DNA Binding and Cleavage by a Cationic Manganese Porphyrin

A cationic manganese porphyrin−peptide nucleic acid (PNA) conjugate has been prepared and used to cleave a double-stranded DNA target. Cleavage ...
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Bioconjugate Chem. 1997, 8, 267−270

267

DNA Binding and Cleavage by a Cationic Manganese Porphyrin-Peptide Nucleic Acid Conjugate Pascal Bigey,† Søren Holst So¨nnichsen,‡ Bernard Meunier,† and Peter E. Nielsen*,‡ Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, F-31077 Toulouse Cedex, France, and Center for Biomolecular Recognition, Department of Biochemistry B, The Panum Institute, Blegdamsvej 3c, DK-2200 N Copenhagen, Denmark. Received November 11, 1996X

A cationic manganese porphyrin-peptide nucleic acid (PNA) conjugate has been prepared and used to cleave a double-stranded DNA target. Cleavage experiments were performed with a 247-base pair restriction DNA fragment containing a 10-base pair homopurine binding target for the PNA. Oxidative activation by this Mn porphyrin-PNA conjugate leads to sequence specific, 3′-staggered cleavage of both DNA strands near the strand displacement junction. Furthermore, the Mn porphyrin-PNA porphyrin conjugates bind over 100-fold better to double-stranded DNA compared to the native PNA.

INTRODUCTION

The sequence specific recognition of double-stranded DNA is an essential biological process performed by DNA-binding proteins and involved in the regulation of transcription, replication, recombination, and DNA repair. The design of synthetic molecules that bind sequence specifically to unique sites on human DNA, thereby to some extent mimicking the action of the natural proteins, may have major implications for the treatment of genetic, oncogenic, and viral diseases. Oligonucleotide (via triple-helix binding), oligopeptide, or protein fragments have been used during the past decade (1-3). More recently, homopyrimidine peptide nucleic acids (PNAs) were shown to form stable triplexes with single-stranded DNA and to invade double-stranded DNA, thereby providing a novel approach to sequence specific DNA recognition (4-6). Encouraged by the work on oxidative DNA cleavage by tetrakis(4-N-methylpyridiniumyl)porphyrinatomanganese(III) activated by potassium monopersulfate (KHSO5) (7-9) and the attachment of trismethylpyridiniumylporphyrinatomanganese(III) motif (Mn-TrisMPyPCOOH) to oligonucleotides (10-12), we found it of interest to prepare a metalloporphyrin-PNA conjugate using the same porphyrin precursor to utilize the DNA targeting properties of the PNA. Here we report the preparation of such a “cationic manganese porphyrinPNA” molecule (see Figure 1 for structure) and its ability to cleave a double-stranded DNA target.

Chart 1. Sequences of DNA Duplex Target (in Bold) and of PNA-n or Conjugate na

a X ) H-(ado) for PNA-n (n ) 1, 2, 3) and MnTrisMPyPn (ado)n for conjugate n [ado ) 8-amino-3,6-dioxaoctanoyl [sHN(CH2CH2O)2CH2COs]; J ) pseudoisocytosine].

EXPERIMENTAL PROCEDURES

Preparation of Mn-TrisMPyP-PNA Conjugate 1 (See Chart 1 and Figure 1 for Structure). The metallated cationic porphyrin precursor MnTrisMPyPCOOH was prepared according to the procedure given in ref 13 and activated by 1,1′-carbonyldiimidazole (CDI) and 1-hydroxybenzotriazole (HOBt) according to the procedure given in ref 10. MnTrisMPyP-COOH (1 mg, 62.5 nmol) was dissolved in 110 µL of dry dimethylformamide (DMF), and CDI (1.5 mg, 9.2 µmol) was added. The mixture was allowed to react for 1 h at room temperature before addition of a solution of HOBt (1.7 mg, 12 mmol in 40 µL of dry DMF). After an extra 1 h * Author to whom correspondence should be addressed. † Laboratoire de Chimie de Coordination duCNRS. ‡ The Panum Institute. X Abstract published in Advance ACS Abstracts, April 1, 1997.

