Peptide Nucleic Acid Delivery to Human Mitochondria

PF Chinnery1, RW Taylor1, K Diekert2, R Lill2, DM Turnbull1 and RN Lightowlers1 1Department of Neurology, The University of Newcastle upon Tyne, UK; and 2Institut fu¨r Zytobiologie der Philipps-Universita¨t Marburg, Marburg, Germany

Peptide nucleic acids (PNAs) are synthetic polynucleobase as therapeutic agents for mtDNA disease, we attempted to molecules, which bind to DNA and RNA with high affinity localise PNAs to the mitochondrial matrix. When attached and specificity. Although PNAs have enormous potential to the presequence peptide of the nuclear-encoded human as anti-sense agents, the success of PNA-mediated gene cytochrome c oxidase (COX) subunit VIII, the biotinylated therapy will require efficient cellular uptake and sub-cellular PNA was successfully imported into isolated organelles in trafficking. At present these mechanisms are poorly under- vitro. Furthermore, delivery of the biotinylated peptide-PNA stood. To address this, we have studied the uptake of bioti- to mitochondria in intact cells was confirmed by confocal nylated PNAs into cultured cell lines using fluorescence microscopy. These studies demonstrate that biotinylated confocal microscopy. In human myoblasts, initial punctate PNAs can be directed across cell membranes and to a staining was followed by the release of PNAs into the cyto- specific sub-cellular compartment within human cells – sol and subsequent localisation and concentration in the highlighting the importance of these novel molecules for nucleus. To determine whether PNAs could also be used human gene therapy.

