Báo cáo khoa học: Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide docx

Enhancement of intracellular concentration and biological activity of
PNA after conjugation with a cell-penetrating synthetic model peptide
Johannes Oehlke
1
, Gerd Wallukat
2
, Yvonne Wolf
1
, Angelika Ehrlich
1
, Burkhard Wiesner
1
, Hartmut Berger
1
and Michael Bienert
1
1
Institute of Molecular Pharmacology, Berlin, Germany;
2
Max Delbru
¨
ck Center for Molecular Medicine, Berlin, Germany
In order to evaluate the ability of the cell-penetrating
a-helical amphipathic model peptide KLALKLALKALK
AALKLA-NH
2
(MAP) to d eliver p eptide nucleic acids
(PNAs) into mammalian cells, MAP was covalently linked
to the 12-mer P NA 5¢-GGAGCAGGAAAG-3¢ directed
against t he mRNA of the nociceptin/orphanin FQ receptor.
The c ellular u ptake of both the naked PNA and its MAP-

conjugate was studied by means of capillary e lectrophoresis
combined with laser-induced fluorescence detection, confo-
cal laser scanning microscopy and fluorescence-activated cell
sorting. Incubation with the fluorescein-labelled PNA–pep-
tide con jugate led to three- and eightfold higher intrac ellular
concentrations in neonatal rat cardiomyocytes and CHO
cells, respectively, than found after exposure of the cells to
the naked PNA. Correspondingly, pretreatment of sponta-
neously-beating neonatal rat cardiomyocytes with the
PNA–peptide conjugate and the n aked PNA slowed down
the positive chronotropic effect elicited by the neuropeptide
nociceptin by 10- and twofold, respectively. The main rea-
sons for the higher bioavailability of the PNA–peptide
conjugate were found to be a more rapid cellular uptake in
combination with a lowered re-export and resistance against
influences of serum.
Keywords: cell-penetrating peptides; cellular uptake; PNA–
peptide conjugates.
The wider applic ation o f p eptide nucle ic acids (PNAs) [1] as
antisense agents a ppears to b e limited mainly by poor
cellular uptake [2,3]. Improved delivery into mammalian
cells and enhanced antisense a ctivity have be en achieved
after c ovalent c oupling of PNAs t o cell-penetrating peptides
(CPPs), which a re able to enter cells in a nonendocytic but
as yet unknown mode [3–8]. The structural requirements for
the delivery activity of peptides h ave been unclear until now.
In order to contribute to an elucidation of structure–
delivery a ctivity relationships we have previously investi-
gated the cellular uptake and biological activity of
CPP–phosphorothioate oligonucleotide conjugates using

the cell-penetrating amphipathic m odel peptide MAP
(KLALKLALKALKAALKLA-NH
2
) [9, 10] as the l ead
compound [11]. The value of the results of this study was
limited, however, by a h igh cell toxicity of t he phosphoro-
thioate o ligonucleotide–peptide conjugates. Therefore, in
the present study we evaluated the suitability of PNA to
serve as the cargo molecule in MAP-based structure–
delivery activity investigations. To this end we investigated
cellular uptake and biological activity of a 12-mer peptide
nucleic acid (5¢- GGAG CAGGA AAG -Lys -3¢; c ompound I;
Table 1) complementary to bases 12–23 of the translated
region of the nociceptin/orphanin FQ receptor, proven
previously to be sensitive to a ntisense attack s [ 12,13], a nd of
its conjugate with MAP (compound II; Table 1). For
assessing the cellular uptake, we developed a protocol based
on capillary electrophoresis with laser-induced fluorescence
detection (CE-LIF) providing absolute quantities of inter-
nalized PNA which was used supplementally with confocal
laser scanning microscopy (CLSM) and fluorescence-acti-
vated cell sorting (FACS).
Material and methods
General
Chemicals and reagents were purchased from Sigma
(Deisenhofen, Germany), Bachem (Heidelberg, Germany)
or PE Biosystems unless specified otherwise. Release of
lactate dehydrogenase was assessed by means of LDH-L
reagent from Sigma.
Synthesis of PNA and PNA–MAP conjugates

