Folding monitored by UV-VIS and CD

First, the thermal stabilities and CD profiles of the ttel24-T₄G-quadruplex were compared to those of the modified sequence ttel24-L²₄. The UV-VIS based melting curves of the ttel24- T₄ sequence exhibit one single transition in KCl buffer atT_m= 54.8 °C, with no observable effect on the melting temperature when CuSO₄ is added. For the denaturation in Na⁺ containing solution, the melting temperature was observed to be quite low, withT_m= 28.2 °C, so all further experiments were conducted using KCl as the salt additive. It should be noted that for the strand ttel24-T₄, one DNA sample batch did not show a single transition but a broad denaturation profile with two transitions. The analytical RP-HPLC traces and the ESI(–) mass spectra were identical for the different stock solutions, so it has to be assumed that the observed differences arose from the sample preparation, like variations in buffer pH, or an unidentified contamination.

A denaturation profile with a double transition is observed for the modified sequence ttel24-L²₄, the overall melting transition is, how-ever, in the same range as the unmodified sequence ttel24-T₄. This observation suggests a more complicated melting behaviour with a de-viation from the simple two-state equilibrium between unfolded and

folded G-quadruplex.^[300] Moreover it also indicates potential forma-tion of higher-order structures or the presence of multiple similar G-quadruplex conformers.^[277]When the melting profile is fitted with a double-sigmoidal function, denaturation temperatures ofT_m= 32.7 °C and 50.8 °C for ttel24-L²₄can be calculated. When the denaturation experiments are repeated in the presence of CuSO₄, the melting pro-files change partially. While the high temperature transition is mainly unaffected, the low temperature transition shifts to higher values. Al-together, the profile now resembles more that of one single transition, rather than two separate ones. The available experimental data do not allow to determine if the high temperature transition corresponds to a separate G-quadruplex topology to which the Cu²⁺ ion cannot bind, or if it belongs to the same topology which is also responsible for the lower temperature transition.

Figure 7.13.:CD spectra for the G-quadruplexes a) ttel24-T4 and b) ttel24-L²4 upon addition of CuSO₄. 10 mmLiCaco pH 7.3, 100 mm KCl, 4.0 µmDNA, 0 or 4.0 µmCuSO₄.

Further insights into the nature of the Cu²⁺binding could be derived from the CD spectra. In case of the unmodified ttel24-T₄ sequence, the CD spectrum exhibits an intense positive band at 293 nm and a less intense negative one at 260 nm. The overall shape of the CD signal resembles that reported for the sequence ttel22 d[GGGG(TTGGGG)₃], measured in Na⁺ containing solution, and that for the (3+1) assembly of the human telomeric sequence discussed above. In contrast, the CD profile of ttel24-L²₄differs considerably, with two intense positive bands at 264 nm and 288 nm, respectively, and a smaller negative signal at

238 nm. This suggests a different folding topology for the modified strand and also potentially the presence of a mixture of different G-quadruplex conformers. Despite the differences between the CD spectra, both can be classified as ‘type 2’, according to Karsisiotis et al.^[279]

The profile is typically found for G-quadruplexes with three G-quartets, which stack in both a homopolar and a heteropolar fashion.

Figure 7.14.:a) Thermal denaturation curves (monitored at 295 nm) con-verted to the fraction folded values and b) CD spectra for the G-quadruplex ttel24-L²4 at three different strand concentra-tions. 10 mmLiCaco pH 7.3, 100 mmKCl, 1.0, 4.0, or 8.0 µm DNA.

To elucidate if higher order structures are present in the G-qua-druplex sample of the modified sequence ttel24-L²₄, concentration de-pendant UV melting and CD experiments were performed. For all three concentrations, namely 1.0, 4.0, and 8.0 µm, the denaturation profiles were superimposable and the CD spectra did also not change significantly, so the presence of higher-order structures like G-wires or stacked G-quadruplexes is unlikely. The co-existence of a mixture of different monomeric, unimolecular G-quadruplex topologies cannot be ruled out by these experiments, however.

Apart from the ttel24 sequence, two other NMR-derived solution state structures of G-quadruplexes with similar sequences compared to ttel24-T₄ have been deposited in the PDB data base, but are not yet published elsewhere. The two sequences are d[TG₃T₃G₃TTG₃T₃G₃] (PDB entry 2MFU)^[301] and d[G₃T₄G₃TG₃T₄G₃] (PDB 2MFT)^[302], respectively, and assemble into (2+2) antiparallel G-quadruplexes in

Figure 7.15.:Schematic G-quadruplex models for the sequences a) ttel24 in Na⁺solution;^[299]b) d[TG3T3G3TTG3T3G3] in Na⁺ solu-tion;^[301]c) d[G3T4G3TG3T4G3] in Na⁺solution;^[302]d) I–III:

examples for potential folding topologies of ttel24-L²4in K⁺ solution.

Na⁺ solution (see figure 7.15b and c). Although this can by no means be generalised, it seems that for these sequences, the long dT₄ sequence favours a diagonal loop, dT₃ lateral loops, and dT₂ propeller-type loops. That the dT₂ connection mostly forms propeller-type loops was also reported by Cheatham III and coworkers.^[30] Unfortunately, no spectroscopic data like CD spectra are available for the 2MFU or 2MFT G-quadruplexes, so it cannot be verified if the antiparallel (2+2) strand arrangement still gives rise to a ‘type 2’ CD spectrum as observed for ttel24-L²₄. The glycosidic bond orientations in the reported structures reveal, however, that the G-quartets do indeed show both homo- and heteropolarity stacking, so a CD spectrum similar to that found for the modified sequence seems very likely. Based on these structures, models for the modified sequence ttel24-L²₄ can be derived (see figure 7.15d I–III). All three potential topologies have one common structural feature:

The ligandL² modifications are not located on the same side of the G-quadruplex, but on opposite ones, meaning that the square-planar Cu²⁺(L²)₄ complex cannot be formed without changes in the strand orientation.