Letters in Peptide Science, 7: 35–39, 2000. KLUWER/ESCOM © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Synthesis of peptide nucleic acid-peptide chimeras carrying the c-myc tag-sequence Dirk Gottschlinga, Hartmut Seligerb , Gema Tarras´onc , Jaume Piulatsc, Marten Wiersmaa & Ramon Eritjaa,∗ a b c
European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany; Universität Ulm, Sektion Polymere, Albert-Einstein-Allee 11, D-89081 Ulm, Germany; Merck Farma-Qu´ımica, Laboratori de BioInvestigaci´o, C/Casp 108, E-08010 Barcelona, Spain
Received 12 May 1999; Accepted 22 June 1999
Key words: nonradioactive labeling, peptide nucleic acids, PNA, PNA-peptide chimera, tag-peptide
Summary The preparation of peptide nucleic acids (PNA) carrying a c-myc tag-peptide sequence is described. These PNApeptide chimeras have higher affinity to complementary DNA than unmodified oligonucleotides. Moreover, they can be used as nonradioactive probes with sensitivity similar to other nonradioactive methods. Abbreviations: Ac, acetyl; Bhoc, benzhydryloxycarbonyl; DIPEA, N,N-ethyldiisoproylamine; DMF, N,Ndimethylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; HATU, N-[(dimethylamino)-1H, 1,2,3-triazolo-[4,5b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; OPC, oligonucleotide purification cartridge; PNA, peptide nucleic acids; TBTU, N-[(1H-benzotriazol-1-yl) (dimethylamino)methylene]-Nmethylmethanaminium tetrafluoroborate N-oxide; TCP, trityl chloride polystyrene; TFA, trifluoroacetic acid. Introduction Peptide Nucleic Acids (PNA) are oligonucleotide analogs, in which the sugar-phosphate backbone has been replaced by N-(2-aminoethyl)glycine units [1]. In spite of the large change on the backbone structure, PNA molecules bind strongly to complementary DNA and RNA sequences [2], they are achiral and uncharged. For these reasons PNA oligomers have a strong potential as therapeutic agents, diagnostic tools and probes in molecular biology [3]. One of the problems encountered with the use of PNA as probes is the labeling and detection of PNA oligomers. The standard radioactive labeling protocols for DNA probes use enzymes such as polynucleotide kinase and terminal transferase, which are not active on PNA oligomers. In order to overcome this problem, a peptide segment, which is a substrate for ∗ To whom correspondence should be addressed. Present address: Centro de Investigaci´on y Desarrollo, C.S.I.C., Jordi Girona 18-26, E-08034 Barcelona, Spain. E-mail:
[email protected]
protein kinase, can be incorporated into PNA oligomers. The resulting PNA-peptide chimeras can be labeled with protein kinase and radioactively labeled ATP [4]. Moreover, some nonradioactive labels such as biotin [5,6] and fluorescein [7,8] were incorporated into PNA in order to determine the cellular uptake and intracellular distribution of the PNA oligomers [5–8] as well as for in situ hybridization experiments [9]. But, to our knowledge, there is very little data on the sensitivity and the use of these nonradioactive labeled PNA oligomers as hybridization probes [10]. We are interested in introducing peptide sequences in nucleic acid derivatives as a nonradioactive labeling system. The great diversity of antibodies raised against peptide sequences led us to consider PNA-peptide chimeras as tools for multiple hybridization experiments. Recently, we showed that oligonucleotidepeptide conjugates carrying the c-myc tag-sequence bind to their complementary sequences and are recognized by an anti c-myc monoclonal antibody [11]. In the present article the preparation of PNA-peptide chi-
36 meras carrying the c-myc tag-sequence is described. The PNA-peptide chimeras have excellent hybridization properties and they are specifically detected by antibodies conjugated with peroxidase and a chemiluminescent substrate.