PNA oligomers were synthesized manually using the t-Boc
strategy [14]. The peptide segments of the conjugates were
synthesized by the solid phase method using standard Boc
chemistry [15], after which the PNA moiety was extended
from the N-terminus of the peptide by manual Boc cou pling
according to Christensen et al. [14]. To introduce the
Correspondence to J. Oehlke, Institute of Molecular Pharmacology,
Robert-Ro
¨
ssle-Str. 10, D-13125 Berlin, Germany.
Fax: + 49 30 94793 159, Tel.: + 49 30 94793 267,
E-mail:
Abbreviations: CE-LIF, capillary el ectrophoresis with laser-induced
fluorescence detection; CLSM, confocal laser scanning microscopy;
CM, spontaneously-beating n eonatal r at cardiom yocytes; C PP ,
cell-penetrating peptide; DPBSG, Dulbecco’s phosphate buffered
saline/glucose; FACS, flu orescence-activated cell sort ing;
Fluos, 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester; MAP,
model amphipathic peptide; PNA, peptide nucleic acid.
(Received 16 March 2004, revised 21 May 2004,
accepted 28 May 2004)
Eur. J. Biochem. 271, 3043–3049 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04236.x
fluorescent label, t he unprotected N-termini of the PNAs or
the PNA–MAP conjugates were reacted in dimethylform-
amide for 3 days at room temperature with 10 equivalents
of 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester
(Fluos; Boehringer, Mannheim, Germany). Purification
was carried out by semipreparative HPLC on Vydac C18
using a 250 · 8 mm column. MALDI-MS (Voyager-DE
STR BioSpectrometry Workstation MALDI-TOF; Persep-

tive Biosystems, Inc., Framingham, MA, USA) provided
the expected [M + H]
+
peaks (3878 and 5609 Da for I
and II, III, respectively).
Cell culture
CHO cells were cultured in 24-well plates (5 · 10
4
cells per
well) or for CLSM on 22 · 22 mm coverslips (2 · 10
4
cells)
at 37 °C in a humidified air environment containing 5%
CO
2
in Ham’s F-12 medium supplemented with
290 mgÆmL
)1
glutamine and 10% (w/v) fetal bovine serum.
Spontaneously-beating, neonatal rat cardiomyocytes (CM)
were obtained from ventricles of 1–2 day-old Sprague–
Dawley rats and cultured as described earlier [16]. Experi-
ments conformed with the Guide for the Care and Use of
Laboratory Animals (NIH) and were approved by the local
government.
The chronotropic response of the CM was measured as
described p reviously [16] on day 4 after seeding, every 5 min
after cumulative a ddition of nociceptin/orphanin FQ
(FGGFTGARKSARKLANQ) [17,18] at 37 °C. Antisense
pretreatment of the heart cells was performed on days 1 and

2 a fter seeding by administration of either 0.2 l
M
PNA o r
PNA–MAP conjugate.
Assessment of cellular uptake by CLSM
The CLSM measurements were performed using a LSM
410 inverted confocal laser s canning microscope (Carl Zeiss,
Jena GmbH, Jena, Germany) as described previously
[19, 20]. In brief, the fluorescent oligonucleotide derivatives
were dissolved in 1 mL prewarmed (37 °C) Dulbecco’s
phosphate bu ffered saline supplemented with 1 gÆL
)1
D
-glucose (DPBSG) and the cells were overlaid with this
solution within 5 min. After 30 min observation, the
viability of the cells was assessed by the addition of trypan
blue. Excitatio n was performed at 488 nm (Fluos) a nd
543 nm (trypan blue) a nd emission was m easured at 515 nm
and 570 nm, respectively. Three regions of interest (16 · 16
pixel; 30 scans with a scan time of 2 s with double
averaging) in the cytosol and one in the nucleus of three
selected cells were chosen such that the intensity of the
diffuse fluorescence could be recorded without substantial
interference from vesicular flu orescence. The intracellular
fluorescence signal was corrected for t he con tribution of the
extracellular fluorescence, arising f rom nonideal confocal
properties o f the CLSM, by estimating the distribution
function of sensitivity in the z direction of the microscope.
Assessment of cellular uptake by FACS
The cells (10

5
per well) were washed three times with
prewarmedDPBSGandthenoverlaidwith0.2mLofa
freshly prepared prewarmed (37 °C) solution of the fluor-
escent PNA derivative in DPBSG (2 l
M
I or 0.1 l
M
II;
Table 1). After 30 min incubation at 37 °C, the cells were
washed two times with NaCl/P
i
and detached by 15 min
trypsination at 37 °C using 0.5 mL 0.05% Trypsin/0.02%
EDTA (v/v) per well. Then 1 mL culture medium was
added and the cell suspension was centrifuged at 1000 g fo r
8 min. Subsequently the cells were resuspended i n 1 mL
NaCl/P
i
and s tored on ice until the m easurement. T he
accumulation of fluorescence was determined at 525 n m
after excitation a t 488 nm using a Becton Dickinson
(Franklin Lakes, NJ, USA) FACS Calibur flow cytometer
with
CELLQUEST
software. Cytograms were acquired with
10
4
cells.
Assessment of cellular uptake by CE-LIF

