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J. Biol. Chem., Vol. 276, Issue 31, 28991-28998, August 3, 2001
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,,,
, and**
From the NIA, National Institutes of Health,
Baltimore, Maryland 21224, the § Novartis Horsham Research
Centre, Horsham, West Sussex, RH12 4AB United Kingdom, ¶ Novartis
Pharma, Ltd., 4002 Basel, Switzerland, and the Department of
Biochemistry and Molecular Biology, School of Hygiene and Public
Health, Johns Hopkins University, Baltimore, Maryland 21205
Received for publication, April 17, 2001, and in revised form, May 23, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Triplex forming oligonucleotides (TFOs) are of
interest because of their potential for facile gene targeting. However,
the failure of TFOs to bind target sequences at physiological pH and Mg2+ concentration has limited their biological
applications. Recently, pyrimidine TFOs with
2'-O-aminoethyl (AE) substitutions were shown to have
enhanced kinetics and stability of triplex formation (Cuenoud, B.,
Casset, F., Husken, D., Natt, F., Wolf, R. M., Altmann, K. H., Martin, P., and Moser H. E. (1998) Angew. Chem. Int.
Ed. 37, 1288-1291). We have prepared psoralen-linked TFOs with
varying amounts of the AE-modified residues, and have characterized
them in biochemical assays in vitro, and in stability and
HPRT gene knockout assays in vivo. The AE TFOs
showed higher affinity for the target in vitro than a TFO
with uniform 2'-OMe substitution, with relatively little loss of
affinity when the assay was performed in reduced Mg2+. Once
formed they were also more stable in "physiological" buffer, with
the greatest affinity and stability displayed by the TFO with all but
one residue in the AE format. However, TFOs with lesser amounts of the
AE modification formed the most stable triplexes in vivo,
and showed the highest HPRT gene knockout activity. We conclude that the AE modification can enhance the biological activity of pyrimidine TFOs, but that extensive substitution is deleterious.
Reagents that recognize and bind specific genomic sequences in
mammalian cells would have broad application for promoter suppression, gene knockout, target validation, and as probes of chromosome structure. Thus the interaction of third strands and intact DNA duplexes to form stable DNA triplexes (1) has been of interest for many
years (2-6). Some of the properties of these structures make triplex
forming oligonucleotides
(TFOs)1 attractive candidates
for development as gene targeting reagents. Triplex formation, which
occurs on polypurine:polypyrimidine sequences, is an inherent property
of nucleic acids and has no protein or enzyme requirement (7, 8). The
binding of the third strand to the duplex is stabilized by hydrogen
bonds between the bases of the third strand and the purines in the
target duplex. The specific interactions can be described as a third
strand binding code (9), and are the basis for the high stringency of
TFO specificity (10, 11). Triplexes can be quite stable with half-lives of many hours or days (12-15). Although current oligonucleotide chemistry limits triplex formation to polypurine:polypyrimidine stretches, these elements are over-represented in the mammalian genome
(16), and most genes contain appropriate target sequences. Furthermore,
data from different experimental systems indicate that both
extrachromosomal and chromosomal target sites in living cells are
accessible to TFOs (17-19).
However, despite 40 years of research, there remain a number of
impediments to the successful employment of TFOs as gene
targeting reagents. Some of these obstacles reflect the properties of
the oligonucleotides. Depending on the nature of the target either purine or pyrimidine TFOs can be used, but there are problems associated with each motif. Under physiological conditions purine TFOs
are often subject to self-structure formation, which is incompatible with triplex formation (20, 21). Cytosine residues in pyrimidine motif
TFOs must be protonated at N-3 in order for hydrogen bond formation at
this position. The pKa of cytosine is 4.5, and most
pyrimidine TFOs are relatively inactive at physiological pH.
Furthermore, triplex formation by TFO in both motifs is often sharply
dependent on Mg2+ concentration (22). Most binding studies
use 5-10 mM Mg2+, which is likely to be higher
than the free intracellular concentration. Obviously, if TFOs are to
become effective gene targeting reagents, they, at a minimum, must be
active at physiological pH and Mg2+ concentration.
Some progress has been made toward overcoming these problems,
particularly in the pyrimidine motif. Replacement of cytosine by
5-methylcytosine partially alleviates the pH restriction on triplex
formation (23, 24). Following the observation that pyrimidine triplexes
with RNA third strands are more stable than the complex with the
corresponding DNA third strand (25-27), it was shown that triplex
stability could be enhanced when the TFOs contained
2'-O-methyl (OMe)-modified sugars (28-30). Structural studies indicate that this modification stabilizes the
C3'-endo,2'-exo configuration of the sugar. This is the
optimal conformation for triplex formation by pyrimidine
oligonucleotides, and provokes the least perturbation of the underlying
duplex (31).
