Specific targeting of a DNA-alkylating reagent to mitochondria
Synthesis and characterization of [4-((11aS)-7-methoxy-1,2,3,11a-tetrahydro-
5
H
-pyrrolo[2,1-
c
][1,4]benzodiazepin-5-on-8-oxy)butyl]-triphenylphosphonium iodide
Andrew M. James
1
, Frances H. Blaikie
2
, Robin A. J. Smith
2
, Robert N. Lightowlers
3
, Paul M. Smith
3
and Michael P. Murphy
1
1
MRC-Dunn Human Nutrition Unit, Wellcome Trust-MRC Building, Cambridge, UK;
2
Department of Chemistry, University
of Otago, Dunedin, New Zealand;
3
Department of Neurology, Medical School, University of Newcastle upon Tyne, UK
The selective manipulation of the expression and replica-
tion of mitochondrial DNA (mtDNA) within mammalian
cells has proven difficult. In progressing towards this goal
we synthesized a novel mitochondria-targeted DNA-
alkylating reagent. The active alkylating moiety [(11aS)-8-

there are no effective therapies [2–8]. Possibilities for
treatment, such as the replacement of the defective gene
by gene therapy, are being explored; however, gene therapy
for mtDNA diseases is even more challenging than for
nuclear gene defects because of the problem of delivering
DNA to mitochondria, the difficulty of generating stable
insertion or expression of exogenous DNA within mam-
malian mitochondria, and the large number of mitochon-
dria and mtDNA molecules per cell [9,10]. Even if this
approach is effective, it will not be practical for the majority
of mtDNA diseases that are caused by mtDNA deletions, or
mutations in RNA genes [9,11] until we extend our
knowledge of potential RNA import processes in mamma-
lian mitochondria [12].
Because of these challenges an alternative ÔantigenomicÕ
strategy has been developed as a potential therapy for
mtDNA diseases [13–16]. This approach does not introduce
a functioning copy of the defective gene; instead it utilizes
the following mtDNA properties: mtDNA is present in
patients at high copy number as a mixture of both normal
and mutated molecules; mtDNA diseases are only pheno-
typically expressed above a threshold proportion of mutated
mtDNA; and mtDNA is continually degraded and resyn-
thesized. Consequently, if the proportion of mutated
mtDNA molecules in a patient can be decreased below this
threshold the disease phenotype may be suppressed. This
could be done by selectively enhancing the degradation, or
inhibiting the replication, of mutated mtDNA molecules
without affecting wild-type mtDNA [17]. The potential of
this approach has been demonstrated for the mtDNA

complementary DNA sequences [19], selectively inhibiting
the replication of mutated mtDNA sequences without
disrupting wild-type sequences that differ by only a single
base pair [20]. Furthermore PNAs can be delivered to the
mitochondrial matrix, by attachment of a mitochondrial
protein import sequence [21], or conjugation to a lipophilic
cation [22]. However, even though both approaches
appeared to lead to the accumulation of a PNA
within the mitochondria of cultured cells, neither affected
the proportion of mutated mtDNA in heteroplasmic
myoclonic epilepsy with ragged-red fibres (MERRF) cells
[23].
Accepting that the PNA has been successfully transpor-
ted to the site of mtDNA replication, these negative results
suggestthatdeliveryofaPNAthatbindstoaparticular
sequence to mitochondria is not sufficient, perhaps because
the PNA does not form a complex with the target mtDNA
sequence that is durable enough to inhibit mtDNA repli-
cation or expression. One approach to increase the duration
of PNA binding to DNA is to conjugate it to a DNA-
alkylating reagent so that the PNA becomes covalently
bound to its target sequence. To do this a DNA alkylating
reagent that reacts relatively slowly with DNA is required to
ensure that the alkylation occurs following binding to the
specific sequence by the PNA. As a first step towards this
goal we set out to develop a mitochondria-targeted DNA-
alkylating reagent to determine whether it was possible to
alkylate mtDNA within intact mitochondria and cells. In
addition to facilitating the development of antigenomic
therapies, such a reagent might also be useful in investi-

