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INTRODUCTION |
Efficient methods for site-directed genome modification are
desirable for research and possibly for gene therapy applications. One
approach utilizes triplex-forming oligonucleotides
(TFOs),1 which bind as third
strands to duplex DNA in a sequence-specific manner, to mediate
directed mutagenesis. Such TFOs can act either by delivering a tethered
mutagen, such as psoralen or chlorambucil (1-5), or by binding with
sufficient affinity to provoke error-prone repair (6).
Another strategy for genome modification involves the induction of
homologous recombination between an exogenous DNA fragment and the
targeted gene. This approach has been used successfully to target and
disrupt selected genes in mammalian cells and has enabled the
production of transgenic mice carrying specific gene knockouts (7).
This approach, however, relies on the transfer of selectable markers to
allow isolation of the desired recombinants. Without selection, the
ratio of homologous to nonhomologous integration of transfected DNA in
typical gene transfer experiments is low, usually in the range of
1:1000 or less (8). This low efficiency of homologous integration
limits the utility of gene transfer for gene therapy.
The frequency of homologous recombination can be enhanced by damage to
the target site from UV irradiation and selected carcinogens (9) as
well as by site-specific endonucleases (8, 11, 12). We and others have
also demonstrated that DNA damage induced by triplex-directed psoralen
photoadducts can stimulate recombination within and between
extrachromosomal vectors (12, 13).
Other work has helped to define parameters that influence recombination
in mammalian cells. In general, linear donor fragments are more
recombinogenic than their circular counterparts (14). Recombination is
also influenced by the length of uninterrupted homology between both
the donor and target sites, with short fragments appearing to be
inefficient substrates for recombination (15). Nonetheless, several
recent efforts have focused on the use of short fragments of DNA or
DNA/RNA hybrids for gene correction (10, 16).
A direct method of targeting donor DNA to the gene of interest is
lacking in many of the above recombination strategies. The process by
which a DNA fragment finds its site of homology in mammalian cells is
not fully understood, but it is thought that recA-like factors (such as
Rad51) catalyze a homology search and mediate DNA pairing in an
intricate, energy-dependent manner. In contrast, a TFO can
find its cognate site within complex DNA without the need for any
associated enzyme activity (5, 17). Indeed, in previous work, we found
that a TFO could find and modify its target site within mouse genomic
DNA in vitro within minutes (18).
The sequence-specific binding properties of TFOs have been used to
deliver a series of different molecules to target sites in DNA. For
example, a diagnostic method for examining triplex interactions
utilized TFOs coupled to Fe-EDTA, a DNA cleaving agent (19). Others
have linked biologically active enzymes like micrococcal nuclease and
streptococcal nuclease to TFOs and demonstrated site-specific cleavage
of DNA (20, 21). We and others have previously shown that site-directed
DNA damage and mutagenesis can be achieved using TFOs conjugated to
either psoralen (1, 4) or alkylating agents (5, 22).
Because TFOs can efficiently deliver reactive molecules to specific
sites within target genes, we asked whether a TFO could also be used to
guide a homologous donor DNA fragment to its intended target site and
position it for efficient information transfer via recombination and/or
gene conversion. Thus, we designed an oligonucleotide that covalently
tethers a TFO to a DNA donor fragment. In addition, because triplex
formation itself can provoke repair (6) activity, we hypothesized that
the TFO, in addition to positioning the donor fragment, may also
enhance strand transfer and recombination by inducing repair activity
at the target site.
Here we describe the design of several TD-TFO reagents. These bind
specifically and with high affinity to DNA via a triplex-forming domain
while simultaneously providing DNA sequence information through an
attached donor domain to revert or induce a mutation in a target gene
(Fig. 1). Using a SV40-based shuttle vector assay, we demonstrate that
these TD-TFOs can mediate specific and directed sequence changes within
an extrachromosomal supF reporter gene in mammalian cells.
Furthermore, we show that the intact TD-TFO molecule is more active
than either of its parts alone or unlinked combinations thereof and is
also more active than control oligonucleotides that substitute either
domain with a control sequence. Using human mutant cell lines, we also
demonstrate that the enhanced activity of the combined TD-TFO is
diminished in cells deficient in nucleotide excision repair (NER),
suggesting that the TFO domain may stimulate gene conversion in part
through the ability of triple helices to provoke repair.
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EXPERIMENTAL PROCEDURES |
Oligonucleotides--
Oligonucleotides were synthesized by
standard phosphoramidite chemistry using materials from Glen Research
(Sterling, VA) and purified by either gel electrophoresis or high
pressure liquid chromatography, followed by Centricon-3 filtration in
distilled water (Amicon, Beverly, MA). The oligonucleotides consisted
primarily of phosphodiester linkages but were modified at the 3' end to resist 3' exonuclease activity by the inclusion of phosphorothioate linkages at the terminal three residues. Exceptions were the
oligonucleotides A, B, B(144), B(Sal), and Y(Rndm), which included a 3'
propylamine (Glen Research) as their only modification. The linker
segment between the donor fragment and the TFO domain consisted of the sequence 9TT9TT9, in which 9 indicates a 9-carbon polyethylene glycol
linker (Spacer 9; Glen Research). The pyrimidine oligonucleotide UC30
was composed of the RNA nucleosides 2'-O-methyluridine and 5-methyl-2'-O-methylcytidine.