Keywords: MtDNA disease; peptide nucleic acids; cell uptake; gene therapy

Introduction interested in their efficacy as anti-genomic therapeutic agents for disorders of the mitochondrial genome. It is expected that human gene therapy will often depend MtDNA defects cause a diverse group of progressive dis- upon the delivery of highly specific and stable anti-sense eases.10 At present there is no effective treatment for these agents to their desired site of action, away from sites of disorders,11 which often result in severe disability and potential toxicity. This requires an understanding of the premature death.12 Each human cell contains between mechanisms of cellular uptake and sub-cellular traffick- 1000 and 100 000 copies of mtDNA which encodes 13 ing of molecules, which can regulate gene expression. essential polypeptides that form part of the mitochon- Peptide nucleic acids (PNAs) are novel, synthetic DNA- drial respiratory chain.13 The majority of patients who like molecules in which the chains of pyrimidine harbour pathogenic mtDNA defects have a mixture of and purine bases are linked by an aminoethyl mutated and wild-type mtDNA (heteroplasmy). On a 1 (pseudopeptide) backbone. This structure confers two cellular level, the phenotypic defect is only expressed important properties on the molecule: (1) highly specific when the percentage of mutated mtDNA exceeds a criti- hybridisation and affinity to complementary DNA and cal threshold,14 and the clinical features of mtDNA dis- 2 3 RNA; and (2) resistance to nuclease and protease attack. ease are intimately dependent upon the ratio of mutated As a result, PNAs have been shown to be important in and wild-type mtDNA in vivo.15 Unlike nuclear DNA, 1,4 various diagnostic and therapeutic applications. mtDNA is continuously being copied and degraded with Despite their enormous potential as therapeutic agents, a half-life estimated to be between 0.7 and 30 days in rat reports of poor cellular uptake, due to their low phospho- tissue.16 By specifically inhibiting the turnover of mutated 5–8 lipid permeability, dampened the initial enthusiasm. mtDNA, it is theoretically possible to correct the bio- However, early studies on PNA import were limited to chemical defect, prevent clinical progression and poten- a small number of different cell lines and import con- tially reverse some of the pathological features of mtDNA ditions. In addition, it is well recognised that oligodeoxy- disease.17 We have shown that sequence-specific PNAs nucleotide uptake can differ markedly between different selectively inhibit the replication of mutated mtDNA in cell types. For example, muscle cells are particularly vitro,18 but future applicability of this approach is depen- amenable to DNA and modified DNA uptake, possibly dent upon the development of a mutation-specific anti- because of the transverse tubule system and specific cell mtDNA agent which can be targeted into mitochondria 9 surface receptors. in vivo. Whilst PNAs have great potential as anti-sense agents Because of the potential importance of PNAs in the to regulate nuclear gene expression, we are also very gene therapy of both nuclear and mtDNA disease we have studied their uptake in a variety of different cell lines and under different conditions. We show that bioti- Correspondence: RN Lightowlers, Department of Neurology, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK nylated PNAs are readily internalised by a variety of cell The first two authors contributed equally to this work lines. A further hurdle in the development of mitochon- Received 22 March 1999; accepted 13 September 1999 drial gene therapy is the delivery of the antigenomic PNA delivery to human mitochondria PF Chinnery et al 1920 agent into double-membrane bound cytoplasmic parison to those exposed to biotinylated-5 ␮m PNA. organelles.17 We report the successful import of PNAs Increasing the concentration to 20 ␮m did not result in a into isolated mitochondria upon conjugation of the bioti- significant increase in intracellular fluorescence after 4 h nylated PNA molecule to a mitochondrial targeting pre- exposure, consistent with saturation of the PNA uptake sequence. Finally, we also report that addition of this pre- mechanism in human myoblasts. sequence successfully targets the PNA to mitochondria in human myoblasts. Intracellular targeting of peptide nucleic acids As shown in the previous sections, biotinylated PNAs are internalised by human myoblasts in culture. Our main Results aim, however, is to determine whether PNAs can be effective agents in treating patients with defects of the PNAs are internalised by cultured cell lines mitochondrial genome. Once internalised, it is therefore We studied the uptake of an 11-mer biotin-labelled PNA. of fundamental importance to target these molecules to This PNA (identical to PNA-MERRF in Ref. 18) was mitochondria and to demonstrate import into the mito- chosen as we have previously shown that it can selec- chondrial matrix. In order to achieve this, we synthesised tively inhibit replication of mutated mtDNA in vitro. Cell the identical biotinylated PNA attached to the C-terminus ␮ lines were exposed to 20 m biotinylated PNA for 4 h. of a known mitochondrial targeting presequence which After fixation, the cells were labelled with streptavidin- directs the import of the nucleus-encoded cytochrome c fluorescein before immediate image collection by con- oxidase (COX) subunit VIII into mitochondria.19 focal fluorescence microscopy. For human fibroblasts, HeLa, HepG2 and SY5Y (Figure 1), the mean intracellular Quantification of the cellular internalisation of peptide– fluorescence relative to background (MIF/B) was signifi- PNA conjugates cantly greater in the PNA-exposed cells compared with One major advantage of the biotinylated peptide–PNA controls (Table 1), consistent with the import seen pre- conjugate over the biotinylated PNA alone is that the con- viously with human myoblasts and repeated here jugate can be resolved by denaturing gel electrophoresis, (Ref. 18, Table 1 and Figure 2). The MIF/B for the biotin- migrating on SDS-PAGE as a function of its molecular treated myoblasts was not significantly greater than con- weight. The biotinylated conjugate can then be visualised = trol values (MIF/B biotin 0.85, s.d. 0.12, n 16, Figure 2), with 35S-radiolabeled streptavidin following Western confirming that the increase seen in the biotinylated PNA transfer. To quantify internalisation, human myoblasts exposed cells was due to PNA uptake and not free biotin were bathed in culture media containing varying concen- following degradation of the biotinylated PNA molecule. trations of the biotinylated conjugate for zero or 180 min. Despite numerous attempts, the NT2 and IMR 32 Cells were washed and several aliquots were trypsinized cell lines did not adhere to the slide during fixation to remove adventitiously bound conjugate before sub- and labelling. jecting cell lysates to denaturing gel electrophoresis as detailed in Materials and methods. Quantification was Three distinct phases following the uptake of PNA into made by comparing the signals from the internalisation human myoblasts experiments (Figure 3a) against a standard curve A time course revealed three distinct phases following (Figure 3b and c). As shown in Figure 3, approximately biotinylated PNA uptake into human myoblasts. Within 5000 myoblasts bathed in 2 ␮m biotinylated conjugate 30 min of exposure, punctate staining was apparent were able to internalise 11.5 pmols in 3 h. This rep- within the cytoplasm (Figure 2c), which increased up to resented an import of approximately 6% of the biotinyl- 4 h exposure (Figure 2d). Between 6 and 8 h exposure, the ated conjugate in the culture media. Internalisation was punctate fluorescence decreased, the cytoplasm became not limited by even the highest (5 ␮m) concentration of more diffusely fluorescent and there was a low level sig- biotinylated conjugate. nal in the nucleus (Figure 2e). In the third phase, after 8– 12 h, intranuclear fluorescence was more intense and was PNA import into isolated mitochondria still visible 24 h after exposure (Figure 2f). A similar tri- Having demonstrated that the biotinylated peptide–PNA phasic pattern was observed with a biotinylated oligo- conjugate can be internalised, we then wished to deter- deoxynucleotide, consistent with PNA and DNA mol- mine whether the conjugate could be targeted to and ecules sharing a similar import pathway (data not imported by mitochondria. Rat liver mitochondria were shown). We saw little residual punctate staining in myo- first tested for their import-competence by demonstrating blasts exposed to 20 ␮m biotinylated PNA for greater import of a 35S-methionine-labeled pSu9-DHFR chimaeric than 12 h. Pulsing the myoblasts with 20 ␮m biotinylated preprotein. Mitochondrial import of this chimaeric pro- PNA for 15 min and 30 min led to a significant reduction tein has been well characterised.20 It contains the first 69

