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Bioconjugate Chem. 2003, 14, 962−966
Synthesis of Trifunctional PNA-Benzophenone Derivatives for Mitochondrial Targeting, Selective DNA Binding, and Photo-Cross-Linking Gu¨nther F. Ross,† Paul M. Smith,† Alistair McGregor, Douglass M. Turnbull, and Robert N. Lightowlers* School of Neurology, Neurobiology and Psychiatry, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. Received March 31, 2003; Revised Manuscript Received July 22, 2003
Mutations in mitochondrial DNA (mtDNA) cause a variety of human pathologies. In many patients, mutated and wild-type mtDNAs coexist in the same cell, a situation termed mtDNA heteroplasmy. In the absence of standard therapies for these disorders, a genetic strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by the selective hybridization of a nucleic acid derivative, allowing propagation of the wild-type genome and correction of the associated defects. To allow for selective binding under physiological conditions, peptide nucleic acids (PNA) are being used. Two other problems, however, have to be resolved: mitochondrial import and attachment of the PNA to the target DNA to inhibit replication. Mitochondrial localization can be achieved by the addition of a caged lipophilic cation and addition of a photo-cross-linking reagent should facilitate covalent attachment. We therefore report the synthesis of benzophenone-PNA derivatives carrying a triphenylphosphonium moiety and demonstrate irreversible binding selectivity between two DNA molecules that differ by a single nucleotide.
INTRODUCTION
Human mtDNA, a 16 569 bp circular genome, encodes 13 polypeptides and 24 RNA molecules. All 13 proteins are believed to be essential components of the enzyme complexes that couple respiration to the generation of ATP. Although clinical syndromes with an underlying mtDNA mutation such as MERRF (myoclonus epilepsy with ragged red fibres) and MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) are rare, when considered as a group, mtDNA mutations are increasingly being recognized as important genetic defects (1, 2). Epidemiological studies in the North East of England showed that as many as 1:8000 individuals carry a pathogenic mtDNA mutation and 1:17 000 have an associated clinical defect (3). There are many copies of mtDNA in each nucleated cell of the body. In the diseased state, the pathogenic allele is often represented by a major subgroup of molecules, a situation termed heteroplasmy. Dependent on the particular mutation, it is normally recessive, with ratios reported as high as 95% before a biochemical deficiency is apparent (4, 5). Mutations may take the form of rearrangements (deletions or duplications), but often the wild-type and mutated mtDNA differ by only a single base pair substitution. In the absence of standard treatments for these disorders, we have suggested that antigenomic molecules could be made to selectively target the replicating mutated mtDNA, preventing normal replication and * Address for correspondence: R. N. Lightowlers, School of Neurology, Neurobiology and Psychiatry, The Medical School, Framlington Place, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom, NE2 4HH. Telephone: (+44)-191-222-8028. Fax: (+44)-191-222-8553. E-mail: r.n.
[email protected]. † Both authors contributed equally to this paper.
allowing propagation of the wild-type molecule with time (6, 7). This antigenomic hypothesis assumes that the resultant alteration in the balance of wild-type to mutated molecule could be sufficient to reverse the biochemical and potentially the clinical defect in patients. Advances in targeting molecules to mixed-sequence duplex DNA are beginning to be made, as illustrated by recent work with pseudocomplementary peptide nucleic acid molecules (8). However, as mtDNA replicative intermediates will be single-stranded for a period during replication, it may not be necessary to target duplex mtDNA. Selective hybridization of antigenomic agents to single-stranded DNA could be effective in inhibiting mtDNA replication, particularly if this binding was irreversible. Our search for an ideal antigenomic agent has focused on peptide nucleic acids (PNAs) and derivatives. These molecules comprise a backbone of repeating N-(2-aminoethyl)-glycine units connected to standard purine or pyrimidine bases which are able to base pair in both Watson-Crick or Hoogsteen forms (9). As PNAs are uncharged, they show increased binding affinity when pairing with complementary oligonucleotides. DNA/ PNA complexes are more sensitive to single base mismatches than the DNA duplex counterpart, and their unusual chemistry makes them resistant to degradation under physiological conditions (10, 11). Initial experiments in vitro, exploring the potential of PNAs as antigenomic agents, were promising. PNAs were able to bind to single-stranded DNA templates and inhibit polymerization by mitochondrial DNA polymerase γ in a sequence-selective manner, even when using an 11-mer PNA (PNA-MERRF) to target templates mimicking heteroplasmy of the common A8344G MERRF point mutation (12). By derivatizing the PNAs with the lipophilic