Vectors--
The mammalian shuttle vectors pSupFG1 and
pSupFG1/G144C (Fig. 2A), which contain a wild-type
supFG1 gene or a supFG1 gene with an inactivating
G-to-C point mutation at position 144, respectively, were prepared by
large-scale DNA preparation (Qiagen, Santa Clarita, CA). The pSupFG1
plasmid has been described previously (3). The mutant pSupFG1/G144C
plasmid was isolated in the course of mutagenesis experiments with
pSupFG1 (3).
The shuttle vector pSupF5/G115T was used in experiments involving
TD-TFOs in the pyrimidine triplex motif. This vector is distinct from
pSupFG1; it contains a modified supF gene in which a 30-bp
A-rich site amenable to triplex formation in the pyrimidine motif has
been placed immediately 5' to the supF gene (Fig.
2B). The gene also contains an inactivating G-to-T point
mutation at position 115.
Cells--
Monkey COS-7 cells were obtained from American Type
Culture Collection (1651-CRL). Transformed XPA fibroblasts from patient XP2OS and XP2OS cells transfected with a vector expressing XPA cDNA
(XP2OS-pCAH19WS) were obtained from K. Kraemer (23). The cells were
grown in growth media (Dulbecco's modified Eagle's medium; Life
Technologies, Inc.), 10% fetal calf serum (Life Technologies, Inc.),
and 1% penicillin/streptomycin (Sigma) at 37 °C in a humidified incubator in the presence of 5% CO2.
Gel Mobility Shift Assays--
2 pmol of a 57-mer
oligonucleotide containing the 30-bp polypurine site in
supFG1 were 5' end-labeled with [
-32P]ATP
(Amersham Corp.) using T4 polynucleotide kinase (New England Biolabs,
Beverly, MA). The labeled oligonucleotide was heated to 85 °C in the
presence of 2 pmol of its 57-mer complement and cooled to room
temperature over a 90-min period. The resulting 57-bp duplex was
diluted to 10 nM in 10 mM Tris, pH 7.5. In a 10 µl final volume, a final concentration of 1 nM labeled
target duplex was incubated with varying concentrations of unlabeled oligonucleotide for 2 h in binding buffer (10 mM Tris,
pH 7.5, 1 mM spermine, 20 mM MgCl2,
and 10% sucrose) at 37 °C. The samples were resolved via
electrophoresis in a native 15% polyacrylamide gel containing 89 mM Tris, pH 7.5, 89 mM boric acid, and 20 mM MgCl2 and run at 10 V/cm overnight at room
temperature. The gel was dried and visualized by autoradiography.
In Vitro Triplex Formation--
3 µg (0.9 pmol) of either the
wild-type or mutant pSupFG1 plasmids were incubated with a 233-fold
molar excess of various oligonucleotides in a 10 µl final volume in
the presence of binding buffer at 37 °C for 2 h. In experiments
using double-stranded donor oligonucleotides, complementary
oligonucleotides were heated to 90 °C for 30 s and annealed at
room temperature. All control oligonucleotides were also subjected to
the same heat-annealing protocol.
Co-transfection Shuttle Vector Assay--
Co-transfection of
oligonucleotide/plasmid DNA complexes was carried out using
LipofectAMINE (Life Technologies, Inc.) per the manufacturer's
instructions. After incubation for 48 h at 37 °C, cells were
harvested, and plasmid vector DNA was isolated using a modified
alkaline lysis procedure (3). Isolated vector DNA was digested with
DpnI (to eliminate unreplicated plasmid that had not
acquired the mammalian methylation pattern) and used to transform
indicator bacteria for genetic analysis of supF gene function as described previously (3). Plasmid DNA was isolated from
randomly selected colonies and subjected to DNA sequence analysis, as
described previously (3).
Intracellular Targeting Protocol--
COS-7 cells were grown to
a culture density of 60-70% in T150 cell culture flasks and
transfected with 9 µg of pSupFG1/G144C using LipofectAMINE. After
34 h at 37 °C, cells were detached via trypsinization, washed
once with growth media, washed twice in DMEM, and resuspended at 2 × 107 cells/ml in DMEM. Selected oligonucleotides were
added to a final concentration of 1 µM in 100 µl of
cells. Cells were transferred to 0.4-cm electroporation cuvettes
(Bio-Rad) on ice, electroporated at 250 V, 200 ohms, and 25 µF (Gene
Pulser; Bio-Rad), immediately placed on ice for 10 min, transferred to
37 °C for 20 min, and then diluted 1:1 with DMEM with 20% fetal
calf serum and incubated at 37 °C for 1 h. Cells were then
plated into 100-mm dishes in growth media. After 48 h, the shuttle
vector was recovered and analyzed as described above.