in the amount of punctate fluorescence 6 h later, but the residues of N. crassa Fo-ATP synthase subunit 9, fused N- effects of a 1 h pulse were indistinguishable from myo- terminal to murine dihydrofolate reductase. When mito- blasts exposed continuously for 6 h (data not shown). chondria were incubated with pSu9-DHFR, the preprotein was cleaved, liberating the mature form (22 kDa) PNA uptake into human myoblasts is concentration which was resistant to proteolysis by added proteinase dependent K (Figure 4a). No preprotein cleavage and no protease- The intracellular fluorescence in cells exposed to 1 ␮m resistant material were observed when the membrane biotinylated PNA was indistinguishable from control potential was dissipated by addition of the mitochondrial values (Table 2), consistent with a threshold level for the uncoupler carbonyl cyanide m-chlorophenylhydrazone detection of uptake. Intracellular fluorescence was greater (CCCP). The uptake of biotinylated peptide–PNA conju- in myoblasts exposed to 10 ␮m biotinylated PNA in com- gate was studied in parallel experiments (Figure 4b). As PNA delivery to human mitochondria PF Chinnery et al 1921 a b

c d

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Figure 1 Confocal microscopy images showing the uptake of the biotinylated 11-mer PNA into cultured cells. Left hand images show cell lines not exposed to the PNA, but otherwise treated in exactly the same way as the cells exposed to 20 ␮m biotinylated PNA for 4 h at 37°C (right hand images). The confocal microscopy settings (aperture, black level and gain) were adjusted for the control cells of each cell type to achieve the image on the left, and kept constant to obtain the image on the right: (a and b) human fibroblasts; (c and d) HeLa; (e and f) HepG2; (g and h) SY5Y. (Magnification, ×1000). discussed in the previous section, experiments with bioti- prevent cleavage of the charged peptide from the conju- nylated PNA molecules that do not carry a peptide prese- gate, as the biotinylated peptide–PNA conjugate could be quence revealed that these small, uncharged molecules freely resolved by gel electrophoresis. Cleavage of the do not enter polyacrylamide gels, even in the presence of presequence in these experiments was prevented by the SDS. To visualise import, it was therefore necessary to addition of 1,10-phenanthrolene and EDTA which inhibit PNA delivery to human mitochondria PF Chinnery et al 1922 Table 1 Uptake of 20 ␮m PNA into cultured cells Table 2 PNA uptake into human myoblasts is concentration dependent Cell line MIF/Ba s.d. n Pb PNA/␮m MIF/Ba s.d. n Pb Human fibroblasts 1.69 0.39 8 Ͻ0.001 HeLa 1.69 0.38 8 Ͻ0.001 1 1.02 0.03 8 0.10 HepG2 3.03 0.42 8 Ͻ0.001 5 1.26 0.06 8 Ͻ0.001 SY5Y 2.97 0.36 8 Ͻ0.001 10 1.39 0.11 8 Ͻ0.001 Human myoblasts 1.52 0.15 8 Ͻ0.001 20 1.42 0.05 8 Ͻ0.001

aMIF/B, mean intracellular fluorescence pixel intensity/control aMIF/B, mean intracellular fluorescence pixel intensity/control (non-PNA treated) after 6-h exposure. (non-PNA treated) after 6-h exposure. bP values: result of two-sample t test between mean intracellular bP values: result of two-sample t test between mean intracellular fluorescence for the cell type and the control (PNA untreated) fluorescence for the cell type and the control (PNA untreated) cells of the same type. cells.

a b

c d

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Figure 2 Uptake of biotinylated 11-mer PNA into cultured human myoblasts. (a) Control cells not exposed to the biotinylated PNA but otherwise treated in an identical fashion. This image was used to set the confocal aperture, black level and gain. (b) 6-h exposure to 20 ␮m free biotin; (c) 1 h exposure to 20 ␮m biotinylated PNA; (d) 4-h exposure to 20 ␮m biotinylated PNA; (e) 8-h exposure to 20 ␮m biotinylated PNA; (f) 24 h exposure to 20 ␮m biotinylated PNA. (Magniﬁcation, ×1000). PNA delivery to human mitochondria PF Chinnery et al 1923