10.1021/bc034050f CCC: $25.00 © 2003 American Chemical Society Published on Web 08/15/2003
Trifunctional PNA Derivatives
cation, triphenylphosphonium (TPP),1 the resultant molecules appeared to be imported into the mitochondrial matrix in intact cells (13). Disappointingly, however, no alteration in the levels of heteroplasmy was noted when using cells heteroplasmic for the A8344G MERRF mutation and the TPP-PNA-MERRF. As the reported data were consistent with the TPPPNA being imported, the lack of effect was due either to the PNA not being able to access mtDNA, or that after binding it was removed. To dissect these possibilities, we set out to synthesize a molecule that could be covalently attached to mtDNA in a sequence-selective manner. Principally, a suitable construct has to be optimized for a three-step process. First, the molecule has to be imported solely into the mitochondrial matrix. Interactions with other organelles and especially with nuclear DNA would cause severe side effects. Second, inside the matrix the agent has to bind to the mutated mtDNA and spare the wild type. Here a single mismatch has to be recognized. In the third step, a covalent cross-link to the mutated mtDNA would be ideal, inhibiting replication of the target. An alternative would be to use bis-PNAs for their extremely tight binding and ability to strand displace DNA duplexes (14). However, it has not been demonstrated that such molecules can be imported into mitochondria, and preliminary data suggest the increased size of these molecules may adversely affect their import (PMS unpublished observation). Further, the elevated pH in the mitochondrial matrix (≈ pH 7.8) would be likely to prove detrimental to the strand invasion potential. To satisfy these three requirements, we report the synthesis of a peptide nucleic acid derivative carrying a TPP moiety for mitochondrial targeting and a benzophenone system as a photoreactive cross-linker. Although unlikely to be of use for treating patients in vivo, the constructs will be essential for experiments with cells in culture to determine whether PNA derivatives do indeed bind to mtDNA in intact organelles. This information will be crucial for the design of novel targeted PNA derivatives that can be used to treat patients with mtDNA disease. EXPERIMENTAL PROCEDURES
Automated Synthesis of PNA Oligomers. PNA oligomers were synthesized on an Expedite 8909 Nucleic Acid Synthesis system (Applied Biosystems) using Fmoc/ Bhoc monomers (Expedite PNA and linker AEEA-OH monomers, Applied Biosystems) and HATU activation (15). Syntheses were carried out using the standard 2 µmol PNA protocol provided by the supplier. Reaction columns packed with Fmoc-PAL-PEG-PS resin as well as all reagents, solvents, and solutions used during the syntheses were also purchased from Applied Biosystems unless stated. Synthesis of PNA-BZP and PNA-BZP-TPP Conjugates. To form 4-benzoylbenzoic acid conjugates (1, 1 Abbreviations: TPP, triphenylphosphonium; Bhoc benzhydryloxycarbonyl; Fmoc, 9-fluorenylmethoxycarbonyl; Bhoc, benzhydryloxycarbonyl; AEEA, 8-amino-3,6-dioxaoctanoic acid; HATU, (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; BZBA, 4-benzoylbenzoic acid; NHS, Nhydroxy succinimide; DIEA, diisopropylethylamine; DMF, N,Ndimethylformamide; DCM, dichloromethane; NMP, N-methylpyrrolidone; CPTPP, carboxypentyl triphenylphosphonium bromide; MMT, monomethoxytrityl; PFP, pentafluorophenyl; TFA, trifluoroacetic acid; IBTP, iodobutyltriphenylphosphonium iodide; ODN, oligodeoxynucleotide.
Bioconjugate Chem., Vol. 14, No. 5, 2003 963
Figure 1. Diagrammatic representation of the PNA derivatives used in this study. Protocols for the synthesis and purification of these molecules are described in Experimental Procedures. Benzophenone-PNA 1 and the mitochondrially targeted derivatives benzophenone-triphenylphosphonium-PNAs 2a,b. O specifies the 8-amino-3,6-dioxaoctanoic acid spacer monomer. The PNA sequences of these molecules are identical except that 2b has a single base truncation at the C-terminus.