In experiments using the pyrimidine TFO-based reagents, the cells were
initially transfected by electroporation (rather than lipofection) with
pSupF5/G115T at 5-10 µg/0.4 ml in 0.4-cm gap cuvettes at 210 V,
infinite ohm, and 1050 µF. After a 10-min incubation at room
temperature, cells were washed twice in growth medium to remove excess
extracellular plasmid DNA and replated in culture. After 24 h,
cells were trypsinized, washed, and resuspended at 2.5-10 × 107 cells/ml. Selected oligonucleotides were added to a
final concentration of 5 µM in a 0.4-ml cell suspension
in the presence of 10 mM MgCl2 and
electroporated as described above. Cells were replated and incubated
for 60 h, and then shuttle vector DNA was harvested as described above.
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RESULTS |
Design and Rationale of the TD-TFO Strategy--
In an endeavor to
promote targeted recombination, we have designed a TD-TFO molecule that
tethers a TFO to a donor DNA fragment homologous to a region of the
target gene via a linker segment (Fig.
1). This arrangement facilitates target
site recognition via triplex formation while positioning the donor
fragment for possible recombination and information transfer. This
strategy also attempts to exploit the ability of the triple helix
itself to provoke DNA repair at the binding site, potentially
increasing the probability of recombination with the tethered donor
DNA.

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Fig. 1.
Schematic diagram of a TD-TFO binding to DNA
via triplex formation, delivering donor sequence information to a
desired region and leading to the change of the original DNA sequence.
X, the base pair to be changed.
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The donor domain is identical in sequence to the target gene, except
for a desired sequence change that may either restore or abolish gene
activity. In designing the tethered donor, we hypothesized that either
a single- or double-stranded donor could potentially transfer sequence
information to the target gene through either direct assimilation into
DNA via recombination or as a template for replicative or reparative
DNA synthesis in a gene conversion-type event.
A series of TD-TFOs were constructed to target two different modified
supF tRNA reporter genes. The first target gene,
supFG1, contains a 30-bp G-rich site at the 3' end of the
gene (Fig. 2A) to which the
purine-rich 30-mer TFO, AG30, binds and forms a triple helix in the
anti-parallel motif (3). The other target gene, supF5/G115T,
contains a 30-bp A-rich site at the 5' end of the gene suitable for
triplex formation in the parallel pyrimidine motif by the TFO
designated UC30 (Fig. 2B).

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Fig. 2.
Schematic diagrams depicting the binding and
positioning of single-stranded TD-TFOs to the supF genes in
two different supF constructs. A, binding of
A-AG30 to its site in the supFG1 gene in the mammalian
shuttle vector pSupFG1/G144C. G at position 144 of the donor domain is
intended to correct the inactivating G144C mutation. B,
binding of C-UC30 to a triplex site found at the 5' end of the
supF5 gene in the shuttle vector pSupF5/G115T. C at position
115 in the donor domain is intended to correct the inactivating G115T
mutation. U, 2'-O-methyluridine; C in the UC30
segment, 5-methyl-2'-O-methylcytidine. In B, L
designates the linker segment that consists of the same sequence
(9TT9TT9) as in A, where 9 indicates the
polyethylene glycol linker Spacer-9 (Glen Research).
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For each target, a series of experimental and control oligonucleotides
or oligonucleotide mixtures were designed (Fig.
3). These include TD-TFOs with either
single- or double-stranded donor domains, TD-TFOs with either domain
substituted with control sequences to prevent function, and lastly,
separate TFO and donor domains, either alone or in unlinked
combinations.

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Fig. 3.
Schematic diagrams, third-strand binding
affinities, and sequences of oligonucleotides. Double-stranded
TD-TFOs are created by heat-annealing complementary A and B or C and D
strands. Kd values were determined by gel mobility
shift assays using a labeled duplex target. Oligonucleotides A, B,
Y(Rndm), B(144), and B(Sal) have a 3' propylamine modification. All
other oligonucleotides have three phosphorothioate linkages at
the 3' end. Underlined sequences represent changes
from wild-type. Linker represents a linking segment with the
sequence 9TT9TT9, in which 9 is the Spacer-9 polyethylene glycol unit.
UC30 consists of the RNA residues
2'O-methyluridine and 5-methyl-2'-
O-methylcytidine.
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For each TD-TFO, a 40- or 44-nt donor domain was connected to a 30-nt
triplex-forming domain by a mixed linker equivalent to approximately 13 nt. Our nomenclature describes each TD-TFO by its donor and TFO domains
written in the 5' to 3' direction. For TD-TFOs targeting the
supFG1 gene, the donor domains are referred to as A
(corresponding, from 5' to 3', to nt 121-160 of the supFG1 gene) and its complement, B (nt 160-121 of supFG1). The
notation also indicates whether the donor region contains any sequence changes. For example, A/B(144)-AG30 describes a double-stranded donor
fragment containing a sequence change at position 144, along with AG30
as the TFO. In targeting the supF5 gene, C (nt 100-143 of
supF5) and its complement, D (nt 143-100 of
supF5), represent the individual strands of the donor domain.