S1043-1802(97)00019-0 CCC: $14.00

Figure 1. Structure of the metalloporphyrin part of the PNA conjugates and of the J-base: pseudoisocytosine.

at room temperature, the excess of CDI was eliminated by addition of 30 µL of 20 mM sodium 4-morpholinopropanesulfonate (MOPS) buffer, pH 7.5, and 5 µL of pyridine was added. The activated ester solution was added to 9 OD units of PNA-1 (57 nmol) dissolved in 30 µL of 20 mM MOPS buffer, pH 7.5. The reaction was allowed to proceed for 45 min at room temperature. One © 1997 American Chemical Society

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Bigey et al.

milliliter of cold ethanol was then added, and the mixture was allowed to precipitate overnight at -20 °C. After centrifugation, the supernatant was discarded. The precipitate was dissolved in water, and conjugate 1 was purified on a C18 Sep-Pak cartridge from Millipore. The yield of the conjugate was 80-90% (based on starting PNA) after purification. Conjugate 1 (and 2 and 3) was characterized by laser desorption mass spectrometry (MALDI-TOF) on a Kratos MALDI-II instrument. The main peak was detected at 6671 (and 6809 and 6962) and corresponds to the molecular weight of conjugate 1 (and 2 and 3). [Calcd M ) 6670 (6815 and 6960) without axial ligand on the metal and with all of the counterions of the 4-N-methylpyridiniumyl residues of the metalloporphyrin moiety being removed. This should give a trication, the metalloporphyrin having three 4-N-methylpyridiniumyl residues. As only a monocation was observed, this probably implies the loss of two protons.] The PNA was synthesized according to the procedures given in refs 14, 15, and 16. Concentrations of PNA were determined at 260 nm (assuming that base extinction coefficients are identical in oligonucleotides and PNAs). DNA Cleavage Experiments. The 32P-labeled EcoRIPvuII restriction fragment of plasmid pA8G2 (se ref 6 for details) was mixed with conjugate 1 (for concentrations, see caption of Figure 3) in a 20 µL volume of TE buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA). The mixture was incubated for 2 h at 37 °C, and then 0.6 µL of a 2 M NaCl solution or 2 µL of salmon DNA was added as desired. The resulting mixture was allowed to equilibrate at room temperature for 1 h. For cleaving experiments, 2 µL of a 5 mM KHSO5 solution was added at room temperature to the hybridization mixture and the reaction was allowed to proceed for 15 min. The reaction was stopped by the addition of 1 µL of 100 mM Hepes buffer (pH 8). Samples were then diluted with 1 µL of yeast tRNA (10 mg/mL) and 100 µL of 0.3 M sodium acetate (pH 5.2), precipitated with 350 µL of absolute ethanol, and finally rinsed with 70% aqueous ethanol and lyophilized. The DNA were analyzed on a 10% denaturing polyacrylamide gel, and radioactive fragments were visualized by autoradiography. Autoradiograms were scanned using a Molecular Dynamics laser densitometry scanner. RESULTS AND DISCUSSION

The manganese porphyrin moiety was linked to the amino end of the PNA molecule, and the hybrid molecule was characterized by mass spectrometry. The DNA target and PNA shown in Chart 1 were used in the present study. We used a bis-PNA that is able to invade doublestranded DNA by strand displacement (4-6, 16, 17) in a bimolecular process. To optimize triplex formation (16), we used cytosines in the antiparallel (Watson-Crick recognizing) strand, while pseudoisocytosines (termed J; see Figure 1 for structure)sthat allow Hoogsteen hydrogen bond formation independent of pH (16)swere used in the parallel (Hoogsteen recognizing) strand. Binding of conjugates 1, 2, and 3 to a 247-base pair duplex DNA restriction fragment containing a 10-base binding site for the PNA (Chart 1) was studied using electrophoretic mobility shift assay (Figure 2), and the pseudo affinity constant Kdps for all three conjugates was 25 nM. This pseudo affinity constant, which more accurately reflects the binding rate constant (18), was over 100-fold higher for the manganese porphyrin-PNA conjugates than for the free PNAs (Kdps ≈ 10 mM). We ascribe this dramatically improved binding efficiency of the conjugate to the high affinity of the metalloporphyrin