The cells were overlaid with 0.2 mL o f a prewarmed (37 °C)
solution of the fluorescent oligonucleotide derivative in
DPBSG (0.5 l
M
I;0.2l
M
II; Table 1) immediately after
addition of the respective aliquot of the sonicated PNA
stock solution to the DPBSG. After 30 min incubation at
37 °C (if not indicated o therwise), the cells were washed four
times with ice-cold NaC l/P
i
andlysedfor2hat0°Cwith
0.2 mL 0.1% (v/v) Triton X-100 containing 10 mmolÆL
)1
trifluoroacetic acid. The lysate, which contained only
negligible amounts of fluorescent PNA derivatives (below
10% of total cell-associated PNA) was used for protein
determination according to the method of Bradford [21].
The wells containing attached cell debris and nuclei along
with bound or precipitated PNA derivative were extracted
by sonication for 5 min at 60 °C with 0.2 mLÆwell
)1
Tris/
borate buffer (20 m
M
, p H 7.5) supplemented w ith 5
M
urea,
0.1% (w/v) SDS and, as an internal standard, 10 n

M
e-fluoresceinyl lysine. The resulting extracts w ere centri-
fuged for 3 min at 3000 g andstoredat)20 °C; immedi-
ately prior to the CE-LIF analysis the extracts were
sonicated for 5 min at 60 °C.
CE-LIF was performed using a P/ACE MDQ system
with a P/ACE MDQ Laser-Induced Fluorescence Detector
(Beckman Coulter, Fullerton, CA, USA) and a CZESep-
600 neutral coated capillary (31 cm, 50 l
M
i.d.; Pheno-
menex, Asch affenburg, Germany). Tris/borate (200 m
M
,
pH 7.5) with 5
M
urea and 0.1% ( w/v) SDS w as used as the
running buffer. The cell e xtracts w ere i njected into the
capillary for 5 s at 0.5 p.s.i. and the separations were
performed a t 650 VÆcm
)1
and 2 5 °C. The peaks of the
references appeared after 1 .8 min (I), 2.1 min (e-fluorescei-
nyl l ysine) and 4.2 min (II). Apa rt from f ree I and II, the cell
extracts contained the largest quantity of compound I or II
in a complex bound form appearing in both cases at
Table 1. Sequences of the PNA derivatives studied.
Compound Sequence
MAP KLALKLALKALKAALKLA-NH
2

I Fluos-GGAGCAGGAAAG-Lys
(antisense)
II Fluos-GGAGCAGGAAAG-MAP
(antisense)
III Fluos-AGGAGCAGGGAA-MAP
(scrambled)
3044 J. Oehlke et al.(Eur. J. Biochem. 271) Ó FEBS 2004
3.9 min. The assignment for this peak is confirmed by its
complete disappearance after addition of an excess of 2 l
M
unlabelled I and the concomitant g eneration of e qually
intensive fluorescent peaks a t t he positions of the pure
reference compounds I and II, respectively.
Quantitation was performed by fluorescence measure-
ment at 520 nm after excitation at 488 nm using an argon
ion laser. The peaks were integrated using the
P
/
ACE
-
SYSTEM
MDQ
software (Beckman Coulter, Fullerton, CA, USA),
and were no rmalized to the area of the internal standard
e-fluoresceinyl lysine in order to eliminate irregularities of
injection and buffer status. Because the exact volume of the
sample injected into the capillary remained unknown, the
references used as calibratio n standards were injected under
essentially the same conditions in order to eliminate this
factor in the subsequent calculations. The concentrations of