Materials and methods General methods High performance liquid chromatography (HPLC) was performed either on a Beckmann System Gold (Beckmann) or on a Pharmacia Biotech 900 (Pharmacia Biotech). Solvent A was 0.1% TFA in water. Solvent B was 0.1% TFA in acetonitrile. The products were purified by using a YMC column (250 mm × 20 mm, 5 µm C18 ) and a gradient from 10 to 70% buffer B in 40 min. Analytical HPLC was performed on a YMC column (250 mm × 4.6 mm, 7 µm C18 ) or on a Nucleosil C-18 (Klaus Ziemer GmbH, Germany). For immunodetection of PNA-peptide conjugates, samples were blotted with a dot-blot manifold (GIBCO, BRL) on a 0.2 µm nitrocellulose membrane (Bio-Rad). Immunodetection was carried out with supernatant from a murine hybridoma anti-c-myc (clone 9E10, 24.6 µg/mL; [12]) and a goat anti-mouse peroxidase-conjugated secondary antibody (IgG + IgM, Jackson Immunoresearch Laboratories), and finally developed with ECL (Amersham). Washes of the membrane between antibody incubations were performed in phosphate-buffered saline containing high salt concentrations (0.69 g of NaH2 PO4 ·H2 O, 1.98 g of Na2 HPO4 , 58.44 g of NaCl per liter) and 0.1% Tween-20 (Merck). Chemicals Anhydrous solvents were obtained from Merck (Germany), Fluka (Switzerland) and PerSeptive Biosystems (U.S.A.). Chemicals for the peptide synthesis were obtained from Aldrich (Germany), Alexis (Switzerland), Merck (Germany), Novabiochem (Switzerland) and Pepchem (Germany). Chemicals for the PNA and oligonucleotide synthesis were purchased from PerSeptive Biosystems. Oligonucleotide purification cartridges (OPC) were obtained from Cruachem (U.K.). Synthesis of the peptide containing the c-myc tag-sequence Peptides were synthesized automatically on a Multisyntech Syro II (Multisyntech) by using a preloaded
trityl (TCP) support (functionalisation 0.6 mmol/g, Pepchem) and the Fmoc strategy. The Fmoc protecting group was removed by treating the resin two times with 20% piperidine/DMF for 15 min. Double couplings were performed with an excess of Fmoc-protected amino acid (3 equiv), Nhydroxybenzotriazole (3 equiv), TBTU (3 equiv) and DIPEA (9 equiv) for 60 min. Peptides were deprotected and released from the resin by treatment with a solution of TFA/triisopropylsilane/water (95/2.5/2.5) for 4 h at room temperature. The resins were filtered and washed two times with TFA. The solutions were combined and evaporated under reduced pressure. The residues were dissolved in water and lyophilized. The following peptide sequences were synthesized on a 24 µmol scale and characterized by mass spectrometry: (P1) Ala-Glu-Gln-Lys-Leu-Ile-Ser-GluGlu-Asp-Leu-Asn-Ala; mass spectrum (ESIMS) m/z = 1459.6 [M + H+ ] (exact mass: 1458.7 calculated for C61 H102 N16 O25 ) and (P2) Glu-Gln-Lys-Leu-IleSer-Glu-Glu-Asp-Leu-Asn-Ala; mass spectrum: m/z = 1388.5 [M + H+ ] (exact mass: 1387.7 calculated for C58 H97 N15 O24 ). Synthesis and purification of PNA-peptide chimeras The PNA-peptide chimeras were synthesized on a PerSeptive Expedite 8900 synthesizer using the Expedite PNA upgrade option and the protocols recommended by the supplier. For the PNA synthesis Fmocprotected PNA monomers were used. The exocyclic amino groups of the bases A, C and G were protected with the benzhydryloxycarbonyl (Bhoc) groups [13]. The fully protected peptides on TCP resin (10 mg) were used as starting solid supports. PNA monomers were assembled using a single 15-min coupling reaction mediated by HATU. The coupling conditions were 5 equiv DIPEA, 7.5 equiv lutidine, 5 equiv PNA monomer and 4.5 equiv HATU in N-methylpyrrolidone/DMF (1:1). After the assembly of the PNA sequences, the amino group of the Nterminus was acetylated. The PNA-peptide chimeras were deprotected and released from the resin by treatment with a solution of TFA/ triisopropylsilane/water (95/2.5/2.5) for 4 h at room temperature. The resins were filtered and washed two times with TFA. The solutions were combined and evaporated under reduced pressure. The residues were dissolved in water, lyophilized and the resulting products were purified by HPLC. Yield (for 2 µmol scale): 12 and 14 OD units at 260 nm. The following PNApeptide chimeras were synthesized on a 2 µmol scale