2a,b Figure 1), the PNA oligomer was synthesized on solid support as described. When the automated synthesis was complete, the N-terminal Fmoc-group was removed by a final deblock step. A solution of 15 mg (46.4 µmol, 23.2 equiv) of 4-benzoylbenzoic acid N-hydroxysuccinimido ester (BZBA-NHS, Molecular Probes) and 11 µL (64.6 µmol, 32.3 equiv) of DIEA in 300 µL of DMF was prepared and passed back and forth through the column, 2 h at room temperature, using two 1-mL Luer slip-tip syringes attached to the ends of the column. The resin was then washed with 3 × 1 mL of DMF and 3 × 1 mL of DCM. For the synthesis of PNA-conjugates 2a,b carrying BZBA on the N-terminal and TPP on the C-terminal, two NMP-solutions were prepared and loaded onto free monomer ports of the Expedite 8909: A 0.2 M solution of carboxypentyltriphenylphosphonium bromide (CPTPP, Fluka) and a 0.2 M solution of N-R-Fmoc-N--MMT-Llysine (Bachem) (16). First, the protected amino acid was attached to the resin on the synthesizer using the standard PNA protocol and the N-R-Fmoc group was removed by a final deblock step. Then the column was removed from the synthesizer. A solution of 25 mg (52.4 µmol, 26.2 equiv) of F-moc-β-Ala-OPFP (Bachem) in 300 µL of DMF was prepared and reacted manually using the two-syringe procedure described above. Subsequently, the column was replaced on the synthesizer and CPTPP was attached. The acid-labile N--MMT group was removed manually by treatment with 30 mg of chloroacetic acid in 800 µL of 4:1 (v:v) DCM/m-cresol for 5 h at room temperature. The resin was washed thoroughly with DCM and dried. The column was then replaced, the desired PNA-sequence was synthesized, and the Nterminal BZBA conjugation was performed as described above. For deprotection and cleavage, dried resin carrying the PNA conjugates was transferred to a 0.2 µm PTFE filtered microcentrifuge tube (Millipore Ultrafree-MC), TFA/m-cresol (200 µL 4:1) added for 90 min and deprotected product released by centrifugation at 1200g for 5 min. Conjugates were precipitated by addition of diethyl ether and resuspended in 0.1% (v:v) TFA water prior to storage at -80 °C. Analysis and Purification of PNA Conjugates. PNA conjugates were subjected to reverse phase HPLC on a C4 analytical column (Jones Chromatography) at a column temperature of 55 °C. A linear gradient water/ 0.1% (v:v) TFA - 80% (v:v) acetonitrile/0.1% (v:v) TFA was used at a flow rate of 1 mL/min. After preparative runs,
964 Bioconjugate Chem., Vol. 14, No. 5, 2003
the collected fractions were dried down in a vacuum desiccator or by blowing nitrogen over the solvent surface in a heat block at 55 °C. MALDI-TOF MS analysis of the PNA conjugates was carried out by N. Harris (Moredun Research Institute, Edinburgh) from a 4-hydroxy-3,5dimethoxycinnamic (sinapinic) acid matrix. The correct product was considered to be confirmed if the difference between the measured and the calculated mass did not exceed 0.1%. Concentrations of PNA derivatives were calculated from measuring the optical absorbance at 260 nm (1-cm light path, 55 °C). For the PNA monomers, the established extinction coefficients given in the Expedite 8909 PNA manual were used. For the CPTPP monomer, an extinction coefficient of 3 mM-1 cm-1 was assumed (IBTP: 3 mM-1cm-1, 268 nm, ethanol (17)), for the BZBA unit 17 mM-1 cm-1 (benzophenone: 17.6 mM-1 cm-1, 252 nm, ethanol (18)). Photo-Cross-Linking Experiments. A 32-mer oligodeoxynucleotide (ODN) 5′-T10GAAAGCTACGAT10-3′ carrying the internal sequence complementary to the PNA, was end-labeled by T4 polynucleotide kinase (Promega) and [γ32P]-ATP (3000 Ci mmol-1 Amersham Pharmacia) following standard protocols, with unincorporated nucleotide removed using a YM3 spin column (Millipore). For part of the experiment described in Figure 2b, a similar protocol was used with a mismatched ODN containing a T-C substitution at position 17. Varying amounts of PNA were added to 0.1 pmol of ODN (of which 15 fmol was radiolabeled) in PBS, total volume 5 µL. Samples were heated to 37 °C for 5 min and then irradiated at 20 °C with a UV light source (Hybec H600 PUVA phototherapy unit) to deliver 5.1 mW cm-2 for the indicated times. The light source was filtered by window glass to