the references were determined on the basis of the optical
density at 2 60 nm and proved linearly c orrelated to the peak
areas in t he range b etween the quantitation limits and
500 n
M
. The quantitation limits (signal-to-noise ratio > 3)
were about 0.5 pmolÆmL
)1
and 1.5 pmolÆmL
)1
for I and II ,
respectively.
Results
Conjugation with MAP leads to an increased intracellular
availability of PNA
In order t o e xamine the ability of M AP to de live r PNA into
intact cells, we investigated the cellular uptake of the
conjugate of I withMAPincomparisonwiththatofnaked
I, b y m eans of FACS, CLSM a nd CE-LIF. T he former two
protocols h ave been most widely used so far in such c ontext.
The results obtained in this way, however, were suspected
recently to be biased b y s urface adsorption or fixation
artefacts [22, 23]. Moreover, due to the environmental
dependence of the fluorescence intensity, these approaches
only enable relative quantitative conclusions. Therefore, we
have developed the third, a CE-LIF based protocol, which
appears capable of s upplementing FACS and CLSM b y
providing absolute quantitation of internalized PNA.
CLSM revealed extensive fluorescence in the cytosol and
nucleus of CM and CHO cells after exposure to both the

naked PNA I and its MAP-conjugate (compound II)
(Fig. 1). The intensity of this fluorescence (outside of
vesicles) in all cases was of the same order as that of the
extracellular PNA solution, indicating extensive nonendo-
cytic uptake for b oth I and II. No diffe rentiation between
the permeation behavior o f I and II appears possible on the
basis of the CLSM data, except that a lower rate of
re-export became apparent for the conjugate (Fig. 1).
FACS, on the other hand, revealed clearly higher cell-
associated fluorescence even after exposure to 100 n
M
conjugate, than found after incubation with 2 l
M
of the
naked PNA (Fig . 2). However, in this case, surface
adsorption of the c onjugate, combined with washout of
the naked PNA due to the peptide tag and the relatively
long time required for the wash and trypsination processes,
respectively, might h ave biased the results.
In addition to the information provided by CLSM and
FACS, the results obtained by the CE-LIF approach enab-
led a quantitative differentiation between the intracellular
concentrations of I and II. If related to the external
concentrations, the intracellular concentration of the con-
jugate measured by CE-LIF in CM exceeded that of the
naked PNA by about eightfold (Fig. 3), which correlates
well with the respective bioactivities (see below).
Complementary uptake experiments performed with more
conveniently available CHO cells analogously revealed a
significantly higher uptake of the conjugate (Fig. 3).

Washout effects should influence t he CE-LIF results o nly
to a negligible extent, considering that 3 min at 0 °Cand
15 min at 37 °C are required for the wash process and for
the efflux of about 50% of the internalized naked PNA
(Fig. 1), respectively. That surface adsorption should also
Fig. 1. Fluorescence intensity measured by CLSM according to [19] in
cytosol and nucleus of CHO cells and cardiomyocytes. Exposure to
0.5 l
M
I or 0.2 l
M
II for 30 min at 37 °C and subsequent re -exchange
(RE) into empty buffer for 15 min at 37 °C, normalized to the fluor-
escence intensity of the external PNA solution. Each bar represents the
mean of th ree sample s ± SE M.
Fig. 2. Cell-associated fl uoresce nce measured by flow cytometry after
exposure of CHO cells for 30 min at 37 °C to empty buffer, com-
pound I and compound II. Empty buffer (thin line; mean fluorescence
intensity 2.6), to 2 l
M
I (b roken l ine; m ean flu orescence in tensity 5.7)
or to 0.1 l
M
II (bold line ; mean fluorescence i nte nsity 7.3). Cell number
is plotted o n the ordinate as a function of t he fluorescence intensity on
the abscissa.
Ó FEBS 2004 PNA–MAP conjugates (Eur. J. Biochem. 271) 3045
not interfere decisively with the quantification of the
internalized PNA by CE-LIF is implied by a comparison
of the uptake results depicted in Fig. 3. As the extent of

surface adsorption should be comparable in all cases, its
contribution to the uptake results should be confined to the
lowest values found, which w ere assessed for the uptake into
CHO cells at 0 °C (Fig. 3). Thus, the bias by surface
adsorption of the other results should amount at maximum
to about 20% and 50% for the naked PNA and the
conjugate, respectively. The real bias, however, should
clearly be lower than these values because the values found
at 0 °C, at least in p art, should a lso reflect e nergy-
independent uptake. Consistent with these notions in the
extracts of cells exposed to an analogous disulfid e bridged
PNA–MAP-conjugate (Y. Wolf, M. Bienert & J. Oehlke,
unpublished observation) no surface bound conjugate
above the quantification limit of CE-LIF could be detected.
In this case exclusively the naked PNA generated by
cleavage of the disulfide bond in the reducing environment
of the cell interior was found.
Energy-dependent and energy-independent mechanisms
are involved in the cellular uptake of both naked PNA
and its MAP conjugate
The cellular uptake of both the naked PNA and the
conjugate proved only p artially sensitive t o lowered
temperature and energy depletion, implying the involve-
ment of nonendocytic mechanisms (Fig. 3). On the other
hand, energy-dependent and -independent mechanisms
contributed differently to the cellular uptake of the naked
PNA and of the conjugate (Fig. 3), suggesting that distinct
modes were f unctioning in the t wo cases. The d ifferent
sensitivity to the presence of serum observed for the
internalization o f t he naked PNA and its conjugate,