Recently, additional sugar modifications have been described which
considerably extend the stability of pyrimidine motif triplexes (32).
Among these the 2'-O-aminoethyl (AE) substitution produced a
3.5 °C increase in thermal stability per modification, measured at
pH 7.0. NMR analysis indicated that a hydrogen bond formed between the
positively charged amino residue and an oxygen of the negatively
charged phosphate of the purine strand of the duplex. The sugar
conformation was C3'-endo,2'-exo and the underlying duplex
showed only slight distortion (33). This substitution also conferred
high nuclease resistance on the TFO (32, 34). Although the AE TFOs were
not tested in bioassays their properties would seem consistent with
activity in cell-based assays.
We are developing TFOs as gene targeting reagents, and have
demonstrated specific gene knockout in mammalian cells using a TFO
linked to psoralen (18). Consequently oligonucleotide modifications that have the potential to solve some of the problems mentioned above
are of considerable interest. Here we describe the preparation and
characterization of psoralen-linked TFOs with different degrees of
2'-AE substitution. We have characterized these in biochemical assays
in vitro, and in stability and gene knockout assays in mammalian cells in vivo. We find that these TFOs have
enhanced affinity for their target sequences in vitro, and
some, but not all, are active in bioassays in vivo.
Reagents--
The
5'-O-(4,4'-dimethoxytrityl)-5-methyluridine-2'-O-methyl-3'-O-( Synthesis of Phosphoramidites--
The synthesis of
5'-O-(4,4'-dimethoxytrityl)-5-methyluridine-2'-O-(2-aminoethyl)-3'-O-( Synthesis of TFOs--
The oligonucleotides were synthesized on
controlled pore glass supports (500 Å) using an Expedite 8909 synthesizer. All protected nucleoside phosphoramidites were dissolved
in anhydrous acetonitrile at a concentration of 0.05 M. The
nucleoside (2'-AE) phosphoramidite solutions were stored for 12 h
over 4-Å molecular sieves prior to use. The activator used for the
synthesis was 0.4 M 5-ethylthio-1-H-tetrazole. All other reagents used for oligonucleotides synthesis were standard and were obtained from Chemgenes, Ashland, MA. The coupling time was
360 s, except for the psoralen phosphoramidite, which was 90 s. The synthesizer was programmed to carry out a capping step, followed
by the oxidation step, followed by another capping step after each
coupling step. The final coupling was that of the psoralen phosphoramidite.
Deprotection and Purification of TFOs--
Standard deprotection
of Pso TFOs with 2'-AE modifications was carried out in 30% ammonium
hydroxide (J. T. Baker) at 55 °C for 1 h. The controlled
pore glass support was filtered and the solution obtained concentrated
to obtain the crude TFO. For the gas phase deprotection the controlled
pore glass support with Pso TFO was taken in a vial closed with a
porous filter cap. The vial was placed in an enclosed steel pressure
chamber with a valve and evacuated by house vacuum. The valve was then
connected to the gas cylinder, keeping the steel chamber under reduced
pressure. The chamber was incubated with anhydrous methylamine gas
(Aldrich) at room temperature for 30 min. This was followed by the
release of the methylamine gas. The TFO was then taken up in distilled water. Analytical and semi-preparative reversed phase (RP)-HPLC was
carried out using a Symmetry 300TM C18 column
(4.5 × 250 mm) from Waters on a Shimadzu HPLC system (LC-10ADvp)
with a dual wavelength detector (SPD-10AVvp) and an autoinjector
(SIL-10ADvp). The column was eluted using linear gradients of
acetonitrile (5-50%) in 0.1 M sodium acetate (pH 7.2) at
a flow rate of 1.0 ml/min and monitored at wavelengths 254 and 315 nm
( Binding Assays--
TFO affinity determinations were performed
by a restriction enzyme protection assay, essentially as described by
Maher et al. (35). The hamster HPRT triplex
target sequence adjacent to a psoralen cross-linking site was embedded
in the supF12 plasmid. The target sequence and cross-link
site overlap an XbaI restriction enzyme site. Triplex
complexes were formed by incubation of 0.15 pmol of the plasmid with
varying concentrations of the TFO in 200 µl of binding buffer A (20 mM Hepes, pH 7.2, 10 mM MgCl2) for
16 h at room temperature. The psoralen on the plasmid-TFO complex
was photoactivated by exposure to 365 nm UVA (in a Rayonet UV chamber)
for 3 min at a dose of 1.8 J/cm2,, after which the samples
were digested with XbaI. The digests were analyzed by 1%
agarose gel electrophoresis, and the relative amount of DNA in the
protected and non-protected bands was quantitated by fluorescence image
analysis. The KD values were determined by Hill's
equation [y = axb/(cb + xb)], and the data were processed with
SigmaPlotTM 5.0 software. Using a similar protocol,
KD was determined for different TFOs at 1 mM MgCl2 using binding buffer B (20 mM Hepes, pH 7.2, 1 mM MgCl2).