37 and characterized by mass spectrometry: (PI) AcCAC CGA CGG CGC-Ala-Glu-Gln-Lys-Leu-Ile-SerGlu-Glu -Asp-Leu-Asn-Ala; mass spectrum (ESIMS) m/z = 1196.0 [M+2H+Na+K]4+ (exact mass: 4722.0 calculated for C189 H260 N88 O60 ) and (PII) Ac-CAC CGA CGG CGC-Glu-Gln-Lys-Leu-Ile-Ser-Glu-GluAsp-Leu-Asn-Ala; mass spectrum (ESIMS) m/z = 1178.0 [M+2H+Na+K]4+ (exact mass: 4650.9 calculated for C186 H255N87 O59 ). Synthesis and purification of oligonucleotides Oligonucleotide sequences (O1) 50 -d (GTG GCT GCC GCG)-30 and (O2) 50 -d (CAC CGA CGG CGC)-30 were synthesized on a 1 µmol scale using a PerSeptive Expedite 8900 synthesizer and the protocols recommended by the supplier. The standard β-cyanoethylprotected phosphoramidite method was used. The oligonucleotides were removed from the solid support by treatment with concentrated ammonia for 6 h at 55 ◦ C. Under these conditions, all base protecting groups were cleaved off. The crude oligonucleotides were purified with OPC cartridges (Cruachem, Great Britain). Overall yield 25–30 OD units at 260 nm. Melting studies Melting studies were performed in an aqueous solution (1 ml) of 0.15 M NaCl, 50 mM Tris-HCl, pH 7.5. Dodecamer duplexes were made by mixing equimolar amounts (0.5 OD units at 260 nm) of two complementary dodecamer strands. Duplexes were annealed by slow cooling from 90 ◦ C to room temperature (4 h). UV-absorption and melting curves of the oligonucleotides were measured at 260 nm in 1 cm pathlength cells on a Varian Cary 3 UV-spectrometer using a temperature gradient from 20 to 90 ◦ C at a heating rate of 0.5 ◦ C/min. Accuracy of the Tm values was ± 1 ◦ C. Immunodetection of the PNA-peptide chimeras Serial dilutions (from 400 pmol) of the PNA-peptide chimeras were applied to a nitrocellulose membrane (0.2 µm, Bio-Rad) as described in the general Methods section. After drying at 70 ◦ C for 30 min, the membrane was UV cross-linked for 2 min. The membrane was blocked with PBS containing 1% of Tween20 for 1 h at room temperature and incubated with culture supernatant from anti-c-myc murine hybridoma clone 9E10 (24.6 µg/ml[12]) for 1 h at room temperature. After washing twice with PBS containing 0.1% of Tween-20 for 5 min, the membrane was incubated with a horseradish peroxidase-conjugated goat antimouse antibody (Jackson, 0.9 mg/ml) at a 1:40 000
dilution for 1 h. Finally, after several washes with PBS containing Tween-20, the membrane was developed in ECL and exposed to X-ray film.
Results The peptide sequences carrying the c-myc tagsequence P1: Ala-Glu-Gln-Lys-Leu-Ile-Ser-Glu-GluAsp-Leu-Asn-Ala and P2: Glu-Gln-Lys-Leu-Ile-SerGlu-Glu-Asp-Leu-Asn-Ala were prepared on a trityl polystyrene support using standard Fmoc-chemistry protocols. Sequence P1 contains the tag sequence and an extra alanine at the N-terminal position. After the assembly of the sequences, a small aliquot of the supports was cleaved with TFA and the resulting peptide was analyzed by HPLC and mass spectrometry. In both cases a major peak was observed which had the correct mass. PNA sequence Ac-CAC CGA CGG CGC- was assembled directly on the N-terminal amino acid of the P1 and P2 peptide sequences using also Fmocchemistry. This sequence is complementary to a mutated Ha-ras oncogene and it has antiproliferative activity [14]. The PNA monomers were activated with HATU following the protocols recommended by the supplier. Coupling efficiencies were estimated by collecting and measuring the UV absorbance at 301 nm of the Fmoc-piperidine adduct formed during deprotection and they were around 95% per step. Prior to this work, we tried polystyrenepolyethyleneglycol supports carrying the same peptide sequence where coupling efficiencies of PNA monomers were less than 50% (data not shown). Polystyrene-polyethyleneglycol supports without the peptide sequence performed well for the assembly of PNA monomers. Probably the peptide moiety might have changed the swelling properties of the polystyrene-polyethyleneglycol support in such a way that coupling reactions were less efficient, but this was not observed in plain polystyrene supports. Upon completion of synthesis a TFA/triisopropylsilane/water solution was used to cleave PNA-peptide chimeras from the resin and to remove side chain and base protecting groups. PNA-peptide chimeras were purified by reverse phase HPLC. In all cases a major peak was observed that was characterized by mass spectrometry (Figure 1). The hybridization of PNA-peptide chimeras to their complementary DNA sequence was evaluated spectrophotometrically. The melting temperatures
38
Figure 1. Purification of PNA-peptide chimera PII. (a) Semipreparative HPLC profile of crude material after deprotection. The first peak is a solvent peak (dimethylsulfoxide). (b) Analytical HPLC profile of purified compound (see conditions in the Materials and methods section).