respectively, probably also s uggests distinct modes of
uptake (Fig. 3), although different association with serum
components appear more likely to b e the reason here.
Intracellular PNA concentration increases more rapidly
after exposure of cells to PNA–MAP-conjugate than to
naked PNA
The quantity of cell-associated PNA increased significantly
faster after exposing CHO cells to II than after incubation
with I ( F igs 4 and 5). This finding unravels a further r eason
for the enhanced bioavailability of the PNA–MAP conju-
gate, b esides reduced re-export and resistance to s erum
influences mentioned above. After 60 min the c ell-associ-
ated conc entration o f t he con jugate re ached a level at which
apparently th e efflux equalled the influx, whereas that of the
naked PNA increased linearly further (Fig. 4). Possible
alternative reasons for the observed arrest of the uptake of
Fig. 3. Amount of cell-associated PNA determined by CE-LIF in the
extracts of CH O cells and cardiomyocytes. Resu lts following exposure
to 0.5 l
M
I or 0.2 l
M
II for 30 min at 37 °C w ithout (c ontrol) and with
energy depletion or in the presence of 10% (v/v) f etal bovine serum
(FBS) and at 0 °C. For energy depletion, the cells were incubated in
DPBS containing 25 m
M
2-deoxyglucose/10 m
M
sodium az ide (DOG/

NaN3) for 60 min at 37 °C an d subsequ ently expo sed to th e PNA
derivative dissolved in the same buffer. To facilitate comparison, the
values of I were normalized to an exposure at 0.2 l
M
according to t he
linear c oncentration dependence shown in Fig. 7. Each b ar represents
the mean of three samples ± SEM.
Fig. 4. Cell-associated PNA determined by CE-LIF in the extracts of
CHO cells after e xposure to 0.5 l
M
I(r)or0.2l
M
II (j), respectively,
at 37 °C for different periods of time. To facilitate comparison, the
values of I were normalized to an exposure at 0.2 l
M
according to t he
linear c oncentration dependence shown in Fig. 7. Each b ar represents
the mean of three samples ± SEM.
Fig. 5. Cell-associated PNA determined by CE-LIF in the extracts of
CHO cells after e xposure to various concentrations of I ( r)orII(j)for
30 min at 3 7 °C. Each bar represents t he mean of three samples
±SEM.
3046 J. Oehlke et al.(Eur. J. Biochem. 271) Ó FEBS 2004
II after 60 min could be aggregation of the conju gate and
peptidase cleavage of the MAP-tag. However, even after 2 h
exposure to the cells no significant loss of II was detectable
by CE-LIF in the supernatants, ruling out aggregation and
enzymatic breakdown from playing a noticeable role in this
context.

Conjugation with MAP significantly augments the
biological activity of a 12-mer antisense PNA directed
against the nociceptin/orphanin FQ receptor
Pretreatment of CM with 0.2 l
M
of the naked PNA
(I; Table 1) and its MAP-conjugate (II; Table 1) lowered
the ch ronotropic effect exerted by the neuropeptide noci-
ceptin [24] by 50% and 90%, respectively ( Figs 6 and 7).
Exposure of CM to conjugates of MAP with a scrambled
PNA containing the same base composition (com-
pound III; T able 1) did not negatively influence the
chronotropic effect (Fig. 7). These results infer, as anticipa-
ted, antisense down-regulation of the nociceptin/orphanin
FQ receptor to be the mechanism of biological activity of
compounds I and II. The antisense pretreatment remained
without any influence on the basal beating rate of the CM,
implying that the PNA derivatives are nontoxic at the
concentration used. Consistent with this notion, no signi-
ficant signs of toxicity were found by means of L DH release
and trypan b lue exclusion throughout all cellular uptake
experiments of the present s tudy.
Discussion
Improved delivery into mammalian cells has been achieved
by covalent coupling of various highly polar bioactive
substances with CPPs [10,25–27]. The s tructural r equire-
ments for the delivery activity of p eptides, however,
remained unclear until now. As a prerequisite for an
elucidation of such structural requirements in the present
work we evaluated the suitability of a conjugate of the