Figure 3 Quantiﬁcation of biotinylated peptide-PNA internalisation by human myoblasts. (a) Cells were subjected to 1 ␮m (lanes 1–3), 2 ␮m (lanes 4– 6) or 5 ␮m (lanes 7–9) biotinylated conjugate for either zero (lanes 1, 4, 7) or 180 min. Following washing, some cells were trypsinized to remove adventitiously bound conjugate (lanes 3, 6 and 9). Cell lysates were separated by denaturing gel electrophoresis, transferred to a nylon membrane and conjugates visualised as detailed in Materials and methods. (b) Known amounts of biotinylated peptide–PNA conjugate were subjected to a similar analysis and the resultant membrane is shown: Lane 1, 0 conjugate; 2, 20.2 pmol; 3, 50.5 pmol; 4, 101 pmol; 5, 202 pmol; 5, 505 pmol. (c) To quantify import, signals from the trypsinized cells were measured and compared with the standards shown in (b) and graphically represented in (c). the metal ion-dependent matrix processing peptidase, the import reaction inhibited the import of the peptide– MPP. Identical import experiments were performed with PNA conjugate into mitoplasts (Figure 4c, lane 3). Taken mitoplasts, in which the outer mitochondrial membrane together, these in vitro studies demonstrate the mem- was selectively permeabilised with digitonin, thereby brane potential-dependent translocation of the biotinyl- allowing proteinase K access to the inter-membrane ated peptide–PNA conjugate into the mitochondrial space. These experiments gave essentially the same matrix. results as those with intact mitochondria in that a proteinase-resistant peptide–PNA derivative was detectable Targeting PNA to mitochondria in cultured human after incubation with mitoplasts (Figure 4c, lane 2), con- myoblasts sistent with the biotinylated peptide–PNA conjugate We compared the uptake of the biotinylated peptide– being imported into the matrix. The addition of CCCP to PNA conjugate to the uptake of the non-conjugated bioti- PNA delivery to human mitochondria PF Chinnery et al 1924

Figure 4 Mitochondrial import of biotinylated peptide–PNA conjugate into isolated organelles. (a) Isolated rat liver mitochondria are import-competent. 35S-methionine-labeled pSu9-DHFR was incubated in the presence or absence of isolated rat liver mitochondria at 30°C for 60 min. Treatment with proteinase K revealed a proteinase-resistant, imported protein. Addition of CCCP to the import reaction disrupts the membrane potential, thereby inhibiting pSu9-DHFR import. (b) Import of biotinylated peptide–PNA conjugate into rat liver mitochondria. Mitochondria (200 ␮g) were preincubated with 1,10-phenanthroline and EDTA to inhibit cleavage of the conjugate (see Materials and methods) before starting import reactions by the addition of biotinylated-peptide-PNA conjugate (200 pmol). Treatment of the import reaction (lane 1) with proteinase K (lane 2) clearly showed a proteinase- resistant product, whilst in the presence of CCCP, proteinase K degraded all non-speciﬁcally bound peptide–PNA conjugate (lane 4). (c) Import of biotinylated peptide-PNA conjugate into rat liver mitoplasts. Import of conjugate into the mitochondrial matrix was conﬁrmed by performing identical import reactions in mitoplasts prepared by digitonin treatment of intact rat liver mitochondria (see Materials and methods). Treatment of the import reaction with proteinase K revealed a proteinase-resistant, imported conjugate (lane 2). This import was sensitive to the addition of CCCP (+PK; lane 3).

nylated PNA by confocal fluorescence microscopy n = 4). Although we noted a qualitative increase in the (Figure 5). Mitochondria were identified by the red flu- intra-nuclear signal in the presence of the biotinylated orescent signal from the dye MitoTracker Red CMX- conjugate, this did not reach statistical significance Ros.21 Both the biotinylated PNA and biotinylated pep- (MNF/B 5 ␮m biotinylated peptide–PNA 0.97, s.d. 0.18, tide–PNA were identified by the blue-green fluorescent n = 4). signal of streptavidin-fluorescein. Mean blue-green fluorescence pixel intensity for the nuclear and mitochon- Discussion drial compartments were expressed relative to background and statistical analyses were performed as We have shown that biotinylated PNAs are readily described previously. For myoblasts treated with 20 ␮m imported from culture medium by a number of different biotinylated PNA for 8 h (Figure 5c and d), no significant cell lines. Internalisation by myoblasts followed three dis- change was observed in the mean mitochondrial fluor- tinct phases: punctate, diffuse cytosolic and then nuclear escence relative to the background (MMF/B 20 ␮m PNA localisation. Why have other investigators failed to show 1.03, s.d. 0.09, n = 4), but the mean intranuclear PNA uptake into cells? The demonstration of uptake is fluorescence (MNF/B) increased with time (MNF 20 ␮m dependent upon the detection methods employed. The biotinylated PNA 1.34, s.d. 0.15, n = 4). By contrast, a uptake of DNA22 and PNA23 are cell-type specific and significant increase in the mean mitochondrial fluor- concentration dependent. Different investigators have escence was noted after 8 h for the 5 ␮m biotinylated pep- studied PNA uptake at lower concentrations and not in tide–PNA-treated myoblasts (MMF/B 1.28, s.d. 0.14, the cell types that we have studied.7 Despite a sensitive PNA delivery to human mitochondria PF Chinnery et al 1925 a b