give a wavelength range of 330-400 nm, removing 99.984% of the total irradiance below 320 nm and 99.9985% below 315 nm. For the specificity experiments using matched and mismatched probes, samples were overlaid with mineral oil and irradiated at 37 °C in a heated water bath. Samples were denatured in 95% formamide and resolved on 16% (w:v) 19:1 polyacrylamide 8 M urea denaturing gel. Dried gels were exposed to a PhosphorImager cassette (Molecular Dynamics) and analyzed using ImageQuant software (Molecular Dynamics). Efficiency of the production of the ODN/PNA monomer following cross-linking was calculated by measuring the counts of the slower migrating species, C (PNA covalently attached to complementary oligomer), and of the faster migrating species, F (free oligo), and expressing C as a percentage of total counts, C + F. Where irradiation caused the production of large complexes, all multimers were counted together, and the counts were expressed as a percentage of the total. Table 1 records single calculations, but a sample of repeat experiments gave results entirely consistent with the reported percentages. Thermal Melt Analysis. Thermal melt profiles were obtained using 1 µM of the ODN and PNA derivatives in 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 14 mM β-mercaptoethanol, and 150 mM KCl. Mixtures were heated to 90 °C and allowed to anneal by slow cooling to room temperature. The mixture was split into two cuvettes (sample and reference). Melt profiles were measured at 260 nm using a Lambda Bio 20 UV/Visible spectrophotometer (Applied Biosystems, Cheshire UK); the temperature of the sample cuvette was modulated by a PTP-1 Peltier System (Applied Biosystems, Cheshire UK). Data were collected and analyzed using TempLab software (PE-Biosystems, Cheshire UK). Samples were heated at a rate of 0.5 °C min-1, and the sigmoidal melt curve was
Ross et al.
Figure 2. Trifunctional PNA derivatives can be designed to bind irreversibly to DNA templates and to retain sequence selectivity. (A) Photo-cross-linking of a PNA/DNA complex can be induced by irradiation of a PNA conjugated to benzophenone. Molecule 1 was incubated with the ODN probe at the indicated molar excesses and irradiated as detailed in Experimental Procedures. In all cases, a single photo-cross-linked product is generated. Irradiation dosage increases from 18 to 72 J cm-2 . Molecule 1 is represented under the gel. (B) Specificity of binding can still be retained by a trifunctional PNA derivative. Molecule 2b carrying a mitochondrial targeting moiety and benzophenone was incubated with one of two ODN probes differing by a single base, as indicated. Specificity is retained at substantial molar excess, although nonspecific multimeric reaction products are noted at the higher molar concentrations. Molecule 2b is represented under the gel.
expressed as the first derivative (∆A260/time against unit time) to provide an accurate measurement of the melting temperature. RESULTS AND DISCUSSION
PNA-conjugates synthesized (Figure 1) had the sequence (N-terminal, 5′)ATCGTAGCTTTC(C-terminal, 3′) (molecules 1, 2a, and 12-mer in Table 1) or the same sequence lacking the C-terminal base (2b or 11-mer in Table 1). They are identical to nt12621-12632 of the rat mitochondrial genome L-strand (19). Once made, the
Trifunctional PNA Derivatives
Bioconjugate Chem., Vol. 14, No. 5, 2003 965
Table 1. Efficiency of Cross-linking between PNA Derivative and Complementary DNA % DNA cross-linked monomer (multimer)a Xb: 1 PNA/DNA irradiation time (h)
10X: 1 PNA/DNA irradiation time (h)
100X: 1 PNA/DNA irradiation time (h)
PNA
mass
Tm (°C)
1
2
3
4
1
2
3
4
1
2
3
4
12-mer 1 2a 11-mer 2b
2923.81 3588.23 4436.96 3235.05 2923.81
54.0 ( 0.1 54.8 ( 0.1 56.9 ( 0.1 51.4 ( 0.3 54.6 ( 0.4
14 23
31 35
38 41
45 47
20 21
41 35
57 48
61 45
20 20(42)
42 12(49)
60 8(73)
64 8(74)
29
34
49
37
22
34
50
55
23(9)
32(14)
36(34)
37(32)
a
Samples of end-labeled oligodeoxynucleotide were mixed with the indicated molar excess of PNA derivative and irradiated at room temp for 1-4 h as shown. The percentage of single PNA/DNA complex or multimers were calculated as described in the Experimental Procedures. b X is defined as 8.7 for PNA 1, 8.5 for 2a, and 8.4 for 2b.