synthetic CPP MAP (KLALKLALKALKAALKLA-
NH
2
) [9,10] with a 1 2-mer p eptide nucleic acid
(5¢-GGAGCAGGAAAG-3¢) directed against the mRNA
of the nociceptin/orphanin FQ receptor [12,13] to serve as
the parent compound in planned structure–delivery activity
relationship investigations.
In distinction t o earlier studied conjugates of MAP with
phosphorothioate oligonucleotides [11] the MAP–PNA
conjugates proved nontoxic in the concentration range up
to 1 l
M
. Consistent with previous reports about substan-
tially enhanced bioavailability of P NA after conjugation
with natural CPPs [3–8], an almost one order of magnitude
higher intracellular PNA concentration was achieved after
exposing cells to the M AP–PNA conjugate II than after
incubation with the naked PNA. Correspondingly, p retreat-
ment of CM with II impaired the chronotropic effect elicited
by the neuropeptide nociceptin [16–18,24] more than five-
fold more than preincubation with the naked PNA.
In line with a nonendocytic mode of uptake regarded to
be typical of CPPs [10,25,27], a faint cytosolic and nuclear
fluorescence of comparable intensity than measured exter-
nally was detected b y CLSM in cells treated with II in
addition to a clearly visible vesicular fluorescence. Surpris-
ingly, however, a similar pattern was observed a fter
exposing the cells to the naked PNA, suggesting nonend-
ocytic as well as endocytic uptake mechanisms to be

involved in both cases. Quantitation of t he intracellular
PNA b y C E-LIF supported this notion. The measured
quantities corresponded to more than 20-fold and fourfold
enrichments in the cell interior for the MAP-conjugate and
the n aked PNA, respectively ( related t o t he external
concentration and a ratio of about 10 lL cell volumeÆmg
protein
)1
[11]). If the internalization had proceeded primar-
ily by an endocytic mechanism, an intracellular concentra-
tion of, a t maximum, about 10% of the external level should
be expected, c onsidering the total volume of endo somes
comprising ma ximally 10% o f the cell volume [28]. Thus the
commonly accepted interpretation of the different cellular
uptake of n aked PNA a nd PNA–CPP conjugates by an
endocytic internalization of the former and a nonendocytic
one of the l atter appears insufficient for e xplaining our
results. As one of the possible explanations, our observation
Fig. 6. Dose–response c urve for t he influence of nociception on the
beating rate of CM measured on day 4 of culture without and with
pretreatment with either 0.2 l
M
ofIorIIonday1and2afterseeding.
The basal beating rate of the CM on day 4 of culture was 191 ± 12
beatsÆmin
)1
(SD; n ¼ 30). Each point represents the mean of 30–40
cellsorcellcluster±SEM.
Fig. 7. Influence of 10
)5

M
nociception o n the beating rate of CM
measured on day 4 of culture without and with pretreatment with either
0.2 l
M
of II or III (Table 1) on day 1 and 2 after seeding. Each point
represents the mean of 30–40 cells or cell cluster ± SEM. The differ-
ence between the control and the asterisk-marked bar is statistically
significant at P £ 0.05 (Student’s t-test).
Ó FEBS 2004 PNA–MAP conjugates (Eur. J. Biochem. 271) 3047
that energy-dependent and -independent mechanisms con-
tribute differently to the cellular uptake of the nak ed PNA
and of the conjugate suggests that distinct modes of
nonendocytic uptake should be operative in both c ases.
Our findings that the MAP–PNA conjugate in contrast to
the naked PNA proved resistant to re-export p rovide a
further explanation of the different intracellular levels
measured for the con jugate and the naked PNA. Analogous
different susceptibilities against re-export were observed for
phosphorothioate oligonucleotides and MAP–phosphoro-
thioate oligonucleotide c onjugates [11], suggesting that such
peptide m ediated effects a re not confi ned to p articular cargo
molecules. In addition, the uptake differences between
conjugate and naked PNA were strengthened by se rum
influences. Similarly Astriab-Fischer et al.[29]foundthat
the presence of serum in the incubation medium did not
affect the uptake of CPP–phosphorothioate oligonucleotide
conjugates in contrast to that of the naked oligonucleotides,
infering that such peptide m ediated resistance t o serum
influences is a more general reason for the improved