mitochondria. However, in spite of its substantial import,
it did not alkylate mtDNA in isolated mitochondria or
cells. This unexpected finding has significant implications
for the development of antigenomic therapies for mtDNA
diseases.
Fig. 1. Selective uptake of mitoDC-81 by mitochondria and subsequent
alkylation of mtDNA. The uptake of mitoDC-81 into cells driven by
theplasmamembranepotential(Dw
p
) followed by the further accu-
mulation of mitoDC-81 into mitochondria driven by the mitoch-
ondrial membrane potential (Dw
m
) is illustrated. The Nernst equation
indicates a 10-fold increase in accumulation for every 61.5 mV of
membrane potential. This leads to a millimolar concentration of
mitoDC-81 within mitochondria on incubation of cells with high
nanomolar to micromolar concentrations of mitoDC-81. This high
local concentration of mitoDC-81 within mitochondria could then
lead to the alkylation and inactivation of mtDNA, as indicated in the
figure.
2828 A. M. James et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Materials and methods
Synthesis of [4-((11a
S
)-7-methoxy-1,2,3,11a-tetrahydro-
5
H
-pyrrolo[2,1-
c

reduced pressure for 3 h yielding 2 as a mustard coloured
solid (12 mg, 0.014 mmol, 14%).
1
HNMRd 7.6–7.9 (m
(16H, P
+
–ArH and H-11),7.46(1H,s,H-6), 6.49 (1H, s,
H-9),4.1–4.2(2H,m,Ar-O-CH
2
), 3.71 (3H, s, O-CH
3
), 3.4–
4.0(5H,m,P
+
–CH
2
, H-11a and H-3), 1.75–2.4 (8H, m,
O-CH
2
-CH
2
,P
+
–CH
2
-CH
2
, H-1andH-2) p.p.m.,
31
PNMR d 25.65 p.p.m. ESMS found (M

M
Tris/HCl
pH 8.0, 1 m
M
EDTA (TE) supplemented with 500 l
M
mitoDC-81, 500 l
M
methyl triphenylphosphonium (TPMP)
or ethanol carrier. DNA was then precipitated with 1 vol.
0.6
M
NaOAc, 20 m
M
EDTA followed by 2 vols cold
ethanol. After centrifugation (13000 g for 30 min at 4 °C)
the pellet was resuspended in 50 lL10m
M
Tris/HCl
pH 8.0, 0.1 m
M
EDTA and 1 lgwasrunat60 Vona0.6%
agarose/ethidium bromide gel. The DNA was electrotrans-
ferred to a positive nylon membrane (Hybond N+,
Amersham Pharmacia Biotech), treated with 0.4
M
NaOH,
and fixed by UV irradiation. The membrane was then
blocked overnight with 1% (w/v) milk powder, 0.05% (v/v)
Tween-20 in TBS (TBST) before incubation for 1 h with a

positive nylon membrane and TPP moieties were detected
using anti-TPP serum as above.
Mitochondrial incubations
Rat liver mitochondria were prepared by homogenization
followed by differential centrifugation [37]. Protein concen-
tration was determined using the biuret assay with BSA as
standard [38]. The uptake of mitoDC-81 by mitochondria
was measured at 30 °C using a mitoDC-81-sensitive ion-
Scheme 1. [4-((11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-
c][1,4]benzodiazepin-5-on-8-oxy)butyl]triphenylphosphonium iodide (2;
mitoDC-81).
Ó FEBS 2003 Mitochondrial DNA alkylation (Eur. J. Biochem. 270) 2829
selective electrode suspended in a stirred chamber open to
the atmosphere [39,40]. To calibrate the electrode, five
stepwise additions of 1 l
M
mitoDC-81 were made to 3 mL
250 m
M
sucrose, 5 m
M
Tris/HCl pH 7.4, 1 m
M
EGTA,
containing isolated rat liver mitochondria (1 mg pro-
teinÆmL
)1
). The mitochondria were energized with 10 m
M
succinate and the membrane potential was dissipated