Figure 2. (a, b) Gel shift analysis of the DNA binding of PNA 1 (a) and metalloporphyrin-PNA conjugate 1 (b). (At present we do not know if the appearance of multiple bands at higher concentrations (>4 mM) is due to complexes of different conformation or structure, but it is unlikely that they represent different binding sites since no other obvious targets are present in the DNA fragment.) (c) Binding isotherms of the DNA binding of PNA 1 (9) and 2 (2) and metalloporphyrin-PNA conjugates 1 (b) and 2 ([). For comparison, the binding isotherms of three other bis-PNAs with total charges of +2 (in the form of one lysine and the terminal amino group) (- ‚ -), +4 (3 lysines) (- - -), or +5 (4 lysines) (- -) are also shown.

moiety for the minor groove of double-stranded DNA, which will effectively increase the local concentration of the PNA moiety close to the DNA helix and thus increase the probability of duplex invasion. Additional charges per se have been shown to increase binding efficiency [Nielsen and Demidov (in preparation), 19, 20], but the manganese porphyrin-PNA conjugates that have three positive charges bind even better than an analogous PNA with four positive charges but without the manganese porphyrin moiety (Figure 2). Oxidation activation of the targeted manganese porphyrin with potassium monopersulfate (see ref 9 for the formation and the reactivity of manganese-oxo porphy-

DNA Cleavage by PNA Conjugate

Bioconjugate Chem., Vol. 8, No. 3, 1997 269

Figure 3. Sequence selective oxidative cleavage of a doublestranded DNA target by the metalloporphyrin-PNA conjugate 1. (Lane 1) A/G sequence reaction; (lane 2) control with KHSO5 and free PNA 1; (lane 3) control with metalloporphyrin precursor (1 µM) and KHSO5 and free PNA 1; (lanes 4-6) cleavage with porphyrin-PNA conjugate 1 (0.1, 0.3, and 1 µM, respectively) and KHSO5; (lane 7) as lane 3 except for the presence of 50 mM NaCl; (lane 8) as lane 7 except for the presence of 1 µM control PNA (without conjugated porphyrin); (lanes 9-11) as lanes 4-6 except for the presence of 50 mM NaCl; (lanes 12 and 13) as lanes 5 and 6 except for the presence of salmon sperm DNA (1 µg/mL).

rin complexes in water solutions) led to sequence specific cleavage of this long DNA fragment proximal to the PNA target. The main cleavage occurred at the duplex-totriplex junction as a discrete band (Figure 3, lanes 4, 9, and 12; Figure 4a), whereas control experiments with the free metalloporphyrin in the presence of free PNA-1 led to very little cleavage at the PNA target (Figure 3, lane 3; Figure 4). Cleavage with conjugate 1 on the purinerich strand under the same conditions occurred primarily at the triplex-to-duplex junction (Figure 4c,d), whereas hardly any cleavage was observed inside the binding site. Some cleavage also took place at the 5′-end of the purine target (Figure 4a,d). This cleavage could be due to the formation of a kinetically trapped complex in which the PNA binds eight bases in the opposite direction (Figure 4f). (Conjugates 1-3 gave virtually identical cleavage results.) It is noteworthy that the cleavage occurs at a 3′-staggered fashion across the two DNA target strands, thereby indicating that the cleavage takes place from the minor groove in full accordance with the expected binding mode of the porphyrin, although it should be considered that the DNA helix is most probably significantly distorted proximal to the strand displacement loop. It is also interesting that conjugates 2 and 3, in which the linker between the PNA and the porphyrin is increased, cleave the DNA at virtually the same positions as conjugate 1. These results indicate that the precise site of cleavage to a significant extent is determined by the altered/distorted DNA structure proximal to the PNA binding loop. Finally, we notice that increased concen-