constructs were assessed to determine whether conjugation had affected the physical characteristics of the PNA moiety and to determine the efficiency of UV-induced photo-cross-linking (gel retardation assays). A Benzoylbenzoic Acid PNA Conjugate CrossLinks to Single-Stranded DNA following UV Irradiation. In this study, we decided to use the benzophenone system as a photoreactive cross-linker for several reasons. Benzophenone derivatives have extensively been used in protein, nucleic acid, and lipid biochemistry for functionalization, labeling, and crosslinking purposes (20). Upon irradiation with UV light, the benzophenone system reaches an n,π* diradicaloid triplet state. Thymidine nucleosides are excellent reaction partners for this highly reactive intermediate, as two different sites are available for cross-linking reactions. The 5,6 CdC bond can be attacked in a [2+2] cycloaddition (Paterno-Buchi reaction) leading to the formation of oxetanes (21, and refs therein). Aliphatic C-H bonds of the DNA backbone are available for C-H insertion reactions; however, we found very low level photo-crosslinking efficiencies were observed using ODN partners flanked with polyguanosine residues, confirming the importance of the cycloadditon (data not shown). For the cross-linking experiments, we have therefore used ODNs complementary in sequence to the PNA and flanked by polythymidine stretches, which is also similar to the natural mtDNA sequence that flanks the PNA target site. The excited triplet state of benzophenone has an estimated lifetime of 80-120 µs; if no suitably orientated reaction partner is found during this time, relaxation to the ground state occurs (22). The cycle of excitationrelaxation can be repeated many times until a reaction takes place. Thus, benzophenone compounds generally allow much higher yields in cross-link reactions than compounds that are only monoexcitable in a photodissociative way, such as aryl azides or diazo compounds (23). The wavelength required for the excitation of the benzophenone is about 350-360 nm. Thus, photochemical damage to DNA and proteins, which mostly occurs at shorter wavelengths, is limited. Initially, the benzophenone-PNA conjugate 1 was synthesized. Benzophenone was linked to the N-terminal of the PNA as a benzoylbenzoic acid amide that can be assembled easily using the amine-reactive BZBA-NHS ester. One 8-amino-3,6-dioxaoctanoic acid (AEEA) linker molecule (O, Figure 1) was inserted between the PNA and the benzophenone to provide maximum access of the photo-cross-linker to reactive sites and avoid interference with PNA/DNA hybridization. To assess the potential problems associated with the generation of nonspecific photo-cross-linking, studies were performed with varying molar excesses of the PNA
derivative as shown in Figure 2a. Irradiation of the ODN probe in the presence of the PNA species results in the production of a PNA/DNA duplex that is retarded in the denaturing gel (Figure 2a). Under all conditions, molecule 1 exclusively created a single cross-linked product. With an 87-fold molar excess of the PNA and an increasing dose of UVA irradiation from 18 to 72 J cm-2, the percentage of cross-linked ODN probe increased from 20 to 61% without the generation of nonspecific multiple adducts (Table 1). Conjugation of PNA with Benzophenone and TPP Moieties in Maximum Spatial Distance Promotes High Efficiency of Photo-Cross-Linking and Binding Specificity. Building on the positive results with molecule 1, the N-terminal benzoylbenzoic acid amide was chosen as the cross-linker for the generation of trifunctional PNAs. Synthesis of a variety of PNA derivatives carrying both the TPP and benzophenone moieties at the N-terminus produced molecules that showed nonspecific binding to ODNs