementation of intermediate and final types of association of the NAs with each other and with the nanoparticle. Our findings suggest that the preservation of dsDNA structures when dsDNA is adsorbed on GNPs should be experimentally confirmed both in the case of SH-free and SH-containing oligonucleotides. Summing up, it must be noted that the scheme of GNP-DNA interaction proposed by us (Figure 9) indicates the complexity of the processes involved, and the role of each of them may be decisive in the rational design of carriers containing DNA duplexes. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app13127324/s1](https://www.mdpi.com/article/10.3390/app13127324/s1)]([https://www.mdpi.com/article/10.3390/app13127324/s1](https://www.mdpi.com/article/10.3390/app13127324/s1)), Table S1: Thermal denaturation temperature of dsDNA; Table S2: Thermal denaturation temperatures of the F-R duplex at different DNA concentrations; Figure S1: Possible secondary structures of (A) FL4, (B) FL2, and (C) R; Figure S2: Thermal denaturation curves of ssDNA in 4 mM Na\({}_{3}\)C\({}_{6}\)H\({}_{5}\)O\({}_{7}\).