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: oehlke@fmp-berlin.des 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 . Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide Johannes. Sequence MAP KLALKLALKALKAALKLA-NH 2 I Fluos-GGAGCAGGAAAG-Lys (antisense) II Fluos-GGAGCAGGAAAG-MAP (antisense) III Fluos-AGGAGCAGGGAA-MAP (scrambled) 3044