bioavailability of CPP conjugates.
In conclusion, our study revealed the ability of the
synthetic CPP MAP to significantly increase the bioavail-
ability and bioactivity of PNA without eliciting enhanced
cell toxicity. These results along with the possibility to
measure absolute quantities of i nternalized PNA by CE-
LIF provide promising preconditions for studies on struc-
ture–delivery activity relationships which are under way.
Acknowledgements
We thank H . Hans, M. Wegener and G. Vogelreiter for excellent
technical a ssistance. This work was supported by t he European
Commission (QLK3-CT-2002-01989).
References
1. Nielsen, P.E., Egholm, M., Berg, R.H. & Buchardt, O. (1991)
Sequence-selective recognition of DNA by strand displacement
with a thymine-subst ituted po lyamid e. Science 254, 1497–1500.
2. Gray, G.D., Basu, S. & Wickstrom, E. (1997) Transformed and
immortalized cellular uptake of oligodeoxynucleo side phosphoro -
thioates, 3¢-alkylamino oligodeoxynucleotides, 2¢-O-methyl oligo-
ribonucleotides, oligodeoxynucleoside methylphosphonates, and
peptide nucleic acids. Bioch em. Ph arma col. 53, 1465–1476.
3. Pooga, M., Land, T., Bartfai, T. & Langel, U. (2001) PNA oli-
gomers as tools for specific modulation o f gene e xpression.
Biomol. Eng. 17, 183–192.
4. Basu, S. & Wickstrom, E. (1997) Synthesis and characterization of
a peptide nuc leic acid conjugated to a
D
-peptide analog of insulin-
like growth facto r 1 for increased cellular uptake. Bioconjug.
Chem. 8, 481–488.

5. Pooga, M., Hallbrink, M., Zorko, M. & Langel, U. (1998) Cell
penetration by tran sportan. FASE B J. 12, 67–77.
6. Simmons, C.G., Pitts, A.E ., Mayfi eld, L.D., Shay, J .W. & Corey,
D.R. (1997) Synthesis and membrane permeability of PNA-pep-
tide conjugates. Bioorg.Med.Lett.7, 3001–3006.
7. Cutrona, G., Carpaneto, E .M., Ulivi, M., R oncella, S., Landt, O.,
Ferrarini, M. & Boffa, L.C. (2000) Effects in live cells of a c-myc
anti-gene PNA lin ked to a nuclear locali zation signal. Nat.
Biotechnol. 18, 300–303.
8. Stetsenko,D.A.,Arzumanov,A.A.,Korshun,V.A.&Gait,M.J.
(2000) Peptide conjugates of oligonucleotides as enhanced anti-
sense agents. Mol. Biol. 34, 852–859.
9. Oehlke, J ., Sc heller, A ., Wie sner, B., Krause, E., Beyermann , M.,
Klauschenz, E., Melzig, M . & Bienert, M. (1998) Cellular uptake
of an alpha-helical amphipathic model peptide with the potential
to deliver polar c ompound s into the cell interior non-endo-
cytically. Biochim. Biophys. Acta 1414, 127–139.
10. Lindgren, M., H a
¨
llbrink, M., Prochiantz, A. & Langel, U
¨
. (2000)
Cell-penetrating peptides. Trends Pharmacol. Sci. 21, 99–103.
11. Oehlke,J.,Birth,P.,Klauschenz,E.,Wiesner,B.,Beyermann,M.,
Oksche, A. & Bienert, M. (2002) Cellular uptake of antisense
oligonucleotides after complexing or conjugation with cell-pene-
trating mod el pe ptides. Eur. J. Biochem. 269, 4025–4032.
12. Tian, J.H., Xu, W., Zhang, W., Fang, Y., Grisel, J.E., Mogil, J.S.,
Grandy, D.K. & Han, J.S. (1997) Involvement of endogenous
orphanin FQ in electroacupuncture-induced analgesia. Neurore-