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Figure 5 The COX VIII presequence localises the PNA to mitochondria in human myoblasts. Left-hand images collected down the red channel showing the distribution of the mitochondrial-specific label MitoTracker. Right-hand images collected down the green fluorescent channel showing the distribution of the biotinylated PNA or the COX VIII peptide–PNA conjugate. (a and b) Control with no biotinylated PNA or conjugate; (c and d) 8 h of 20 ␮m biotinylated PNA; (e and f) 8 h of 5 ␮m biotinylated peptide–PNA conjugate (magnification, ×1000); (g and h) higher power images of 8 h of 5 ␮m biotinylated peptide–DNA conjugate (magnification, ×1000); (g and h) higher power images of 8 h of 5 ␮m biotinylated peptide–PNA conjugate (magnification, ×2500). PNA delivery to human mitochondria PF Chinnery et al 1926 semi-quantitative detection method, relatively high con- facilitated mitochondrial targeting and import, this was centrations of PNA (Ͼ1 ␮m) were needed to demonstrate not entirely efficient, as a fraction of the molecules were uptake in our study. PNA uptake has been shown to still localised to the nucleus. In our studies, however, we occur in transformed and immortalised cell lines,24 prob- did not detect any cytotoxicity in cells grown for 10 days ably by fluid-phase endocytosis. Unlike the fluorochrome in 20 ␮m biotinylated PNA. tagged PNAs used by Bonham and colleagues,7 these When coupled with our in vitro studies,18 these obser- investigators radiolabelled the PNA with 14C- vations highlight the importance of PNAs as potential formaldehyde.24 It is therefore possible that these differ- anti-sense agents. Although there are many uncertainties ent labelling techniques influence the rate and pattern of surrounding the precise mechanisms of uptake and intra- PNA uptake in these studies. For example, extracellularly cellular trafficking, the evidence presented in this study delivered fluorescent-labelled PNA remained within supports the use of PNAs as potential tools for nuclear cytoplasmic endosomes, and did not localise within the and mitochondrial gene therapy. nucleus.7 By contrast, nuclear uptake was clearly demonstrated with the radiolabelled PNA molecules24 and with both biotinylated PNA and biotinylated peptide–PNA in Materials and methods our study.

