Fluorescence Spectroscopic Analysis of β -PNA Interaction on Bi-

Since the complementary nucleobase interaction motif GTC/GAC showed the most pro-nounced interaction in solution, dimerization of β-PNA strands on lipid bilayers were investigated with the corresponding myristyl-modified β-PNA strands 85/87+89. Anal-ysis was performed via FRET with the same donor-acceptor-pair used in solution, NBD and TAMRA, which only showed slight shifts in presence of lipid bilayers compared to the excitation and emission spectra in solution(Figure 5.24). First, time-resolved emis-sion spectra were recorded to assess how fast theβ-PNA interaction occurs. For that, the NBD-labelled and the TAMRA-labelled β-peptides were sequentially added to previously prepared vesicle suspensions while the NBD fluorescence was monitored at 530 nm.

Frel / %

0 20 40 60 80 100

λ / nm

300 400 500 600 700

TAMRA ex TAMRA em NBD ex NBD em

b)

Figure 5.24. Normalized excitation and emission spectra of NBD and TAMRA measured at pH 7.5 in vesicle suspensions with 10 mM TRIS-HCl buffer.

When the complementary β-PNA strands were mixed in DOPC vesicle suspensions, a precipitate was formed rapidly even when the β-peptide and lipid concentrations were highly diluted. It was assumed that the precipitate was formed due to β-PNA dimer formation between β-PNA strands located on different vesicles surfaces (Figure 5.25(a)).

Therefore, the negatively charged lipid DOPS was added (20 mol%) to hinder the dimer-ization between different vesicles by electrostatic repulsion. Although the measurements with DOPC/DOPS vesicles showed no precipitation, there was also no detectable FRET (Figure 5.26). This could be attributed to the electrostatic repulsion also inhibiting nucle-obase pairing. These findings also imply that interaction between β-PNA strands located on different membranes might be favored over interaction betweenβ-PNA strands located on the same lipid bilayer due to the trilateral structure of the 14-helix. In combination

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5.5. β-PNA Interaction on Bilayer Surfaces with the membrane curvature of the vesicle, the trilateral geometry might impede dimer formation of β-PNA strands located on the same bilayer surface because the nucleobases are misaligned to form the required hydrogen bonds (Figure 5.25(b)). Another expla-nation could be that the β-PNA strands are able to interact while located on the same vesicle, even though the curvature is unfavorable for the interaction, rupturing the vesicle membrane in the process due to tensions resulting from bending the membrane. However, further studies are required to substantiate the described hypotheses.

Figure 5.25. Schematic illustration of the proposed favored interaction between β-PNA units located on different vesicles (a) and the angular constraints impeding interaction between β-PNA units located on the same vesicle (b).

Bicelles were chosen for further measurements because they offer a planar membrane model system due to their disk-like shape.^[61,76] Bicelles with a q_DMPC/DHPC-ratio of 2 were prepared from the long-chain phospholipid DMPC and the short-chain phospholipid DHPC with 5 mM TRIS-HCl buffer (pH 7.5) according to a protocol described in Sec-tion 8.4.2.^[78] To keep the bicelles stable, they were stored on ice prior to measurements and diluted shortly before to yield a bicelle concentration of 10 mM. Then the labelled β -peptides were added to yield a concentration of 0.5 µM respectively. Time-resolved NBD emission measurements revealed that for the complementary β-PNA strands87+89, the NBD fluorescence of89decreased quickly upon addition of the TAMRA-labelled87 (Fig-ure 5.27(a)). In contrast, the same meas(Fig-urements with91showed a lower decrease of NBD fluorescence intensity after87 addition (Figure 5.27(b)). These findings indicate that the

5. Monofacial β-Peptide Nucleic Acids

Figure 5.26. Time-resolved NBD emission of89(GAC) and91(no Nb) (added at point A) with or without 87(GTC) (added at point B) with a concentration of 0.5 µM each, measured in DOPC-LUV suspensions (20 mol% DOPS) with 10 mM TRIS-HCl buffer at pH 7.5.

myristyl-modifiedβ-PNA strands interact while bound to lipid bilayers and that the dimer formation is a rapid process. Additionally, measurements of the NBD-labelled β-peptides 89 and 91 with and without bicelles showed an increase of NBD fluorescence when they were added to bicelles in contrast to buffer or DHPC alone demonstrating rapid binding to the lipid bilayers.

F / a.u.

Figure 5.27. Time-resolved NBD emission of89(a) and91(b) (added at point A) with or without 87 (added at point B) 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 ℃.

Comparing the emission spectra of89before and after addition of the TAMRA-labelled 87 also showed that the decrease of NBD fluorescence intensity was the result of FRET and not any other effect, since it was associated with increased TAMRA fluorescence

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5.5. β-PNA Interaction on Bilayer Surfaces (Figure 5.28(a)). As expected, for the negative control 91 no significant FRET was observable because neither a significant decrease of NBD fluorescence nor increase of TAMRA-fluorescence was observed (Figure 5.28(b)).

Frel / %

0 20 40 60 80 100 120

λ / nm

500 520 540 560 580 600 620 640

a) ₈₉₊₈₇

89

Frel / %

0 20 40 60 80 100 120

λ / nm

500 520 540 560 580 600 620 640 91+87 91 b)

Figure 5.28. Emission spectra of 89 (a, GAC) and 91 (b, no Nb) with or without 87 (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 ℃.

Moreover, time-resolved NBD emission measurements were repeated with TAMRA-labelled82, which is the methyl-modified soluble counterpart to87, to investigate whether myristyl-modified 85 attached to lipid bilayers would interact with 83 in solution. The decrease of NBD fluorescence upon addition of the TAMRA-labelled β-peptide occurred at a much slower rate for 89(Figure 5.29(a)). In the case of91 even no statistical FRET was observed, since the NBD fluorescence intensity showed no significant difference to the measurement without addition of 82 (Figure 5.29(b)).

Consequently, also the emission spectra of89and 91showed a much lower influence of 82 on the NBD fluorescence intensity as well as a reduced TAMRA fluorescence intensity (Figure 5.30). While residual FRET can be observed for89+82, no FRET occurs in case of 91+82

Apparently, the attachment of both interaction partners to the lipid bilayer and the resulting alignment on a two-dimensional plane seems to greatly facilitate duplex forma-tion. In contrast, the interaction is greatly diminished when the preorganization on the membrane surface is omitted for one interaction partner because the chance of the solu-ble β-peptide 82 approaching lipid-bound β-peptide 91 in the right orientation is much lower. Also these findings contradict the previously described hypothesis that preferen-tial binding occurs between β-PNA strands located on different lipid bilayer surfaces and corroborates that duplex formation mainly occurs between β-PNA strands located on the

5. Monofacial β-Peptide Nucleic Acids

0 200 400 600 800 1,000 1,200

89+82

0 200 400 600 800 1,000 1,200

91+82 91

b) _{A B}

Figure 5.29. Time-resolved NBD emission of89(a) and91(b) (added at point A) with or without 82 (added at point B) 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 ℃.

same lipid bilayer surface. Nonetheless, FRET could be detected between 89 and 82 indicating that dimer formation also occurs when one β-PNA strand is attached to lipid bilayers and the other β-PNA strand is in solution.

Frel / %

500 520 540 560 580 600 620 640 89+82

500 520 540 560 580 600 620 640 91+82 91