intact since sodium dithionite cannot penetrate the vesicle
membrane. A calculated 56% overall decrease of NBD
fluorescence for 100 nm vesicles was expected.[22] Indeed, in
our system we measured a fluorescence decrease of about
60% upon addition of sodium dithionite (Figure 2b). Effi-
cient lipid mixing, as indicated by FRET, was also observed
for vesicles pretreated with sodium dithionite for PNA
recognition motifs with both parallel and antiparallel orien-
tations (Figure 2b). This FRET effect requires the participa-
tion of the NBD fluorophores in the inner leaflet and thus
proves that not only the outer leaflets (hemifusion) but also
the inner leaflets had merged (full fusion). However, notably
the kinetics of total lipid mixing (Figure 2a) and lipid mixing
in the inner leaflet (Figure 2b) are different. It is conceivable
that in our model system the lipid mixing was at least partly
terminated at the stage of hemifusion such that only the outer
leaflets of the vesicles merged. Interestingly, the efficiency
and kinetics of lipid mixing in the inner leaflet proved to be
similar for both parallel and antiparallel orientations of PNA
duplex.
In order to support our hypothesis of partial hemifusion,
we performed content-mixing experiments (Figure 2c). The
fluorescence self-quenching of sulforhodamine B (SRB) at
high concentrations (> 10 mm) was used in content-mixing
experiments.[23] Two types of vesicles were prepared, one
containing the encapsulated SRB and another one not labeled
at all. Fusion of these vesicles was expected to lead to an
increase in fluorescence caused by dilution of SRB (Fig-
ure 2c). Nevertheless, the efficiency of content mixing was
determined to be very low, and no difference between parallel
and antiparallel orientations of the PNA duplex was detected.
Thus, based on both lipid-mixing and content-mixing experi-
ments, it was concluded that synthesized TMD/PNA SNARE
mimics lead to partial termination of the fusion process on the
hemifusion stage.
Figure 3. Total lipid mixing for vesicles containing PNA/peptide con-
structs. Unlabeled vesicles were mixed with NBD/Rh-labeled vesicles.
a) Parallel orientation of PNA oligomers in PNA/peptide constructs:
&
PNA3-SxTMD/PNA1-SybTMD ( ), PNA3-SxTMD/PNA1-SybTMD (")
after treatment of labeled vesicles with 5% egg lysophosphatidylcho-
line (LPC); PNA3-SxTMD/PNA1-SxTMD (&), PNA3-SxTMD/PNA1-
SxTMD (") after treatment of labeled vesicles with 5% LPC; b) anti-
parallel orientation of PNA oligomers in PNA/peptide constructs:
*
PNA2-SxTMD/PNA1-SybTMD ( ), PNA2-SxTMD/PNA1-SybTMD (3)
The lipid-mixing experiments were further used to inves-
tigate the potential influence and participation of the TMDs
in the fusion process. TMD/PNA-mediated vesicle fusion was
examined by comparison of PNA recognition complexes with
two identical (both Sx) and two different TMDs (Sx and Syb).
If the TMD units contribute to the fusion process, a difference
in the efficiency should be recognized. Lipid-mixing experi-
ments were performed for the parallel (PNA1-SxTMD or
PNA1-SybTMD with the complementary PNA3-SxTMD;
Figure 3a) and the antiparallel recognition (PNA1-SxTMD
or PNA1-SybTMD with the complementary PNA2-SxTMD;
Figure 3b). Interestingly, for both orientations lipid mixing
when identical Sx TMDs were employed was less efficient
than with the natural Sx Syb TMD pair. Apparently, the TMD
not only functions as a membrane anchor, but also contributes
to the fusion process.
Addition of lysophosphatidylcholine (LPC) to the vesicle
membrane leads to spontaneous positive membrane curva-
ture, which inhibits the formation of the hemifusion inter-
mediate, and therefore, prevents membrane fusion in cells
and model systems.[16,24,25] In a control experiment, TMD/
PNA-mediated vesicle fusion was carried out in the presence
of 5 mol% LPC (Figure 3). Indeed, no lipid mixing was
observed in the presence of LPC.
after treatment of labeled vesicles with 5% LPC; PNA2-SxTMD/PNA1-
*
SxTMD ( ), PNA2-SxTMD/PNA1-SxTMD(3) after treatment of labeled
vesicles with 5% LPC. For details, see the Supporting Information.
The experiments described above show that both parallel
and antiparallel strand alignment causes fusion, with the
parallel orientation being somewhat more efficient. To shed
further light on the role of strand recognition, we took
advantage of the different melting temperatures of parallel
and antiparallel strands.[17] Lipid-mixing experiments were
conducted at various temperatures, spanning the range
characteristic for PNA duplex formation to the formation of
single strands (Figure 4). At 258C, where PNA1/PNA2 and
PNA1/PNA3 in both orientations form stable duplexes, we
observed higher lipid-mixing efficiency for the parallel
orientation, suggesting that this orientation is preferable for
inducing vesicle fusion. The lipid-mixing efficiency for both
types of PNA orientations becomes equal at 408C, because
close to the melting temperature of Tm = 468C, the parallel-
orientated PNA duplex (PNA1/PNA3) is partially unpaired;
the stability of antiparallel-orientated PNA duplex (PNA1/
PNA2) remains almost intact (Tm = 708C). At 608C, lipid
mixing was detected only for the antiparallel orientation of
Angew. Chem. Int. Ed. 2011, 50, 8597 –8601
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Lygina, Antonina S.