Cell incubations
Human 143B osteosarcoma cells were cultured in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 10%
(v/v) foetal bovine serum, glucose (4.5 gÆL
)1
), streptomycin
sulfate (100 mgÆL
)1
), penicillin G (100 000 UÆL
)1
), uridine
(50 mgÆL
)1
) and pyruvate (100 mgÆL
)1
) unless otherwise
stated. Cells were grown in plastic flasks or on glass
coverslips at 37 °C in a humidified atmosphere of 5%
CO
2
/95% air until confluent.
For confocal microscopy, cells were grown overnight on
glass coverslips then treated for 6 h with 500 n
M
(4-iodo-
butyl)triphenylphosphonium (IBTP), 500 n
M
TPMP, or
500 n
M

and fluorescence detected using a 570LP filter. The green
and red channels were acquired using Biorad Lasersharp
2000 and subsequently merged to indicate colocalization.
The laser intensity, iris and gain settings were identical for
all images shown.
To assess whether mitoDC-81 could completely deplete
mtDNA and thereby create q° cells, 143B cells were grown
in DMEM/foetal bovine serum supplemented with
50 mgÆL
)1
uridine and passaged twice per week in the
presence of either 500 n
M
mitoDC-81 or 500 n
M
TPMP for
a period of 17 days [24]. Cells were cloned by dilution and
after a further 28 days their rate of oxygen consumption
respiring on succinate was measured after digitonin-perme-
abilization [41].
Results
Synthesis of mitoDC-81
The compound of interest, mitoDC-81 2, was prepared by
reaction of the phenoxide anion derived from 1 with IBTP
(Scheme 1). This approach was found to be more effective
than creating a bromoalkyl side chain on 1 followed by
reaction with triphenylphosphine. The product 2 (Scheme 1)
was isolated and characterized as described for previously
synthesized complex triphenylphosphonium salts [30,31].
Accumulation of mitoDC-81 by isolated mitochondria

mitochondrial volume is about 0.5–0.9 lLÆmg protein
)1
under these conditions [42–44] this corresponds to an
intramitochondrial mitoDC-81 concentration of 2.5–5 m
M
,
although the steady-state free matrix concentration is likely
to be about 60–80% lower than this due to binding to
the matrix-facing surface of the inner membrane [45]. When
the membrane potential was dissipated by addition of the
uncoupler FCCP, mitoDC-81 was rapidly released from the
mitochondria (Fig. 2). Therefore mitoDC-81 is accumu-
lated about a thousand-fold within energized mitochondria,
driven by the membrane potential.
Alkylation of isolated DNA by mitoDC-81
To determine if the conjugation of a lipophilic cation to
DC-81 disrupted its ability to alkylate DNA, we determined
whether mitoDC-81 could alkylate DNA in vitro.Todothis
we incubated 500 l
M
mitoDC-81 with linearized DNA
molecules of different sizes (i.e. a standard DNA ÔladderÕ
used as a molecular weight marker). After incubation the
DNA was separated by electrophoresis, electrotransferred
to a nylon membrane and probed for mitoDC-81 covalently
bound to the DNA by immunoblotting using anti-TPP
serum [35] (Fig. 3A,B). This showed considerable alkylation
of the DNA ladder by mitoDC-81 (Fig. 3B, lane 1).
Analysis of the gel prior to electrotransfer showed that the
extent of alkylation was proportional to the amount of

from the immunoblot was due to poor transfer from the gel
to the membrane, as was confirmed by Southern blotting.
The presence of relaxed-circular mtDNA after digestion
with ClaI may indicate alkylation of the restriction site as
untreated mtDNA, or mtDNA isolated from mitoDC-81-
treated mitochondria, was always fully linearized by ClaI
under identical conditions. MtDNA that had been ran-
domly broken during isolation or incubation with mitoDC-
81 was also extensively alkylated and migrated as a smear
when subsequently cut with ClaI( 4–8 kb; data not
shown).
We conclude that conjugation of the lipophilic cation to
DC-81 does not disrupt its ability to alkylate DNA and high
micromolar concentrations of mitoDC-81 are sufficient to
alkylate linear, circular and supercoiled DNA. As these
concentrations of mitoDC-81 are easily achieved within
the mitochondrial matrix on incubation with mitoDC-81
(Fig. 2), mitoDC-81 should also alkylate mtDNA within
mitochondria and cells.
MitoDC-81 does not alkylate mtDNA in isolated
mitochondria
Having ascertained that mitoDC-81 was sequestered by
isolated mitochondria and that it could covalently modify
Fig. 3. MitoDC-81 alkylates linear, relaxed-circular and supercoiled
DNA in vitro. A 1-kb ladder of linear DNA (1 lgÆmL
)1
) or plasmid
DNA (1 lgÆmL
)1
; 6.8 kb) was incubated for 6 h at 37 °Cwith500 l

