CD-Spectroscopic Analysis of β -PNA Interaction on Bilayer Surfaces 82

Figure 5.30. Emission spectra of 89 (a, GAC) and 91 (b, no Nb) with or without92 (GTC) with a concentration of 0.5 µM each, measured in DMPC/DHPC bicelle solutions with 5 mM TRIS-HCl buffer (pH 7.5) at 20 ℃.

5.5.3. CD-Spectroscopic Analysis of β -PNA Interaction on Bilayer Surfaces

To further validate β-PNA dimer formation on lipid bilayers, CD spectroscopy was per-formed with the myristyl-modified β-peptides in DMPC/DHPC bicelle solutions with 5 mM TRIS-HCl buffer (pH 7.5) at 20 ℃. Annealing of the samples was omitted because

82

5.5. β-PNA Interaction on Bilayer Surfaces the preceding CD spectroscopic analysis of β-PNA interaction in solution showed that an annealing procedure was not necessary for dimer formation. CD spectra were recorded for 85+88and compared to the spectra of the individualβ-peptides as well as the calculated average from the single strands (Figure 5.31). Just as the CD spectrum of the combined β-PNA strands80+83measured in solution, the CD spectrum of85+88showed a signif-icantly higher maximum of around 273 nm in comparison to the individual measurements or the calculated average demonstrating thatβ-PNA dimerization apparently occurs in a lipid-bilayer-bound state (Figure 5.31(a)).

Additionally, CD spectra were recorded for 85+83 to substantiate the results of the interaction between myristyl-modified 85 attached to lipid bilayers and 83 in solution.

Comparing the CD spectra of 85+83to the calculated average showed an increase of the maximum of around 273 nm although the increase was not as distinct as compared to the measurement with both β-PNA strands attached to lipid bilayer surfaces. These findings demonstrate that β-peptide interaction also occurred if only one interaction partner is located at the lipid bilayer surface.

[θ] / 103 deg∙cm2∙dmol-1

−400

−200 0 200 400

λ / nm

200 220 240 260 280 300

85+88 85+88 calc.

8388 a)

[θ] / 103 deg∙cm2∙dmol-1

−600

−400

−200 0 200 400

λ / nm

200 220 240 260 280 300

85+83 85+83 calc.

8385 b)

Figure 5.31. Combined, individual and calculated average CD spectra at 20 ℃ of85+88 (a, GTC/GAC) and 85+83 (b, GTC/GAC) measured in DMPC/DHPC bicelle solutions with 5 mM TRIS-HCl buffer (pH 7.5).

The CD spectra also revealed that the signal minima and maxima characteristic for the 14-helix correspond to the minima and maxima found for the β-peptides in solution, indicating that the β-PNA strands are not immersed into the lipid bilayer themselves but are still in the aqueous phase as intended. In the case of membrane constitutedβ-peptides, it has been found that the CD signals were shifted to shorter wavelengths.[107,109,145]

Temperature-dependent CD spectroscopy was not possible for the samples because a precipitate was formed upon heating. Therefore, no melting curves could be determined for the β-PNA/β-PNA dimer formation on lipid bilayer surfaces.

5. Monofacial β-Peptide Nucleic Acids

5.6. Lessons learned from the Monofacial β -PNA System

The monofacialβ-PNA model system described in this chapter gave an idea of the factors required for β-PNA/β-PNA interaction as well as β-PNA/membrane interaction. Dimer formation with the reduced minimal nucleobase interaction site could successfully be detected by fluorescence and CD spectroscopy showing the influence of sequence variety on the stability of duplex formation. Moreover, it was demonstrated that dimer formation is highly selective not tolerating any mismatched nucleobases in the sequences.

The interaction of β-PNA strands and lipid bilayers was shown by fluorescence spec-troscopy as well. The attachment of the β-peptides to the membrane surface appears to occur rapidly which is mediated by the hydrophobic myristyl modification. It was also shown by CD spectroscopic analysis that the myristyl-modified β-PNA strands are lo-cated above the lipid bilayer surface and not immersed into the membrane. Preliminary results of leakage assays with sulforhodamine B (SRB) and DLS measurements indicated that attachment of individual myristyl-modified β-peptides to DOPC-LUVs might cause the membranes to become porous but apparently do not cause the vesicles to rupture (Figure 5.32).

Frel / %

0 20 40 60 80 100

t / s

0 500 1,000 1,500

6960 91 90 H2O Blank a)

dh / nm

140 145 150 155 160 165

t / min

0 5 10 15 20

69 60 91 90 H2O

b)

Figure 5.32. Time-resolved SRB emission measurements (a) in vesicle leakage assays of the indicated β-peptides added after 300 s (ex=583 nm, em=583 nm) with DOPC-LUVs containing 20 mM SRB and DLS measurements (b) of the same β-peptides measured with DOPC-LUVs in 10 mM TRIS-HCl buffer (pH 7.5).

After demonstrating that the interaction of the β-peptides with lipid bilayer surfaces is feasible, duplex formation on bicelles could also be detected by fluorescence and CD spectroscopy when bothβ-PNA strands are attached to the lipid bilayer or when only one

84

5.6. Lessons learned from the Monofacial β-PNA System

β-peptide is located at the membrane surface. Additionally, because precipitate formation occured when complementary β-PNA strands were added, the reasons for this observa-tion should be investigated further being possibly caused by omnidirecobserva-tional aggregaobserva-tion of vesicles or fusion. Preliminary fluorescence microscopic studies of immobilized vesicles validated dimer formation on lipid bilayer surfaces because FRET between the donor-acceptor pair NBD and TAMRA could be detected (Figure 5.33). Moreover, the results indicated that DOPC-LUV coated with 89 tend to form aggregates which might be am-plified when the complementary β-PNA strand is present. However, further studies have to be performed to validate these results.

F / a.u.

0 20 40 60 80 100

λ / nm

450 500 550 600 650 700

89+87 89

c)

Figure 5.33. Composite fluorescence micrographs of immobilized DOPC-LUV coated with 89 before (a) and after (b) addition of 87 as well as representative fluorescence emission spectra of the two conditions (c). Measurements were performed in collaboration with the Steinem group in 10 mM TRIS-HCl buffer (pH 7.5).

The findings described in this chapter reveal the prerequisites for for expanding the design and establishing a β-PNA model system with multiple nucleobase interaction sites

5. Monofacial β-Peptide Nucleic Acids

as well as myristyl modifications for an aggregational network on lipid bilayer surfaces which mimics the membrane skeleton.

86

6. Monofacial β -PNA with