Triplex Formation Is Not Altered by the Presence of an Attached
Donor Domain--
Cheng and Van Dyke (24) reported the destabilizing
effect of a 3' or 5' nucleotide tail on triplex formation in certain sequence contexts, with a 3' tail being most detrimental. We were therefore concerned that an attached donor fragment might substantially destabilize TFO binding. However, unlike the oligonucleotides tested in
the Cheng and Van Dyke study, the TD-TFO molecules were designed to
incorporate a highly flexible linker region between the TFO and the
donor tail, with the linker and donor segments extending from the 5'
end of the TFO (AG30). To determine the influence of the attached
linker and donor domains on the affinity of third-strand binding by the
TFO region, the binding of selected oligonucleotides to the target
sequence in the supFG1 gene was measured by gel mobility
shift assay (Fig. 4). As shown, the
single-stranded purine TD-TFOs, A-AG30 and A(144)-AG30, bind with high
affinity to a 57-bp duplex containing the polypurine target site in the supFG1 gene. Calculation of the equilibrium dissociation
constants (Kd values) for third-strand binding from
the quantified band intensities revealed only a very small difference
in binding affinity between A-AG30 (Kd = 5 × 10
8 M) and AG30 alone (Kd = 3 × 10
8 M). These measurements
demonstrate that a single-stranded donor fragment attached via a
flexible linker does not significantly alter the binding affinity of
the 30-nt AG30 TFO domain to the target site. Similar results were
observed using double-stranded TD-TFOs (data not shown). Further gel
shift analyses also show, as expected, that oligonucleotides lacking
the specific TFO domain, AG30, do not bind to the duplex target site
(A-MX30 (Fig. 4) or A and A/B (data not shown)). In the pyrimidine
motif, some decrease in the binding affinity of C-UC30 was seen as
compared with UC30, although the Kd values were
still within the 10
8 M range (see the
Kd values given in Fig. 3).

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Fig. 4.
Gel mobility shift assays comparing the
relative binding affinities of four different oligonucleotides. A
57-bp radiolabeled duplex incorporating the polypurine third-strand
binding site found in the supFG1 gene was incubated at a
concentration of 1 nM in the presence of increasing
concentrations of selected oligonucleotides, as indicated. Listed
oligonucleotide concentrations apply to the bottom panel for
A-MX30 and AG30 as well. Bands of reduced mobility indicate the
formation of triple helices. The oligonucleotides AG30, A-AG30, and
A(144)-AG30 demonstrate roughly equivalent binding affinities, with
Kd values in the 3-5 × 10 8
M range. A-MX30, as expected, does not bind the target up
to concentrations of 1 µM.
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Reversion of a Point Mutation at Position 144 in the supFG1
Gene--
The single-stranded TD-TFO, A-AG30, and its double-stranded
counterpart, A/B-AG30, were designed to correct a single point mutation
(G-to-C point mutation at position 144) in the supFG1 reporter gene of the shuttle vector, pSupFG1/G144C. Within the TD-TFO,
position 144 corresponds to the center of the 40-nt donor domain, which
is otherwise homologous to bp 121-160 of the gene (Fig.
2A). Shuttle vector DNA was incubated in vitro
with selected oligonucleotides, transfected into COS-7 cells, and
recovered after 48 h for analysis of the supFG1 gene in
indicator bacteria. Reversion frequencies (Fig.
5A) were calculated as the
number of blue colonies (revertants) among white ones. The presence of a corrected wild-type supFG1 sequence was confirmed by DNA
sequencing in randomly selected blue colonies (n = 15 for A-AG30; n = 15 for A/B-AG30).

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Fig. 5.
Directed sequence modification of the
supFG1 gene induced by various oligonucleotides. The
shuttle vector DNA was incubated in vitro with the indicated
oligonucleotides and then transfected using cationic lipids into COS-7
cells. After 48 h, the shuttle vector DNA was isolated and
electroporated into indicator bacteria for genetic analysis of the
supFG1 gene. A schematic diagram of each oligonucleotide or
oligonucleotide combination is diagrammed to the left.
Error bars represent standard error of proportions. The
results are cumulative data from at least three experiments. + indicates that the different oligonucleotides are mixed together but
unlinked. indicates that the various oligonucleotides are connected.
A, reversion frequency of mutant pSupFG1/G144C mediated by
the indicated oligonucleotides. The number of blue colonies/the total
number of colonies is presented to the right of each
bar. * indicates that the frequency of reversion is less
than or equal to the value indicated. B, frequency of
mutations induced in wild-type pSupFG1 by the indicated
oligonucleotides. The number of white colonies/the total number of
colonies is listed to the right of each
bar.