(Fig. 2), the
concentration of mitoDC-81 should not be a limiting
factor. Respiration rate measurements indicated that
isolated mitochondria incubated at 30 °C for 1 h could
still be stimulated with uncoupler, but longer incubations
led to the loss of mitochondrial coupling (data not shown).
Therefore it was important to determine if a 1-h incubation
was sufficient to lead to detectable DNA labelling by
mitoDC-81. To do this, isolated plasmid DNA was
incubated with various concentrations of mitoDC-81 for
1 h (Fig. 4A,B). Extensive alkylation of the DNA was
noted after 1 h incubation at concentrations as low as
50 l
M
mitoDC-81 (Fig. 4A,B). That the concentration of
mtDNA within mitochondria is also unlikely to be limiting
under these conditions is supported by the following rough
calculations. There are about 18.7 · 10
9
mtDNA mole-
culesÆmg protein
)1
in rat liver mitochondria [47], giving about
3.05 · 10
14
bpÆmg protein
)1
, or 335 ng DNAÆmg protein
)1
.

with cultured cells, which can be incubated with mitoDC-81
indefinitely without disrupting their mitochondrial mem-
brane potential. Therefore we next investigated whether
mitoDC-81 alkylated mtDNA within intact cells. Cells
couldbeincubatedwithupto500n
M
mitoDC-81 indefin-
itely, although higher concentrations (1–5 l
M
)weretoxic
over 2–24 h. Using 500 n
M
mitoDC-81 will generate an
ample intramitochondrial concentration of mitoDC-81
because mitoDC-81 will be accumulated within the cyto-
plasm driven by the plasma membrane potential and then
be further accumulated within the mitochondria due to the
mitochondrial membrane potential [11]. From the known
plasma and mitochondrial membrane potentials and the cell
and mitochondrial volumes of 143B cells [48] we estimate an
intramitochondrial concentration of  450 l
M
mitoDC-81
for 143B cells incubated with 500 n
M
mitoDC-81.
To see if long-term incubation with mitoDC-81 did result
in alkylation of mtDNA, cells were incubated with 500 n
M
mitoDC-81, IBTP, or TPMP for 24 h and probed using

mitoDC-81, were included as positive
controls and molecular mass markers.
2832 A. M. James et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the same way as mitoDC-81, and once there it binds slowly
but irreversibly to protein thiols, enabling it to be detected
by anti-TPP serum [35]. If mitoDC-81 binds to mtDNA it
will also be retained within mitochondria after fixation and
should be visible by immunocytochemistry in the same way
as IBTP. To see if this was the case, cells were incubated
with 500 n
M
IBTP,TPMPormitoDC-81for24h,then
fixed and dual-labelled with antibodies to the mitochondrial
enzyme cytochrome oxidase (red) and to TPP (green;
Fig. 5). The images were then merged, so yellow indicates
colocalization of the TPP and cytochrome oxidase, while
red indicates that only the cytochrome oxidase was detected
and that there was no TPP immunoreactivity. This experi-
ment showed that IBTP colocalized with cytochrome
oxidase due to its accumulation by mitochondria and
subsequent irreversible reaction with protein thiols
(Fig. 5A). Although TPMP will also accumulate within
mitochondria, it did not generate any TPP-labelling as it is
not covalently bound inside the matrix and is lost on
fixation (Fig. 5B). Confocal microscopy of cells incubated
with mitoDC-81 (Fig. 5C) gave a pattern of fluorescence
that resembled that of TPMP, rather than IBTP. This
suggests that mitoDC-81 is not covalently bound to
mtDNA within mitochondria. Incubations from 2 h to
3 days with mitoDC-81 concentrations ranging from

