Fluorescence-Spectroscopic Analysis of β-PNA Interaction

Since the results of temperature-dependent UV spectroscopy were inconclusive regarding β-PNA dimerization, Förster resonance energy transfer (FRET) measurements were conducted to investigate if β-PNA strands with complementary nucleobase motifs in-teract in solution. The fluorophores 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) and 5(6)-carboxytetramethylrhodamine (TAMRA) were chosen as a FRET donor-acceptor-pair since they have successfully been employed in FRET analysis of aggregating β-peptides before.^[145]As it has been described in Section 2.5, FRET can take place when the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor. As illus-trated in Figure 5.12, this condition is met by the chosen fluorophores since the emission spectrum of NBD coincides with the excitation spectrum of TAMRA.

Frel / %

0 20 40 60 80 100

λ / nm

300 400 500 600 700

TAMRA ex TAMRA em NBD ex NBD em

a)

Figure 5.12. Normalized excitation and emission spectra of NBD and TAMRA measured at pH 7.5 in 10 mM TRIS-HCl buffer.

Fluorescence measurements of the β-PNA were conducted as described in Sec-tion 8.3.7. While the concentration of the NBD-labelled donor β-peptide was kept constant at 4 µM, the mole fraction χ_A of the TAMRA-labelled acceptor β-peptide was varied from 0.0 to 0.5. To keep the total peptide concentration constant at 8 µM, the corresponding acetylated β-peptide was added. In addition to the complementary β-PNA strands 59/62+65 (TTA/TAA), 76/77+79 (GTA/TAC) and 80/82+84 (GTC/GAC), every possible mismatch combination59/62+79 (TTA/TAC), 59/62+84 (TTA/GAC), 76/77+65 (GTA/TAA), 76/77+84 (GTA/GAC), 80/82+65 (GTC/TAA) and 80/82+79 (GTC/TAC) was tested to investigate the sequence speci-ficity of the system. Furthermore, all TAMRA-labelled and acetylated β-PNA strands

5. Monofacial β-Peptide Nucleic Acids

were combined with the negative control 90 (no Nb). Before measurements, the required amounts of β-peptides were mixed in 10 mM TRIS-HCl buffer (pH 7.5) and annealed by incubation at 80 ℃ for 5 min followed by slow cooling to room temperature. Then, emission spectra were recorded in a wavelength range of 470 nm to 650 nm.

Since initial FRET measurements at room temperature utilizing a plate reader showed high variations of the spectra and did not indicateβ-peptide interactions, further measure-ments were performed at a lower temperature of 10 ℃ with a fluorescence spectrometer and a quartz cuvette (complete data sets are shown in Appendix A.6). The results for the complementary combinations 59/62+65 (TTA/TAA), 76/77+79 (GTA/TAC) and 80/82+84 (GTC/GAC) as well as one negative control 80/82+90 (GTC/no Nb) are shown in Figure 5.13. All complementary combinations show a more pronounced FRET with increasingχ_A of the TAMRA-labelledβ-PNA strand than the negative control mea-surements indicating that at 10 ℃ all complementary nucleobase motifs exhibit inter-action. Additionally, the higher the GC content in the sequences the more pronounced interaction could be observed, which was expected.

Since the fluorescence measurements at 10 ℃ showed promising results, they were repeated at 20 ℃. As it is illustrated in Figure 5.14, also at elevated temperature FRET was more pronounced for the complementary β-peptide combinations in comparison to the negative control, even though overall intensity was reduced. Therefore, it can be concluded that β-PNA strand dimerization still occured at 20 ℃ but was less stable than at 10 ℃.