port 8, 497–500.
13. Zhu, C.B., Cao, X.D., Xu, S.F. & Wu, G.C. (1997) Orphanin FQ
potentiates formalin-induced pain behavior and antagonizes
morphine analgesia in rats. Neurosci. Lett. 235, 37–40.
14. Christensen, L. , F itzpatrick, R., Gildea, B ., Petersen, K.H.,
Hansen, H.F., Koch, T., Egholm, M., Buchardt, O., Nielsen, P.E.,
Coull, J., et al. (1995) Solid-phase synthesis of peptide nucleic
acids. J. Pept. S ci. 1, 175–183.
15. Stewart, J.M. & Young, J.D. (1984) Solid Phase Peptide Synthesis.
Pierce Chemical Comp., Rockford, IL, USA.
16. Wallukat, G., Nemecz, G., Farkas, T., Kuehn, H. & Wollenber-
ger, A. (1991) Modulation of the beta-adrenergic response in
cultured rat hea rt cells. I . Beta-adrenergic supersensitivity is
induced by lactat e via a ph ospholipase A2 and 15-lip oxygenase
involving pathway. Mol. Cell. Biochem. 102 , 35–47.
17. Meunier, J.C., Mollereau, C., Toll, L., Suaudeau, C ., Moisand, C.,
Alvinerie, P., Butour, J.L., Guillemot, J.C., Ferrara, P. &
Monsarrat, B., et al. (1995) Isolation and structure of the endo-
genous a gonist o f o pioid r ecep tor-like O RL1 r ecep tor. Nature 377,
532–535.
18. Reinscheid, R .K., Nothacker, H.P., Bourson, A., Ardati, A.,
Henningsen, R.A., Bunzow, J.R., Grandy, D.K., Langen, H.,
Monsma, F J.J. & Civelli, O. (1995) Orphanin FQ: a neuro-
peptide that activates an opioid like G protein-coupled receptor.
Science 270, 792–794.
19.Lorenz,D.,Wiesner,B.,Zipper,J.,Winkler,A.,Krause,E.,
Beyermann, M., Lindau, M. & Bienert, M. (1998) M echanism of
peptide-induced mast cell degranulation – Translocation and
patch-clamp studies. J. Gen. Physiol. 112, 577–591.
20. Wiesner, B., Lorenz, D., K rause, E., Beyermann, M. & Bienert,

M. (2 002) Measurement of intracellu lar fluorescence in the pre-
sence of a strong extracellular fluorescence using Confocal Laser
Scanning Microscopy. LAMSO 3, 1–17.
21. Bradford, M.M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-d ye bindin g. Anal. Biochem. 72, 248–254.
22. Richard, J.P., Melikov, K., Vives, E., Ramos, C., Verbeure, B.,
Gait, M.J., Chernomordik, L.V. & Lebleu, B. (2003) Cell-pene-
trating peptides. A r eevaluatio n of the mechanism of cellular
uptake. J. Biol. Chem. 278, 585–590.
23. Thoren, P.E., Persson, D ., Isakson, P ., Goksor, M., Onfelt, A. &
Norden, B. (2003) Uptake of analogs of penetratin, Tat(48–60)
and o ligoarginine in live cells. Biochem. Biophys. Res. Commun.
307, 100–107.
24. Berger, H., Albrecht, E., Wallukat, G . & Bi enert, M. (1999)
Antagonism by acetyl-RYYRIK-NH
2
of G protein activation in
rat brain pre parations and of c hronotropic effect on rat cardio-
myocytes evoked by n ociceptin/or phanin FQ. Br.J.Pharmacol.
126, 555–558.
25. Prochiantz, A. (1996) Getting hydrophilic compounds into
cells: Lessons from homeopeptides. Curr. Opin. N eurobiol. 6,
629–634.
3048 J. Oehlke et al.(Eur. J. Biochem. 271) Ó FEBS 2004
26. Hawiger, J. (1997) Ce llular import of functional peptides t o block
intracellular signaling. Curr. Opin. Im muno l. 9, 189–194.
27. Langel, U
¨
. (2002) Cell-Penetrating Peptides.CRCPressLLC,

Boca Raton, FL, US A.
28. Guillot, F.L., A udus, K.L. & Raub, T.J. (1990) Fluid-phase
endocytosis by primary cultures of bovine brain microvessel
endothelial cell mono layers. Microvasc. Res. 39, 1–14.
29. Astriab, F.A., Sergu eev, D.S., F isher, M., Shaw, B.R. & Juliano,
R.L. ( 2000) A ntisense inhibition of P-glyco protein expression
using pept ide-oligonucleotide conjugates. Biochem. Pharmacol. 60,
83–90.
Ó FEBS 2004 PNA–MAP conjugates (Eur. J. Biochem. 271) 3049