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The data show that the frequencies of reversion due to the
single-stranded A-AG30 (0.17%) and the double-stranded A/B-AG30 (0.68%) are significantly above the frequency of spontaneous reversion (0.003%), representing 57-fold and 227-fold increases, respectively (Fig. 5A). In comparison, lower levels of reversion were
produced by the single- and double-stranded donor segments alone in the absence of an associated TFO (A, 0.051%; B, 0.062%; A/B, 0.17%), and
there is essentially no reversion above background mediated by the TFO
domain alone (AG30, 0.014%). Furthermore, A/B-MX30, which has the same
donor domain as A/B-AG30 but cannot form a triplex with
supFG1/G144C due to substitution of AG30 with a
non-triplex-forming mixed sequence (MX30), also showed a lower
reversion frequency (0.15%) than A/B-AG30 and almost the same
reversion frequency as the A/B donor alone. As another control,
X/Y(Rndm)-AG30, whose donor domain is replaced with a randomized
sequence unrelated to supF, also demonstrates little
activity (
0.006%). Additionally, to rule out the potential for
bacterially derived recombinants, 210 pmol of A-AG30 or A/B-AG30 were
electroporated directly into bacteria along with supFG1/G144C DNA
recovered from COS-7 cells. These experiments showed reversion
frequencies that were not significantly above background (background,
0.003 ± 0.003%; A-AG30, 0.009 ± 0.006%; A/B-AG30,
0.008 ± 0.005%). Also, when pSupFG1/G144C plasmid DNA prepared
in Escherichia coli was incubated with a 233-fold excess of either A/B-AG30 or A-AG30 and electroporated directly into
bacteria, reversion frequencies of
0.003% (0 blue colonies/39,156 colonies) and
0.002% (0 blue colonies/44,859 colonies) were seen, respectively.
These results demonstrate that both the TD-TFO composite molecules and
the corresponding unlinked donor fragments are active in the assay.
However, whereas the single- and double-stranded donor fragments each
yield revertants on their own, their activity is increased roughly 3- to 4-fold in each case by linkage to the AG30 TFO. In contrast, the
activity of the donor domains is not enhanced by linkage to the mixed
sequence oligonucleotide, MX30. Hence, the TFO segment plays an
important role in TD-TFO activity and can significantly enhance the
activity of the donor segment.
Introduction of Specific Mutations into Wild-type supFG1--
We
further tested the activity of TD-TFO molecules by assaying their
ability to introduce a variety of sequence changes into a wild-type
supFG1 gene in a forward mutation assay. One set of TD-TFOs
was designed to introduce a specific inactivating G-to-C point mutation
at position 144 in the wild-type supFG1 gene. Using the
protocol described above, we found that A/B(144)-AG30 induced a
mutation frequency of 0.64% (Fig. 5B), approximately 9-fold higher than the spontaneous forward mutation frequency of 0.07% and
also higher than that of the single-stranded A(144)-AG30 (0.30%). (Note that the background frequency in this forward mutation assay is
much higher than that seen in the reversion assay because many nonspecific sequence changes can inactivate the gene). Furthermore, A/B(144)-AG30 generated a higher mutation frequency than any of the
components alone (AG30, 0.076%; A/B(144), 0.15%; A/B(144) + AG30,
0.31%). Whereas the majority of spontaneous mutations in the absence
of oligonucleotide exposure were deletions upon sequencing, the mutants
induced by A/B(144)-AG30 carried the specific 144 G-to-C base change in
73% of the mutants tested (n = 15). Random deletions
and scattered point mutations were found in 20%, similar to the type
and frequency of mutations produced by AG30 alone (data not shown) and
consistent with our previous observation of triplex-induced mutagenesis
(6). The remaining mutation was a short tandem duplication (11 bp; nt
121-131) corresponding to the 5' end of the donor domain, suggesting a
possible abortive strand-exchange event.
To determine whether a more complex mutation could be reproducibly
introduced into the target gene by this strategy, we also designed a
TD-TFO to insert a SalI restriction site into the
supFG1 gene. This requires changing 5 of 7 bases from
position 138 to 144. With this reagent, A/B(Sal)-AG30, we observed a
mutation frequency of 0.40% (versus 0.07% background and
0.11% for A/B(Sal); Fig. 5B). Of six randomly picked
colonies, five were found by restriction analysis and DNA sequencing to
have incorporated the SalI site; the sixth was a deletion
mutant, again consistent with the presence of some background-related
or AG30-induced mutants in the sample. The introduction of a complex,
novel mutation such as this expands the potential utility of the TD-TFO
approach. It also provides evidence that the results cannot be
attributed to simple contamination, because the SalI
site-containing plasmid did not previously exist in the laboratory.