All measurements were conducted in triplicates and the relative change of NBD fluores-cence intensity of theβ-peptide combinations at 530 nm (F/F₀) was then plotted against the increasing mole fraction of the TAMRA-labelled β-peptide species. In Figure 5.15 the results of the complementary β-PNA strands are shown. At 10 ℃ (Figure 5.15(a)), a decrease of the NBD fluorescence for all three complementary nucleobase motifs is observ-able with 80/82+84 (GTC/GAC) exhibiting the strongest decrease, while the decrease of fluorescence intensity is lower for 76/77+79 (GTA/TAC) and lowest for 59/62+65 (TTA/TAA). For the measurements at 20 ℃ (Figure 5.15(b)), the β-PNA strands show a similar behavior albeit less pronounced. These findings indicate that the stability of the β-PNA dimerization is highly affected by sequence composition and temperature.

Moreover, when the matching β-PNA combinations were compared to mismatching combinations, it became apparent that the mismatched β-peptides showed significantly reduced changes of NBD fluorescence intensity just like the negative control in contrast to the matching β-peptides. This is especially notable in case of 80/82+84 indicating that the mismatch combinations do not interact whereas the matching combination does

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5.4. β-PNA Interaction in Solution

500 520 540 560 580 600 620 640 TTA/TAA

500 520 540 560 580 600 620 640 GTA/TAC

500 520 540 560 580 600 620 640 GTC/GAC

500 520 540 560 580 600 620 640 GTC/no Nb

Figure 5.13. Emission spectra at 10 ℃ of the complementaryβ-PNA strands59/62+65 (TTA/TAA) (a),76/77+79(GTA/TAC) (b),80/82+84(GTC/GAC) (c) as well as a negative control measurement 80/82+90 (GTC/no Nb) (d) measured with the indicated χ_A of the TAMRA-labelledβ-PNA strand at pH 7.5 in 10 mM TRIS-HCl buffer.

5. Monofacial β-Peptide Nucleic Acids

500 520 540 560 580 600 620 640 TTA/TAA

500 520 540 560 580 600 620 640 GTA/TAC

500 520 540 560 580 600 620 640 GTC/GAC

500 520 540 560 580 600 620 640 GTC/no Nb

Figure 5.14. Emission spectra at 20 ℃ of the complementaryβ-PNA strands59/62+65 (TTA/TAA) (a),76/77+79(GTA/TAC) (b),80/82+84(GTC/GAC) (c) as well as a negative control measurement 80/82+90 (GTC/no Nb) (d) measured with the indicated χ_A of the TAMRA-labelledβ-PNA strand at pH 7.5 in 10 mM TRIS-HCl buffer.

70

5.4. β-PNA Interaction in Solution

Figure 5.15. Relative change of NBD fluorescence intensity (F/F₀) at 530 nm of theβ -PNA combinations 59/62+65 (TTA/TAA),76/77+79(GTA/TAC) and 80/82+84 (GTC/GAC) at 10 ℃ (a) and 20 ℃ (b) as a function of in-creasing χ_A of the TAMRA-labelled β-PNA strand measured at pH 7.5 in 10 mM TRIS-HCl buffer.

(Figure 5.16). These results show that the nucleobase interaction is highly specific and that no unspecific aggregation occurs.

F/F0

Figure 5.16. Relative change of NBD fluorescence intensity (F/F₀) at 530 nm of the matching and mismatchingβ-PNA combinations80/82+84(GTC/GAC), 80/82+90 (GTC/no Nb), 80/82+65 (GTC/TAA) and 80/82+79 (GTC/TAC) at 10 ℃ (a) and 20 ℃ (b) as a function of increasingχ_Aof the TAMRA-labelled β-PNA strand measured at pH 7.5 in 10 mM TRIS-HCl buffer.

In an attempt to quantify the stability of dimer formation, MST measurements were conducted to determine a k_D value for the matching β-PNA strands 58+66 (see Sec-tion 8.3.8 for experimental details). However, the FAM fluorophore showed unexpected variation in fluorescence, the results varied widely and no sigmoidal curve shape could

5. Monofacial β-Peptide Nucleic Acids

be obtained from the data. Therefore, no kD value could be determined. Repeating the MST assay with a different fluorophore, however, could give better results.