Mitochondrial targeting and import Synthesis of PNA and peptide–PNA conjugate Mammalian mitochondria, unlike many plant, yeast and The biotinylated peptide nucleic acid (PNA) was syn- protozoan counterparts, do not need to import nucleic thesised by PerSeptive Biosystems (Framingham, MA, acids for efficient intra-mitochondrial protein translation. USA) from Fmoc expedite monomers. The full construct There is some evidence to suggest that the RNA moiety Bio-O-O-GTTGGCTCTCT-O-Lys-CONH2 was HPLC of a ribonucleoprotein possibly involved in maturing purified and the correct synthesis was confirmed by RNA primers for mtDNA replication may need to be MALDI-TOF. Synthesis of the peptide–PNA conjugate imported, but the mitochondrial location of this RNP is was performed by using standard Fmoc protection chem- 25,26 still disputed. Schon and colleagues have recently istry with TBTU as activator (ALTA Biosystems, Univer- reported that a 5S ribosomal RNA may be imported, but sity of Birmingham, UK). The PNA monomers and the 27 its function has yet to be determined. It is therefore Fmoc amino acids were purchased from PerSeptive possible that mammalian mitochondria do not possess an Biosystems. The biotinyl-lysine and the PNA component efficient import mechanism for nucleic acids. Vestweber were synthesised manually on a Biotech Instruments 28 and Schatz, however, were able to demonstrate that by BT7600. Resin was removed and added to the reactor col- subjecting import-competent, isolated mitochondria to umn of a PerSeptive Biosystems Pioneer instrument for DNA-conjugated mitochondrial precursor proteins, the the automated addition of the peptide. Cleavage from the DNA became nuclease or phosphodiesterase resistant, solid phase was achieved with a mix of trifluoroacetic consistent with the mitochondria importing nucleic acids acid:water:ethane dithiol (95:2:5:2.5). The full conjugate, via the general protein import pathway. A similar obser- containing the PNA with an identical sequence to the 29 vation was more recently reported by Seibel et al, non-conjugated PNA, was MSVLTPLLLRGLTGSARR although their experiments involved a less direct analysis LPVPRAKIHSL-PNA-Lys(biotin). This was HPLC pur- of the putatively imported DNA moiety. Our in vitro ified and correct synthesis confirmed by N-terminal studies with isolated rat liver mitochondria now show sequence analysis. that biotinylated PNAs can also be delivered across an intact mitochondrial membrane into the matrix compartment by the addition of a mitochondrial targeting pep- Cell culture and import conditions tide. The import of the biotinylated PNA-COX VIII conju- Human cell lines were grown in multi-chamber micro- gate required nucleoside triphosphates and an intact scope slide wells until 70% confluent (myoblasts, fibro- inner mitochondrial membrane potential. We have now blasts, Ntera 2, IMR32, HeLa, and HepG2 cell lines). Mus- extended these in vitro observations by confirming mito- cle cells were grown in Hams F10 medium (Gibco BRL) chondrial localisation of the peptide–PNA conjugate supplemented with 20% (v:v) foetal calf serum (FCS) and within intact cells by confocal fluorescence microscopy. 1% (v:v) chick embryo extract. Fibroblast explant cultures The resolution of this technique was not sufficient to were grown in Earle’s modified Eagle’s medium (EMEM, determine the exact intra-mitochondrial localisation of Sigma-Aldrich, Poole, UK) supplemented with 10% (v:v) the PNA, but taken together with our in vitro import FCS. Human Ntera 2 cell lines were grown in EMEM sup- assays, it is reasonable to suggest that the PNA conjugate plemented with 10% FCS, 1% (w:v) non-essential amino was imported to the mitochondrial matrix. Although it is acids, and 1% (w:v) l-glutamine. HeLa cells were grown not possible to comment on the degree of processing of in EMEM supplemented with 5% (v:v) FCS and 2 mml- the biotinylated peptide–PNA conjugate, we have shown glutamine. Hep G2 cells were grown in EMEM sup- that cleavage is not necessary for the sequence-specific plemented with 10% (v:v) FCS, 2 mml-glutamine, and inhibition of mutated mtDNA replication (Taylor et al, non-essential amino acids. Twelve hours before transfec- unpublished observations). tion the cells were transferred to serum-free medium. In conclusion, we have shown that biotinylated PNA Various concentrations of the biotin-labelled 11-mer molecules are readily internalised by various cell types. PNA, a 5Ј biotin-labelled 14-mer oligodeoxynucleotide, Ultimately the PNA localises to the nucleus, but conjugat- and the biotin-labelled peptide–PNA construct were ing the molecule with a mitochondrial-specific targeting added to the medium in concentrations ranging from 1 ␮ sequence directs at least a subset of the molecules to to 20 m. The cells were returned to a 5% CO2 incubator mitochondria. Although addition of the presequence at 37°C for the required time period. PNA delivery to human mitochondria PF Chinnery et al 1927 Cell fixation, fluorescent labelling and confocal SDS-polyacrylamide gel electrophoresis (PAGE) and microscopy autoradiography. Import reactions (100 ␮l total volume) After a variable time period, the cells were rinsed for were performed using 200 ␮g mitochondrial protein in a 3 min on three occasions in phosphate buffered saline buffer (220 mm mannitol, 70 mm sucrose, 10 mm Hepes- (pH 7.4) before fixation with freshly prepared 2.5% para- KOH, pH 7.4, 1 mm MgCl2,1mm DTT, 1 mm EDTA) sup- formaldehyde at 4°C