14
basesÆmg
protein
)1
, 19.3% of which are guanines corresponding to
1.18 · 10
14
guanine residuesÆmg protein
)1
or about
200 pmol GÆmg protein
)1
. Therefore we would predict that
mitoDC-81alkylationofafewpercentoftheavailable
mtDNA guanine residues should have been detectable by
confocal microscopy after 24 h. Even so, we cannot entirely
exclude the possibility that mitoDC-81 did alkylate a small
proportion of the guanine residues in mtDNA, but that the
amount alkylated was below the threshold for detection, or
that alkylated mtDNA is rapidly degraded or repaired. In
summary, we found no evidence for alkylation of mtDNA
by mitoDC-81 within cells.
Long-term incubation with mitoDC81 did not impair
cellular respiration
For mitoDC-81 to be of potential use in preventing
mtDNA replication it might require only a few molecules
of mitoDC-81 bound per mtDNA molecule. Thus, even
though we could not directly detect alkylation of mtDNA
by mitoDC-81, it remained possible that mitoDC-81 was
bound to mtDNA, but at concentrations below the

(cytochrome oxidase) mAb and anti-TPP serum, respectively.
Ó FEBS 2003 Mitochondrial DNA alkylation (Eur. J. Biochem. 270) 2833
of mitoDC-81 as a bulk culture of 143B cells grown in
500 n
M
mitoDC-81 for 6 weeks had normal rates of
mitochondrial respiration (data not shown). Concentra-
tions of mitoDC-81 >500 n
M
were too toxic for long-term
culture and 500 n
M
mitoDC-81 slowed cell growth slightly
relative to 500 n
M
TPMP or no additions (data not
shown), indicating that the DC-81 moiety of mitoDC-81
was affecting the cells. Interestingly, this growth inhibition
was not related to effects on mtDNA as 500 n
M
mitoDC-
81 completely prevented the growth of, although did not
necessarily kill, a previously established 143B-derived q°
cell line (data not shown). This effect was specific to
mitoDC-81 and was not due to nonspecific disruption by
the lipophilic cation as q° cells cultured with 500 n
M
TPMP grew at the same rate as control incubations (data
not shown). One possible interpretation is that the higher
mitochondrial membrane potential of 143B cells causes

ample to alkylate isolated mtDNA, and despite the fact that
with cells it was possible to incubate for far longer periods
(24 h), than was possible with isolated mitochondria.
Finally, alkylation of mtDNA by mitoDC-81 would be
expected to disrupt mitochondrial biogenesis and lead to
depletion of mtDNA, but there was no generation of q° or
respiratory-deficient cells on long-term incubation with
mitoDC-81. Therefore we found no evidence that mitoDC-
81 alkylates mtDNA in mitochondria or cells.
The reasons for the lack of alkylation of mtDNA
within mitochondria by mitoDC-81 are unclear. The local
concentrations of mitoDC-81 and DNA, and the duration
of the experiments were ample to alkylate isolated DNA.
One possibility is that mitoDC-81 does alkylate mtDNA
within mitochondria, but this modified DNA is then
rapidly degraded. This seems unlikely, as the amount of
mtDNA isolated from treated and untreated mitochon-
dria was similar. In addition, such a scenario would have
been expected to readily generate q° clones, which were
not found. Alternatively, upon alkylation the mtDNA, or
the mitochondria themselves, may have become difficult
to isolate. However, the similar yields of mtDNA from
isolated mitochondria treated with mitoDC-81 again
make this explanation unlikely. Furthermore, the lack of
labelling of mtDNA by mitoDC-81 in intact cells, as
explored by immunocytochemistry, makes it unlikely that
mtDNA was alkylated in situ, but was then selectively lost
in subsequent manipulations during which unmodified
mtDNA was retained. Alternatively, mitoDC-81 may
react with RNA, nucleotides, nucleosides or other biolo-

increase in nuclease resistance in vitro [54]. Similar
targeting of PNAs to mitochondria also failed to show
inhibition of mtDNA replication in intact cells [21,23],
suggesting that access of the PNA to mtDNA in the
matrix was also limited.
In summary, even though an active DNA alkylating
reagent could be delivered to mitochondria there was no
evidence for its reaction with mtDNA in situ.These
unexpected findings suggest that the accessibility of
mtDNA to some alkylating reagents may be constrained.
It may be that more reactive alkylating reagents could be
used to modify mtDNA, but nonspecific reactions with
mtDNA or modification to nuclear DNA could limit this
approach. A better understanding of the selective alky-
lation of mtDNA in situ is required in order to develop
therapeutic strategies to deplete mutated mtDNA mole-
cules selectively.
2834 A. M. James et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Acknowledgement
We thank P. Howard (School of Pharmacy, University of London) for
thegiftofDC81.
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