Dependence of the Activity of the TD-TFOs on Nucleotide Excision
Repair--
In previous work, we had found that triplex formation can
induce mutagenesis in a pathway dependent in part on NER (6). In
addition, in vitro studies in human cell extracts revealed that high affinity intermolecular triple helices could provoke DNA
repair of an otherwise undamaged substrate as measured by the
stimulation of DNA repair synthesis (6). Based on these observations,
we hypothesized that triplex-provoked NER might play a role in TD-TFO
activity. To test this, we assayed for reversion of the pSupFG1/G144C
vector after co-transfection of selected oligonucleotide/plasmid
complexes into a human mutant cell line (XP2OS) derived from a patient
with Xeroderma pigmentosum, group A, and deficient in the NER damage
recognition factor, XPA (23). In comparison, we also transfected the
complexes into a subclone of this cell line stably expressing the XPA
cDNA after gene transfer (23). The results of these reversion
assays (Fig. 6) show that the activity of
A/B-AG30 is reduced to 0.29% in the XPA-deficient cells, which is only
marginally greater than the activity of the non-triplex-forming control
A/B-MX30 (0.19%) in these cells. In contrast, in the corrected cell
line, the differential between A/B-AG30 (0.48%) and A/B-MX30 (0.15%)
is increased and approaches that seen in the COS-7 cells (Fig. 5).
Hence, much of the extra stimulation provided by the TFO domain depends
on XPA function and presumably on NER activity. Because A/B-MX30 is
somewhat active in both the mutant and corrected cell lines, there
otherwise appears to be near normal recombination activity in the XPA
mutant, a result consistent with other studies of recombination in such cells (9). Thus, whereas the XPA cells can carry out recombination, the
extra stimulation provided by the TFO domain is reduced in these
cells.

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Fig. 6.
Directed sequence modification of the
supFG1 gene in the SV40 shuttle vector induced by selected
oligonucleotides in either XP2OS (XPA mutant) or XP2OS-pCAH19WS (XPA
corrected) cells. The shuttle vector DNA and oligonucleotides were
co-incubated in vitro and transfected using cationic lipids
into each cell line. After 48 h, shuttle vector DNA was recovered
and electroporated into indicator bacteria to assess supFG1
function. The number of blue colonies/the total number of colonies is
presented to the right of each bar. * indicates
that the frequency of reversion is less than or equal to the value
indicated. Error bars represent the standard error of
proportions. The results represent cumulative data from at least three
experiments.
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However, these data should not be taken to suggest that the full
benefit of the tethered TFO domain is mediated through the ability of
triplexes to stimulate repair. These experiments were carried out by
transfection of pre-incubated oligonucleotide/plasmid complexes, thus
they may underestimate the potential role of the TFO in mediating a
homology search in cells.
Intracellular Targeting of the pSupFG1/G144C Vector and Correction
of a Point Mutation at Position 144--
To demonstrate in
vivo targeting of the supF gene by the TD-TFO
molecules, cells were pre-transfected with the shuttle vector DNA,
followed 34 h later by electroporation of the oligonucleotides into the cells. After another 48 h, the shuttle vector DNA was harvested for genetic analysis of the supF gene (Table
I). In this protocol, A/B-AG30 was found
to mediate reversion at a frequency of 0.014% or 20-fold over
background (0.0007%; Table I). The TD-TFO with the single-stranded
donor domain A-AG30 was also active, yielding a reversion frequency of
0.036%, 51-fold above background. The presence of a wild-type
supFG1 sequence in randomly selected blue colonies
(n = 10 for each) was confirmed by DNA sequencing. The
controls yielded few, if any, revertants, indicating that the triplex
domain is important for intracellular targeting (Table I; compare A,
A-MX30, and A-AG30 as well as A/B, A/B-MX30, and A/B-AG30). Also, the
covalent attachment of the donor and triplex-forming domains is
required, because A + AG30 and A/B + AG30 showed little activity in
this assay. These results suggest that target gene recognition and
information transfer can be mediated by the TD-TFOs within cells.
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Table I
Specific reversion of supF gene mutations mediated by transfection
of TD-TFO conjugates into cells that already contained the shuttle
vector target
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Considering the 34-h interval between the shuttle vector transfection
into COS-7 cells and the subsequent TD-TFO transfection, it is highly
likely that the interaction of the TD-TFO with the shuttle vector is
occurring intracellularly. Also, because SV40 replicons become rapidly
covered with histones to form mini-chromosomes (25), these data suggest
that the TD-TFOs can target chromatinized DNA. However, the chromatin
structure of the SV40 vectors is quite dynamic, because they replicate
actively in COS-7 cells and are highly transcribed. This feature of the
vectors may be important in facilitating targeting by the TD-TFOs.
Whereas both A/B-AG30 and A-AG30 were more active in the intracellular
targeting assay than any of the controls (including the donor fragments
alone), A-AG30 was found to be 2.5-fold more active than its
double-stranded counterpart, A/B-AG30. However, in the co-transfection
assay (Fig. 5), A/B-AG30 was 4-fold more active than A-AG30. Also, in
intracellular targeting experiments with the pSupF5 target (see below),
the TD-TFO with the double-stranded donor domain was found to be more
active than that with the single-stranded one. Whether these
differences between the single- and double-stranded TD-TFOs have
functional significance is not yet clear, and further studies are
needed to optimize the design of the donor domain. It is clear,
however, that the composite TD-TFO molecules are consistently more
active than the corresponding controls in all of the experiments
conducted thus far.