Figure 4. Densitometric scanning and schematic representations of the cleavage results: (a) cleavage of the pyrimidine strand by the metalloporphyrin-PNA conjugate 1 (corresponding to lane 4 of the autoradiogram presented in Figure 3); (b) control (corresponding to lane 3); (c) cleavage positions on the double-stranded DNA target; (d) cleavage of the purine strand by the metalloporphyrin-PNA conjugate 1 (analogous to the experiment of lane 4 in Figure 3, but performed on a DNA fragment with the 32P-label at the 5′-end of the EcoRI site; X denotes a background band also present in the control samples); (e) schematic structure of the PNA DNA strand displacement complex showing the position of the metalloporphyrin group (por); (f) alternative complex (minor) that may account for the slight cleavage at the 5′-end of the pyrimidine strand of the target.

trations of the porphyrin-PNA conjugate decrease the site specific cleavage (Figure 3, lanes 5 and 6) and that addition of carrier DNA both restores this cleavage and suppresses unspecific cleavage at other sites of the DNA fragment (Figure 3, lanes 12 and 13), as would be expected since lower affinity sites are titrated out by the carrier DNA without affecting the high-affinity target. No efforts to optimize the cleavage reaction with the metalloporphyrin-PNA conjugates have been undertaken. Thus, it is likely that the relatively low yields (estimated as 5-10%) observed in the experiments presented here can be improved once the mechanism of the cleavage reaction and the DNA binding of these metalloporphyrin-PNA conjugates are better understood. Furthermore, the properties and efficiencies of the present DNA cleavers should be directly compared with

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other analogous PNA conjugates based on Fe (20) or Ni (21) redox chemistry. In conclusion, conjugation to the cationic porphyrin dramatically enhances the duplex invasion potency of the PNA. Furthermore, the oxidative activation of a manganese porphyrin-PNA hybrid molecule allows the cationic metalloporphyrin to efficiently create irreversible damages on the double-stranded DNA target at the expected sites. Thus, further studies of such metalloporphyrin-PNA hybrid molecules are of interest in the development of sequence specific gene targeting and cleaving reagents. ACKNOWLEDGMENT

P.B. is indebted to CNRS and Re´gion Midi-Pyre´ne´es for a fellowship. This work was supported by the French Agency for AIDS Research (ANRS), Re´gion Midi-Pyre´ne´es, and CNRS (B.M.) and by The Danish National Research Foundation (P.E.N.). LITERATURE CITED (1) Nielsen, P. E. (1991) Sequence Selective DNA Recognition by Synthetic Ligands. Bioconjugate Chem. 2, 1-12. (2) Moser, H., and Dervan, P. B. (1987) Sequence specific cleavage of double helical DNA by triplex formation. Science 238, 645-650. (3) Thuong, N. T., and He´le`ne, C. (1993) Sequence specific recognition and modification of of double-helical DNA by oligonucleotides. Angew. Chem., Int. Ed. Engl. 32, 666. (4) Nielsen, P. E., Egholm, M., Berg, R. H., and Buchardt, O. (1991) Sequence selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497-1500. (5) Cherny, D. Y., Belotserkovskii, B. P., Frank-Kamenetskii, M. D., Egholm, M., Buchardt, O., Berg, R. H., and Nielsen, P. E. (1993) DNA unwinding upon strand displacement of binding of PNA to double stranded DNA. Proc. Natl. Acad. Sci. U.S.A. 90, 667-1670. (6) Nielsen, P. E., Egholm, M., and Buchardt, O. (1994)Evidence for (PNA)2/DNA triplex structure upon binding of PNA to dsDNA by strand displacement. J. Mol. Recognit. 7, 165-70. (7) Pitie´, M., Pratviel, G., Bernadou, J., and Meunier, B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3967. (8) Pratviel, G., Duarte, V., Bernadou, J., and Meunier, B. (1993) Nonenzymatic Cleavage and Ligation of DNA at a Three A‚T Base Pair Site. A Two-Step “Pseudohydrolysis” of DNA. J. Am. Chem. Soc. 115, 7939. (9) Pitie´, M., Bernadou, J., and Meunier, B. (1995) Oxidation at Carbon-1′ of DNA Deoxyriboses by the Mn-TMPyP/KHSO5 System Results from a Cytochrome P-450-Type Hydroxylation Reaction. J. Am. Chem. Soc. 117, 2935.