for 15 min. Cells were then permea- plemented with 1 mm ATP, 5 mm NADH, 20 mm sodium bilised with 0.5% (v/v) Triton X-100 in 2.5% paraformal- succinate, 0.1 mm GTP, 1.5 mm creatine phosphate and dehyde for a further 15 min at 4°C before three additional 15 ␮g/ml creatine kinase. The import was initiated by the 3-min PBS rinses at room temperature. Cells were then addition of 5 ␮l of labelled pSu9-DHFR and incubated exposed to streptavidin-fluorescein (1:50, Boehringer at 30°C for 60 min under conditions in which the inner Mannheim, Lewes, UK) for 1 h. Excess label was membrane potential was either maintained or disrupted removed with three further PBS rinses before embedding by the inclusion of 50 ␮m carbonyl cyanide m-chloro- in glycerol/PBS (1:2 v/v). Images were collected immedi- phenylhydrazone (CCCP) in the import buffer. Protein- ately on a BioRad-MRC 600 confocal microscope with ase resistance of the imported protein was assessed by fixed laser and photomultiplier parameters (Kallman fil- incubating samples with 5 ␮g proteinase K for 30 min on ter factor 5). Control chambers were prepared in exactly ice. The proteinase was inactivated by the addition of the same way in an adjacent chamber on the microscope 1mm phenylmethylsulfonyl fluoride, mitochondrial slide. For the mitochondrial uptake studies, the mito- membranes washed in 1 ml of isolation medium and pel- chondrial-specific fluorescent dye dihydrotetramethylro- leted (13 000 g for 10 min). Mitochondrial pellets were samine20 (Mito Tracker, Molecular Probes, Eugene, OR, resuspended in sample gel-loading buffer, and proteins USA) was added to the culture medium to give a final analysed by SDS-PAGE and autoradiography. concentration of 2 ␮m, 45 min before fixation. In vitro import of peptide–PNA conjugate Image and statistical analysis Isolated rat liver mitochondria were pre-incubated with Digital image analysis was performed (COMOS). Semi- 2mm 1,10-phenanthroline and 5 mm EDTA for 15 min on quantitative analysis of mean pixel intensity within the ice before initiating import reactions by the addition of cells and subcompartments of the cells were measured 200 pmol peptide–PNA conjugate. Imports were perfor- from randomly chosen areas. The mean pixel intensity med at 30°C for 60 min, again in the presence and for each subcellular compartment was expressed relative absence of CCCP. Following proteinase K treatment (as to the background levels (mean fluorescence above detailed above), mitochondria were pelleted, solubilised background). Different values were compared by two- and separated through 16.5% tris-tricine gels. Separated sample t tests and analysis of variance when appropriate. proteins were transferred (200 mA for 16 h) on to PVDF membranes, and the biotinylated peptide–PNA conjugate Quantification of internalised peptide–PNA conjugate detected following incubation in 35S-streptavidin by Approximately 5–10 000 human myoblasts were seeded Phosphorimage analysis. Preincubation of mitochondrial into the wells of a 96-well microtitre tray. After 24 h, ␮ fractions with 1,10-phenanthroline and EDTA inhibits the varying concentrations of conjugate were added to 100 l activity of the matrix MPP, thereby allowing the peptide– serum-free medium and cells were either immediately PNA conjugate to remain unprocessed and maintain its washed or left for 3 h to internalise the biotinylated con- electrophoretic property. Proteinase treatment of the jugate. After washing in PBS, some aliquots were tryp- ␮ ° mitochondria after import allows the detection of pro- sinized (5 g trypsin 15 min 0 C) before all aliquots were teinase-resistant, imported PNA molecules. To confirm lysed by sample loading buffer and separated through a the presence of the biotinylated peptide–PNA within the 16.5% tris-tricine gel. Separated proteins were transferred mitochondrial matrix compartment, similar import (200 mA for 16 h) on to PVDF membranes (Immobilon- experiments were performed using mitoplasts prepared PSQ, Millipore, Watford, UK), and the biotinylated pep- 35 by treating mitochondrial aliquots with increasing tide–PNA conjugate detected following incubation in S- amounts of digitonin for 15 min on ice. Those aliquots streptavidin (Amersham Pharmacia Biotech, Amersham, that had been treated with the highest amount of digi- UK; 200–1000 Ci/mmol) by Phosphorimage analysis tonin to result in the release of less than 5% of the total (Molecular Dynamics, Sevenoaks, UK). citrate synthase activity were deemed to be mitoplasts. Aliquots (200 ␮g) were washed, pelleted (6000 g for Isolation of rat liver mitochondria 10 min), and import reactions performed as described Rat liver mitochondria were prepared as previously above. described30 and the protein concentration31 adjusted to 20 mg/ml. Spectrophotometric assay of the mitochondrial matrix marker citrate synthase in the presence and Acknowledgements absence of Triton X-10032 was performed to demonstrate the intactness (92–95%) of mitochondrial membranes. PFC is a Wellcome Trust Clinical Research Fellow. RWT is funded by the Muscular Dystrophy Campaign. We are In vitro mitochondrial preprotein import very grateful to Dr ZMA Chrzanowska-Lightowlers for The ability of isolated rat liver mitochondria to import her help with the various cultured cell lines. KD and RL precursor proteins was routinely confirmed using the acknowledge funding by grants of the Sonderforschungs- fusion protein pSu9-DHFR.22 The fusion protein was syn- bereich 286 of Deutsche Forschungsgemeinschaft and the thesised in rabbit reticulocyte lysate (Promega, Sou- Fonds der Chemischen Industrie. DMT and RNL are thampton, UK) using 35S-methionine (ICN Radiochem- indebted to the Muscular Dystrophy Campaign and the icals, Basingstoke, UK; 1000 Ci/mmol), and analysed by Wellcome Trust for continuing support. PNA delivery to human mitochondria PF Chinnery et al 1928 References 18 Taylor RW, Chinnery PF, Turnbull DM, Lightowlers RN. Selec- tive inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids. Nat Genet 1997; 15: 212–215. 