Table 1. Melting temperatures (Tm ) of the duplexes containing PNA-peptide conjugates (0.15 M NaCl, 0.05 M Tris·HCl, pH 7.5, 1 mM EDTA). The PNA sequence is given in italics Sequence (50 →30 )
Tm (◦ C)
CACCGACGGCGCa Ac-CAC CGA CGG CGC-AEQKLISEEDLNAa Ac-CAC CGA CGG CGC-EQKLISEEDLNAa
62 80 79
a Complementary strand 50 -GCGCCGTCGGTG-30 .
(Tm ) for the various duplexes are shown in Table 1. PNA-peptide chimeras bind better to their complementary than unmodified oligodeoxynucleotides DNA (79–80 ◦ C versus 62 ◦ C). This increase is similar to that described for unmodified PNA [2]. Also, both PNA-peptide chimeras have a similar Tm . For these reasons we conclude that the peptide part has little influence on the hybridization properties of the PNA. This was expected, because the peptide moiety is attached on a terminal position of the PNA. The same peptide sequence, linked through an internal C residue of an oligodeoxynucleotide, induced a destabilization of approximately 10 ◦ C [11]. Nonradioactive detection of PNA-peptide chimeras was assessed in a dot-blot experiment. Serial dilutions of PNA-peptide chimeras (from 400 pmol) were applied to a nitrocellulose membrane. After immobilization, the samples were incubated with an antic-myc monoclonal antibody [12], then incubated with a secondary antibody conjugated to horseradish perox-
idase, and finally developed with a chemiluminescent substrate (ECL, Amersham). Figure 2 shows the autoradiography of the detection of PNA-peptide chimeras together with different controls such as a PNA, an oligonucleotide and an oligonucleotide-peptide conjugate carrying an unrelated peptide sequence. Positive dots were obtained only for the PNA-peptide chimeras. The sensitivity of the detection was approximately 6 pmol in both PNA-peptide chimeras. This result is similar to that obtained with oligonucleotidepeptide conjugates [11].
Discussion and conclusions Recently, it has been shown that the incorporation of specific peptide sequences into PNA oligomers results in the addition of new and interesting properties that may increase the potential applications of PNA. For example, the addition of a peptide substrate for protein kinase A opens the possibility for radioactive labeling of PNA oligomers [4]. The addition of the Antennapedia (43–58) peptide sequence [5,6] or a sequence related to the insulin-like growth factor [7] improves the antisense properties of PNA due to an enhanced cellular uptake. Moreover, the incorporation of cationic peptides into PNA increases the rate of strand invasion of double stranded DNA as well as the affinity for single stranded DNA [15–17]. Finally, oligohistidine-PNA chimeras are useful for sequence-specific purification of nucleic acids [18].
39 1
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A B C D E F Figure 2. Dot-blot analysis of PNA-peptide chimeras. Serial dilutions of PNA-peptide chimeras and controls were applied on a nitrocellulose membrane, incubated with anti-c-myc murine hybridoma clone 9E10 followed by a horseradish peroxidase-conjugated goat anti-mouse antibody and developed with an ECL system (Amersham) as described in the Materials and methods section. In each line: 1/2 dilutions from 400 pmol; 1 (400 pmol), 2 (200 pmol), 3 (100 pmol), 4 (50 pmol), 5 (25 pmol), 6 (12.5 pmol), 7 (6 pmol), 8 (3 pmol), 9 (1.5 pmol), 10 (0.7 pmol). (A) PNA-peptide chimera PI; (B) PNA-peptide chimera PII; (C) oligonucleotide O2-peptide conjugate having a nuclear localization sequence not related with the c-myc tag-sequence; (D) PNA oligomer; (E) oligonucleotide O2; (F) water.
Acknowledgements We thank Dr. O. Massó for his generous gift of culture supernatants of anti-c-myc murine hybridoma clone 9E10.
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3. 4.
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The introduction of peptide sequences to PNA oligomers can be achieved easier than in oligonucleotides. Both PNA and peptide monomers can be protected with similar groups, have similar activation chemistry and similar stability towards acids. This opens the possibility to use the same deprotection conditions for both moieties. For these reasons, peptide epitopes are a logical alternative as nonradioactive labels for PNA oligomers. The large diversity of antibodies raised against peptides opens the possibility to use peptide sequences as labels for specific PNA oligomers in multiple hybridization experiments. We have shown that PNA oligomers carrying a peptide epitope can be prepared and the resulting PNA-peptide chimeras have higher affinity to complementary DNA compared to unmodified oligonucleotides maintaining their immunoreactivity. We believe that such chimeras will be extremely useful not only as hybridization probes but also in the study of cellular uptake of PNA derivatives.
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