In addition, the differences in reversion frequencies seen when the
oligonucleotides are incubated with the target vector in
vitro and co-transfected (Fig. 5) versus when they are
introduced into cells already containing the vector (Table I) reflect
the challenges involved in achieving intracellular oligonucleotide delivery and triplex formation under physiologic conditions.
Furthermore, the in vivo protocol may underestimate the true
frequency of intracellular targeting because electroporation delivers
oligonucleotides into only a fraction of the cells containing the
supF plasmid (transfected 34 h earlier using cationic
lipids). Hence, much of the rescued shuttle vector DNA used for
analysis is derived from cells that did not receive the oligomers and
in which no recombination could have occurred. Nonetheless, revertants
were detected in the in vivo protocol, suggesting that the
TD-TFO molecules can successfully target an episomal gene inside cells.
These initial results raise the possibility that improvements in
oligonucleotide delivery and reagent design may enhance the
effectiveness of this approach.
Targeting by a TD-TFO Molecule in the Pyrimidine Triple Helix
Motif--
The work presented above focuses on TD-TFOs that form
triplexes in the purine motif, where the third strand is typically
G-rich and binds anti-parallel to the purine-rich strand of the duplex. For comparison, we also tested the activity of a TD-TFO whose pyrimidine-rich TFO domain was designed to bind to a different target
site in the pyrimidine motif. In this motif, the third strand binds in
a parallel orientation relative to the purine strand of the duplex, and
we considered the possibility that these differences in target site and
polarity might influence our results.
To this end, TD-TFOs C-UC30 and C/D-UC30 were designed to correct a
G-to-T point mutation at position 115 in the supF5 gene in
vector pSupF5/G115T (Fig. 2B). The supF5/G115T
gene contains an A-rich polypurine site at its 5' end, suitable for
third-strand binding in the pyrimidine motif. This vector has an
extremely low spontaneous rate of reversion in our assay (
0.0003%).
In the reagents tested, the triplex-forming segment (UC30) was
synthesized to contain the RNA residues 2'-O-methyluridine and
5-methyl-2'-O-methylcytidine. These modifications provide for increased
nuclease resistance and decreased pH sensitivity of third-strand binding.
When the oligonucleotides and target vector were pre-incubated in
vitro and transfected into cells, results similar to those with
the purine motif TD-TFOs were obtained, with reversion frequencies in
the 0.1% range (data not shown). In experiments in which pSupF5/G115T was pre-transfected into the cells and electroporated 24 h later with the oligonucleotides, C/D-UC30 led to a reversion frequency of
0.0051% as compared with a background of
0.0003% (Table I), representing at least a 17-fold increase in activity over background. Also, UC30 by itself was ineffective (
0.0001%), as were the donor fragments alone (C,
0.0001%; D, 0.0004%) or together as a duplex (C/D, 0.0004%). The presence of the wild-type supF5 gene
was confirmed by sequencing randomly selected revertants
(n = 18 for C/D-UC30). In these experiments, the
double-stranded C/D-UC30 was more active than the single-stranded
C-UC30 (0.0004%), consistent with the trend seen when the
oligonucleotides and the shuttle vector DNA were co-incubated in
vitro and subsequently co-transfected into cells (Fig. 5).
In general, the pyrimidine motif TD-TFOs were not as effective as their
purine counterparts (A/B-AG30 was nearly 3-fold more active than
C/D-UC30). This may be due to differences in several factors, including
strand polarity relative to the target duplex, binding characteristics
in vivo (such as pH and ion dependence), intracellular
stability of the TD-TFOs, the experimental protocol, the mechanisms of
information transfer, and the suitability of the target site. For
example, C-UC30 was designed to target a site in the supF5
gene where the sequences corresponding to the TFO and donor domains are
directly adjacent (Fig. 2B). In contrast, the regions of the
supFG1 gene that correspond to the donor and TFO domains of
A-AG30 or A/B-AG30 are separated by 6 bp (Fig. 2A). This
spacing may ultimately be important for efficient information transfer
by TD-TFO-type molecules. As a result, caution should be exercised in
making a direct comparison between the efficiency of gene correction by
A/B-AG30 and C/D-UC30. However, within each series, the TD-TFOs are
consistently more active than their component parts.
 |
DISCUSSION |
The work presented here demonstrates the ability of selected
TD-TFO molecules in both the purine and pyrimidine triple helix motifs
to mediate targeted sequence alterations within a SV40 shuttle vector
in mammalian cells. Successful reversion of the supF target
genes was seen after in vitro co-incubation of the target
vector with the TD-TFOs and also after an in vivo protocol in which cells already containing the shuttle vector were transfected with the oligonucleotides. Oligonucleotides consisting of covalently linked donor and triplex-forming domains were more effective than either of the individual segments alone and more effective than the
unjoined segments mixed together, suggesting that the two domains work
synergistically when present in a single molecule. Furthermore, we
found that a TD-TFO could introduce a complex mutation into the
wild-type supFG1 gene to generate a novel SalI restriction site, demonstrating the potential ability of TD-TFOs to
correct mutations involving more than a single base pair substitution. To our knowledge, this has not been reported previously using such
short fragments (40 bp or less).
