*3.6. Fluorescence Quenching Experiments*

The dyes in the vicinity of micellar arrangements can displace a complex physicochemical and photophysical behavior. Some studies evidenced that the fluorescence intensity of fluorescein derivatives may depend not only on the pH value [45], but also on the complicated intermolecular, hydrophobic/electrostatic interactions between the fluorophore, and the lipids in the microenvironment. It is important to gain insights into these non-specific interactions of the LOCs with the dyes that are widely used to detect intracellular localization of NA-based constructs. The fluorescence quenching of some dyes can be induced by dynamic (or collisional), static, or combined dynamic and static complex mechanisms. Self-quenching and fluorescence resonance energy transfer (FRET) can often occur, due to molecular interactions between the molecules of fluorophores themselves in proximity to each other [46].

To explore if self-assembly affects the fluorescence of 6-carboxyfluorescein (FAM) residue combined with DOCs, we investigated fluorescence intensities (FI) and absorption spectra of FAM-labeled oligomers with dodecyl groups. Dodecyl-free oligonucleotide FAM-17 served as a control. The obtained results evidence for 86–87% fluorescence quenching of FAM-D-17 under the conditions of micelle formation in TAM buffer, in comparison with FI value in TA buffer lacking Mg2<sup>+</sup>, where 17-mer DOC did not reach CAC at the used concentration (Figure 6). At the same time, in the case of dye-labeled control oligonucleotide FAM-17- , the addition of Mg2<sup>+</sup> did not affect the fluorescence intensity (Figure 6).

**Figure 6.** Fluorescence intensity values of 1.5 μM fluorescently labeled FAM-17- , FAM-17-D, FAM-D-17, and D-17-FAM oligomers in TAM (black columns) or TA (grey columns) buffer conditions. For excitation and emission scanning parameters, see Section 2.9. We hypothesized that during the FAM-D-17 self-assembly, FAM residues are encapsulated into the micelle core, thereby interacting with each other in close proximity and providing significant fluorescence quenching. For instance, fluorescence from Oregon green residues in dye-conjugated titin molecules quenched in the native folded state of the protein due to the proximity of dye residues [47].

Further, we compared the FI values of FAM-17-D and D-17-FAM conjugates in the presence/absence of magnesium ions. These DOCs bear dodecyl chains and the FAM residue at the opposite ends of the 17-mer oligonucleotide, which is assumed to exclude dye encapsulation in the micellar inner core. We supposed that if interactions inside the core cause the fluorescence decrease, these conjugates will demonstrate the smaller quenching degree. Interestingly, the fluorescence intensity of the D-17-FAM, as expected, was quenched less (by 67–72%), while the FI value of the other conjugate FAM-17-D with 5- -terminal dye residue surprisingly decreased by 89–91%. We attribute this difference to the existence of multiple mechanisms of molecular interactions involved in fluorescence quenching. Fluorescence of fluorescein and its derivatives are extremely sensitive for the quenching, even to the type of the binding linker [48]. An earlier finding of fluorescence quenching of 6-carboxyfluorescein in liposomes was reported decades ago [49]. The authors concluded that dimerization of the dye and energy transfer to nonfluorescent dimers made a major contribution to the mechanisms of this concentration quenching phenomenon. The absorbance spectra of FAM-D-17, FAM-17-D, and D-17-FAM DOCs also contain shoulders at about 468 nm (Figure S21). Furthermore, these spectra show differences in absorbance of the conjugates in solutions with or without Mg2<sup>+</sup> (Figure S21), so a possibility of ground-state complex formation also cannot be excluded. It has been reported that Mg2<sup>+</sup> ions up to 15 mM do not cause appreciable fluorescence quenching of 5- -fluorescein-labeled DNA oligonucleotides [50]. On the other hand, 5-FAM and TAMRA-modified oligonucleotides significant fluorescence quenching during hybridization with a complementary strand is caused by photoinduced electron transfer between the fluorophore and nucleotide base [51]. It has been revealed that guanine bases can strongly quench the fluorescence of the dyes mentioned above. The presence of two guanines at 5- -end of the FAM-D-17 and FAM-17-D DOCs can add one more step to complex molecular interactions of quenching mechanisms. These underlying diverse and probe-dependent mechanisms are still exactly unknown and require careful use of fluorophores in LOC-based systems.