Bigey et al. (10) Pitie´, M., Casas, C., Lacey, C. J., Pratviel, G., Bernadou, J., and Meunier, B. (1993) Selective Cleavage of a 35-mer Single-stranded DNA Containing the Initiation Codon of the TAT Gene of HIV-1 by a Tailored Cationic Manganese Porphyrin. Angew. Chem., Int. Ed. Engl. 32, 557-559. (11) Casas, C., Lacey, C. J., and Meunier, B. (1993) Preparation of Hybrid “DNA Cleaver-Oligonucleotide” Molecules based on a Metallotris(methylpyridiniumyl)porphyrin Motif. Bioconjugate Chem. 4, 366-271. (12) Bigey, P., Pratviel, G., Meunier, B. (1995) DNA cleavage by a metalloprophyrin-spermidine-oligonucleotide” molecule. J. Chem. Soc., Chem Commun., 181-182. (13) Casas, C., Saint-Jalmes, B., Loup, C., Lacey, C. J., and Meunier, B. (1993) Synthesis of Cationic Metalloporphyrin Precursors Related to the Design of DNA Cleavers. J. Org. Chem 58, 2913. (14) Dueholm, K. L., Egholm, M., Behrens, C., Christensen, L., Hansen, H. F., Vulpius, T., Petersen, K., Berg, R. H., Nielsen, P. E., and Buchardt, O. (1994) Synthesis of peptide nucleic acid monomers containing the four natural nucleobases: thymine, cytosine, adenine and guanine, and their oligomerization. J. Org. Chem. 59, 5767-5773. (15) Christensen, L., Fitzpatrick, R., Gildea, B., Petersen, K. H., Hansen, H. F., Koch, T., Egholm, M., Buchardt, O., Nielsen, P. E., Coull, J., and Berg, R. H. (1995) Solid-phase synthesis of peptide nucleic acids (PNA). J. Peptide Sci. 3, 175-183. (16) Egholm, M., Christensen, L., Dueholm, K., Buchardt, O., Coull, J., and Nielsen, P. E. (1995) Efficient pH independent sequence specific DNA binding by pseudoisocytosine-containing bis-PNA. Nucleic Acids Res. 23, 217-222. (17) Griffith, M. C., Risen, L. M., Greig, M. J., Lesnik, E. A., Sprangle, K. G., Griffey, R. H., Kiely, J. S., and Freier, S. M. (1995) Single and Bis Peptide Nucleic Acids as Triplexing Agents: Binding and Stoichiometry. J. Am. Chem. Soc. 117, 831-832. (18) Demidov, V. V., Yavnilovich, M. V., Belotserkovskii, B. P., Frank-Kamenetskii, M. D., and Nielsen, P. E. (1995) Kinetics and mechanism of PNA binding to duplex DNA. Proc. Natl. Acad. Sci. U.S.A. 92, 2637-2641. (19) Veselkov, A. G., Demidov, V. V., Nielsen, P. E., and FrankKamenetskii, M. (1996) A new class of genome rare cutters. Nucleic Acids Res. 24, 2483-2487. (20) Lohse, J., Hui, C., So¨nnichsen, S. H., and Nielsen, P. E. (1996) Sequence selective DNA cleavage by PNA-NTA conjugates. In DNA and RNA Cleavers and Chemotherapy of Cancer and Viral Diseases, NATO ASI Series (Meunier, B., Ed.) pp 133-141. (21) Footer, M., Egholm, M., Kron, S., Coull, J. M., and Matsudera, P. (1996) Biochemical evidence that a D-loop is part of a four-stranded PNA-DNA bundle. Nickel-mediated cleavage of duplex DNA by a Gly-Gly-His bis-PNA. Biochemistry 35, 10673-10679.

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