The tethered donor approach differs from our previous efforts to use
triplex-targeted DNA damage to induce homologous recombination (13). In
that work, the TFO was used to introduce site-specific psoralen
photoadducts to stimulate recombination between two separate supF genes. In the work presented here, no mutagen other
than the triplex itself is involved, and the recombination is intended to occur not between two intact genes but between a target gene and a
donor fragment tethered to the TFO.
The data from the in vitro co-incubation and transfection
experiments (Fig. 5) show that sequence alteration of the
supF gene in the SV40 vector can also be mediated by short
donor fragments alone. The unlinked single- or double-stranded donor
fragments achieved reversion frequencies that were 17- and 57-fold
above background, respectively. These results are somewhat consistent with other reports that short nucleic acid constructs can mediate directed sequence changes (10, 16). However, we found that short donor
molecules alone were not effective in the in vivo protocol,
suggesting that, at least for the SV40/supF target, the attached TFO
domain is functionally important in cells.
We propose two mechanisms to explain the apparent synergy between the
donor and TFO domains. The first mechanism relies on the ability of an
attached TFO to conduct a functional homology search for a target site
in an ATP- and enzyme-independent manner and bind specifically to the
DNA as a third strand, thereby positioning the donor domain near the
sequence to be changed. This increases the local concentration of the
donor fragment and may increase the likelihood of an information
transfer event from the TD-TFO to the gene. Supporting this hypothesis
is the 9-fold increase in activity of A-AG30 over A alone in the
in vivo protocol in which the oligonucleotides must find the
target plasmid that was previously transfected into the cells. In
contrast, in the in vitro co-incubation and co-transfection
protocol in which the oligonucleotides and the plasmid are already
mixed together in solution at relatively high concentrations, there is
less difference between A-AG30 and A alone (3.4-fold). Further
supporting this hypothesis is the demonstrated ability of TFOs to
deliver other moieties, such as a psoralen conjugate, to specific DNA
sites in cells (3, 26). Theoretically, an alternative mechanism for the
activity of the TFO domain could be strand invasion and the formation
of a Watson-Crick double helix with one of the strands of the target
DNA. We consider this unlikely, because the AG30 sequence is
anti-parallel in polarity to the purine strand of the duplex and thus
is not complementary to the pyrimidine-rich strand of the target site.
Also, no species other than the expected triplexes were seen in the gel
mobility shift experiments (Fig. 4).
The second mechanism involves the finding that triplex formation can
provoke DNA repair at or around the triplex target site (6). This
prompted the experiments reported here to test whether part of the
benefit of the TFO domain in the TD-TFO molecules might come from the
stimulation of DNA repair. Such induced repair could potentially lead
to recombinogenic strand breaks or could possibly involve the
recruitment of proteins important in homologous pairing, strand
exchange, and/or recombination. The results of experiments using the
XPA-deficient cell line and its corrected derivative are consistent
with this model in that the activity of the TD-TFOs was diminished in
the absence of NER but was restored when the NER defect was corrected.
Hence, taken together, the TD-TFO molecules may enhance the frequency
and specificity of oligonucleotide-mediated mutagenesis by making
specific sequence information available during a process of induced
repair, facilitating a pathway of recombinational repair. The coupling
of repair and recombination as well as the ability of the third strand
to mediate target site recognition would therefore be a plausible
mechanism to explain the effectiveness of the tethered donor molecules.
The TD-TFO molecules described here represent a first-generation
design. Improvements may be possible by elucidating a mechanism of
action and then exploiting this knowledge to guide refinements in
reagent structure and composition. For example, the data on mutation
induction using TD-TFOs indicate that these reagents can introduce a
specific mutation at an efficiency of nearly 75%. However, in 20% of
the sequenced mutations, we observed deletions and other point
mutations that are consistent with mutagenesis induced by the triplex
alone (6). We previously observed that triplex-induced mutagenesis may
be related to either TFO length or affinity or both (6) and that
triplexes of different length may differentially interfere with NER
(27). We are currently exploring means of reducing nonspecific
mutagenesis by changing these TFO parameters and by trying to improve
the efficiency of the information transfer from the donor.
Also, the work presented here involves targeting an extrachromosomal
SV40-based vector. Whereas such a vector provides a chromatinized target, it may not fully reflect a chromosomal gene. The ability of
psoralen-conjugated TFOs to target a chromosomal site has recently been
demonstrated (26), but the effectiveness of TD-TFOs to mediate sequence
changes at chromosomal sites remains to be determined.
Finally, the TD-TFO approach remains constrained by the restriction of
high-affinity triplex formation to polypurine regions of DNA. Expansion
of the third strand binding code through either the development of
nucleotide analogs or the use of novel oligonucleotide backbones may
greatly enhance the effectiveness of these reagents. Nonetheless, the
TD-TFO approach as a method for DNA sequence modification has the
potential to be a useful research tool and may eventually provide the
basis of a gene therapy strategy.