and a UV-induced production of DNA/PNA multimers (data not shown). To prevent the abundant formation of multiple adducts, a maximum distance between benzophenone and TPP was necessary. Thus, TPP was conjugated to the C-terminal of the PNAs 2a,b. N-R-Fmoc-N--MMT-Llysine provides two orthogonally protected amino functions that can be manipulated independently. After the attachment of the amino acid to the resin, the R-nitrogen was deprotected, followed by the addition of β-alanine and CPTPP. The β-alanine facilitates the automated assembly of CPTPP because its β-amino function is more reactive than the R-amino function of lysine. After the assembly of the TPP-carrying moiety, the -amino function of the lysine was liberated by mild acidolytic removal of the MMT-group using the medium-strength chloroacetic acid. Subsequently, the PNA sequence was synthesized and the N-terminal BZBA was added. The molecules 2a,b differ only in the length of their PNA sequence. The TPP is therefore positioned distal to an -amino and two added spacers at the C-terminal of the molecule, with benzophenone on the N-terminal. Following confirmation of synthesis by MALDI-TOF analysis, thermal melt assays were conducted to assess the effect on the binding affinity of the molecules. Both molecules formed a PNA/DNA complex with a slightly elevated TM than the underivatized PNA of similar length (Table 1), possibly due to the addition of the caged positive charge from the TPP. To determine their crosslinking capabilities, assays were performed as previously described. 2a showed increased cross-linking efficiency at both 8.5:1 and 85:1 molar ratios when irradiation doses were increased (18-72 J cm-2, Table 1). Molecule 2b also showed similar increases under identical conditions, but
966 Bioconjugate Chem., Vol. 14, No. 5, 2003
the percentage of monospecific cross-link remained the major product, even in the presence of large (840-fold) molar excess of PNA. To evaluate mismatch discrimination, molecule 2b was tested with matched ODN probe as well as ODN carrying a single mismatch in the middle of the target sequence and was performed under physiological temperatures. As shown in Figure 2b, molecule 2b retains an impressive selectivity even when measured with high (84-fold) molar ratios of PNA derivative, although longer irradiation times and greater PNA molar excess do begin to induce multicomplexes (data not shown). As the long term aim of this project is only to modulate the levels of heteroplasmy, this marked selectivity would appear to be ideal for such purposes. In summary, we have been able to design and synthesize a trifunctional molecule that retains specificity of binding and efficient cross-linking after irradiation. We now intend to assess this molecule in isolated organelles and cultured cells to determine whether covalent attachment is sufficient to inhibit the de novo synthesis of mtDNA and to investigate the potential of other PNA derivatives as efficient antigenomic molecules. ACKNOWLEDGMENT
R.N.L. and D.M.T. wish to thank the Wellcome Trust for funding this project (Programme Grant 056605). LITERATURE CITED (1) Chinnery, P. F., and Turnbull, D. M. (1999) Mitochondrial DNA and disease. Lancet 354 (Suppl. 1), 117-121. (2) DiMauro, S., Bonilla, E., Davidson, M., Hirano, M., and Schon, E. (1998) Mitochondria in neuromuscular disorders. Biochim. Biophys. Acta 1366, 199-210. (3) Chinnery, P. F., Johnson, M. A., Wardell, T. M., Singh-Kler, R., and Hayes, C., et al. (2000) The epidemiology of pathogenic mitochondrial DNA mutations. Ann. Neurol. 