1 Corey DR. Peptide nucleic acids: expanding the scope of nucleic acid recognition. Trends Biotechnol 1997; 15: 224–229. 19 Rizzuto R et al. A gene specifying subunit VIII of human cyto- 2 Demidov VV, Yavnilovich MV, Frank-Kamenetskii MD. Kinetic chrome c oxidase is localised to chromosome 11 and is analysis of specificity of duplex DNA targeting by homopyrim- expressed in both muscle and non-muscle tissues. J Biol Chem idine peptide nucleic acids. Biophysical J 1997; 72: 2763–2769. 1989; 264: 10595–10600. 3 Demidov VV et al. Stability of peptide nucleic acids in human 20 Rapaport D, Neupert W, Lill R. Mitochondrial protein import: serum and cellular extracts. Biochem Pharmacol 1994; 48: 1310– Tom40 plays an important role in targetting and translocation 1313. of preproteins by forming a specific binding site for the prese- 4 Norton JC et al. Inhibition of human telomerase activity by pep- quence. J Biol Chem 1997; 272: 18725–18731. tide nucleic acids. Nature Biotechnol 1996; 14: 615–619. 21 Whittaker JE, Moore PL, Haugland RP. Dihydrotetramethylros- 5 Hanvey JC et al. Antisense and antigene properties of peptide amine: a long wavelength, fluorogenic peroxidase substrate nucleic acids. Science 1992; 258: 1481–1485. evaluated in vitro and in a model phagocyte. Biochem Biophys 6 Nielsen PE, Egholm M, Berg RH, Buchardt O. Peptide nucleic Res Commun 1991; 751: 387–393. acids (PNAs): potential antisense and anti-gene agents. Anti- 22 Gewirtz AM, Stein CA, Glazer PM. Facilitating oligonucleotide Cancer Drug Des 1993; 8: 53–63. delivery: helping antisense deliver on its promise. Proc Natl Acad 7 Bonham MA et al. An assessment of the antisense properties of Sci USA 1996; 93: 3161–3163. RNase H-competent and steric-blocking monomers. Nucleic 23 Good L, Nielsen PE. Antisense inhibition of gene expression in Acids Res 1995; 23: 1197–1203. bacteria by PNA targeted to mRNA. Nat Biotechnol 1998; 16: 8 Wittung P et al. Phospholipid membrane permeability of pep- 355–358. tide nucleic acid (corrected and republished with original pag- 24 Gray GD, Basu S, Wickstrom E. Transformed and immortalized ing, article originally printed in FEBS Lett 1995; 365: 27–29). cellular uptake of oligodeoxynucleoside phosphorothioates, 3Ј- FEBS Lett 1995; 375: 27–29. alkylamino oligodeoxynucleosides, 2-O-methyl oligoribonucleo- 9 Dowty ME, Wolff JA. Possible mechanisms of DNA uptake into tides, oligodeoxynucleoside methylphosponates, and peptide skeletal muscle. In: Wolff JA (ed). Gene Therapeutics: Methods and nucleic acids. Biochem Pharmacol 1997; 53: 1465–1476. Applications of Direct Gene Transfer. Birkhauser: Boston, 1994, 25 Kiss T, Filipowicz W. Evidence against a mitochondrial location pp 83–98. of the 7-2/MRP RNA in mammalian cells. Cell 1992; 70: 11–16. 10 Chinnery PF, Turnbull DM. Mitochondrial medicine. Q J Med 26 Li K et al. Subcellular partitioning of MRP RNA assessed by 1997; 90: 657–666. ultrastructural and biochemical analysis. J Cell Biol 1994; 124: 11 Taylor RW et al. Treatment of mitochondrial disease. J Bioenerg 871–882. Biomemb 1997; 29: 195–205. 27 Magalhaes PJ, Andreu AL, Schon EA. Evidence for the presence 12 Chinnery PF, Turnbull DM. The clinical features, investigation of 5S rRNA in mammalian mitochondria. Mol Biol Cell 1998; 9: and management of patients with mitochondrial DNA defects. 2375–2382. J Neurol Neurosurg Psych 1997; 63: 559–563. 13 Larsson N-G, Clayton DA. Molecular genetic aspects of human 28 Vestweber D, Schatz G. DNA–protein conjugates can enter mito- mitochondrial disorders. Ann Rev Genet 1995; 29: 151–178. chondria via the protein import pathway. Nature 1989; 338: 14 Attardi G, Yoneda M, Chomyn A. Complementation and segre- 170–172. gation behavior of disease causing mitochondrial DNA 29 Seibel P et al. Transfection of mitochondria: strategy towards a mutations in cellular model systems. Biochim Biophys Acta 1995; gene therapy of mitochondrial DNA diseases. Nucleic Acids Res 1271: 241–248. 1995; 23: 10–17. 15 Chinnery P, Howell N, Lightowlers R, Turnbull D. Molecular 30 Taylor RW et al. The control of mitochondrial oxidations by pathology of MELAS and MERRF: the relationship between complex III in rat muscle and liver mitochondria. Implications mutation load and clinical phenotype. Brain 1997; 120: 1713– for our understanding of mitochondrial cytopathies in man. J 1721. Biol Chem 1994; 269: 3523–3528. 16 Gross NJ, Getz GS, Rabinowitz M. Apparent turnover of mito- 31 Peterson GL. A simplification of the protein assay of Lowry et chondrial deoxyribonucleic acid and mitochondrial phospholip- al which is more generally applicable. Ann Biochem 1977; 83: ids in the tissues of the rat. J Biol Chem 1969; 244: 1552–1562. 346–356. 17 Chrzanowska-Lightowlers ZM, Lightowlers RN, Turnbull DM. 32 Shepherd D, Garland PB. Citrate synthase from rat liver. Meth Gene therapy for mitochondrial DNA defects: is it possible? Enzymol 1969; 13: 11–16. Gene Therapy 1995; 2: 311–316.