48, 188-193. (4) Chomyn A., Martinuzzi A., Yoneda M., Daga A., Hurko O., et al. (1992) MELAS mutation in mtDNA binding site for transcription termination factor causes defects in protein synthesis and in respiration but no change in levels of upstream and downstream mature transcripts. Proc. Natl. Acad. Sci. U.S.A. 89, 4221-4225. (5) Boulet, L., Karpati, G., and Shoubridge, E. A. (1992) Distribution and Threshold Expression of the tRNAlys Mutation in Skeletal Muscle of Patients with Myoclonic Epilepsy and Ragged Red Fibres (MERRF). Am. J. Hum. Genet. 51, 1187-1200. (6) Chrzanowska-Lightowlers, Z. M., Lightowlers, R. N., and Turnbull D. M. (1995) Gene therapy for mitochondrial DNA defects: Is it possible? Gene Ther. 2, 311-316. (7) Taylor, R. W., Chinnery, P. F., Clark, K. M., Lightowlers, R. N., and Turnbull, D. M. (1997) Treatment of mitochondrial disease. J. Biomembr. Bioenerg. 29, 195-205. (8) Demidov, V. V., Protozanova, E., Izvolsky, K. I., Price, C., Nielsen, P. E., and Frank-Kamenetski, M. D. (2002) Kinetics and mechanism of the DNA double helix invasion by
Ross et al. pseudocomplementary peptide nucleic acids. Proc. Natl. Acad. Sci. U.S.A. 99, 5953-5958. (9) Egholm, M., Buchart, O., Christensen, L., Behrens, C., and Freier, S. M., et al. (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365, 566-568. (10) Orum, H., Nielsen, P. E., Egholm, M., Berg, R. H., Buchardt, O., and Stanley, C. (1993) Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Res. 21, 5332-5336. (11) Demidov, V. V., Potaman, V. N., Frank-Kamenetskii, M. D., Egholm, M., Buchardt, O., et al. (1994) Stability of peptide nucleic acids in human serum and cellular extracts. Biochem. Pharmacol. 48, 1310-1313. (12) Taylor, R. W., Chinnery, P. F., Turnbull, D. M., and Lightowlers, R. N. (1997) Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids. Nat. Genet. 15, 212-215. (13) Muratovska, A., Lightowlers, R. N., Taylor, R. W., Turnbull, D. M., Smith, R. A. J., et al. (2001) Targeting of peptide nucleic acid (PNA) oligomers to miitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease. Nucleic Acids Res. 29, 1852-1863. (14) Kosaganov, Y., Stetsenko, D., Lubyako, E., Kvitko, N., Lazurkin, Y., and Nielsen, P. E. (2000) Effect of temperature and ionic strength on the dissociation kinetics and lifetime of PNA-DNA triplexes. Biochemistry 39, 11742-11747. (15) Casale, R., Jensen, I., and Egholm, M. 1999. Synthesis of PNA oligomers by Fmoc chemistry. In Peptide Nucleic Acids: Protocols and Applications (P. E. Nielsen, M. Egholm, Eds.) pp 39-50, Horizon Scientific Press, Wymondham, UK. (16) Dubowchik, G., and Radia, S. (1997) Monomethoxytrityl (MMT) as a versatile amino protecting group for complex prodrugs of anticancer compounds sensitive to strong acids, bases and nucleophiles. Tetrahedron Lett. 38, 5257-5260. (17) Coulter, C. V., Kelso, G. F., Lin, T.-K., Smith, R. A. J., and Murphy, M. P. (2000) Mitochondrially targeted antioxidants and thiol reagents. Free Rad. Med. Biol. 28, 1547-1554. (18) Perkampus H.-H. (1992) UV-Vis Atlas of Organic Compounds, VCH, Weinheim, Germany. (19) Gadaleta, G., Pepe, G., De Candia, G., Quagliariello, C., Sbisa`, E., and Saccone, C. (1989) The Complete Nucleotide Sequence of the Rattus novegicus Mitochondrial Genome: Cryptic Signals Revealed by Comparative Analysis between Vertebrates. J. Mol. Evol. 28, 497-516. (20) Dorman, G., and Prestwich, G. (1994) Benzophenone photophores in biochemistry. Biochemistry 33, 5661-5673. (21) Nakatani, K., Yoshida, T., and Saito, I. (2002) Photochemistry of benzophenone immobilised in a major groove of DNA: formation of thermally reversible interstrand crosslink. J. Am. Chem. Soc. 124, 2118-2119. (22) Prestwich, G., Dorman, G., Elliott, J., Marecak, D., and Chaudhary, A. (1997) Benzophenone photoprobes for phosphoinositides, peptides and drugs. Photochem. Photobiol. 65, 222-234. (23) Brunner, J. (1993) New photolabeling and cross-linking methods. Annu. Rev. Biochem. 62, 483-514.
BC034050F