Following psoralen cross-linking the plasmid-TFO complex was separated
from unbound oligonucleotide by precipitation (36) so as to prevent
reassociation of the free TFO with the plasmid in the restriction
digest conditions with higher levels of MgCl2.
In Vitro Triplex Stability Assay--
The plasmid TFO complex
was formed by incubation of saturating amounts (2 µM) of
TFO with the 0.15 pmol of supF12 plasmid in 200 µl of
buffer A for 16 h at room temperature. The plasmid-triplex complex
was separated from the unbound oligonucleotide by precipitation. The
plasmid-triplex complexes were resuspended in physiological buffer C
(140 mM KCl, 10 mM NaCl, 1 mM
MgCl2, 20 mM Hepes, pH 7.2), at 37 °C at a
concentration of 7.5 nM. The resuspension buffer also
contained a 35-mer duplex oligonucleotide with the triplex target
sequence at a concentration of 75 nM. This was to trap any
free TFO dissociating from the triplex complex in the physiological
buffer. Control experiments showed that the 35-mer duplex was effective
in trapping free oligonucleotide. At various times after resuspension
aliquots were removed and irradiated with UVA to activate the psoralen.
The plasmid complex was precipitated as described above. The samples
were digested with XbaI to determine the extent of
protection by psoralen cross-links. The digests were analyzed and quantitated.
In Vivo Triplex Stability Assay--
A shuttle vector plasmid
supF12 was constructed with the hamster HPRT
intron 4/exon 5 triplex target sequence embedded in the pre-tRNA region
of a variant supF tRNA gene. The first two bases of the
mature tRNA gene sequence were changed to 5' TA, the preferred target
for psoralen intercalation and cross-linking (for a discussion of the
general strategy for construction of functional variant supF
mutation marker genes see Ref. 37). In vivo stability assays
were performed as described previously (36). Briefly, the plasmid was
incubated with individual Pso-TFOs under conditions that supported
triplex formation (20 mM Hepes, pH 7.2, 10 mM
MgCl2, 2 µM TFO, 0.6 pmol of plasmid),
unbound TFO removed, and the plasmid-TFO complexes were electroporated
into Cos-1 cells. At various times after electroporation the cells were
exposed to UVA to activate the psoralen. The cells were plated for an
additional 48 h, during which time the psoralen cross-links were
repaired and/or mutagenized, and the plasmid replicated. Progeny
plasmids were then harvested, treated with DpnI to remove nonreplicated input plasmids (38), and introduced into the
Escherichia coli indicator strain MBM 7070 (39). The
bacteria were spread on indicator plates containing
isopropyl-1-thio- Cells and HPRT Mutagenesis Protocol--
Chinese hamster ovary
cells were grown in Dulbecco's modified Eagle's medium supplemented
with penicillin and streptomycin and 10% fetal bovine serum. Prior to
an HPRT knockout experiment cells were cultured in HAT
medium (10
A similar protocol was observed when the frequency of cells with
mutations in APRT (adenosine ribosyltransferase) was
determined. Chinese hamster ovary cultures were cleared of
APRT-deficient cells by growth in azaserine. Selections for
APRT colonies were done in medium containing aza-adenine.
TFOs and Targets--
The sequence and substitution patterns of
the Pso-TFOs in this study are displayed in Fig.
1A. The TFOs were designed to
form a triplex with a target sequence found in intron 4, immediately adjacent to Exon 5 in the Chinese hamster HPRT gene (18). We prepared TFOs designed to target the 17-base uninterrupted
polypurine:polypyrimidine element which ends in a T and is followed by
an A, providing an appropriate site for psoralen cross-linking. We were
interested in determining the biochemical and biological activity of
TFOs with different amounts and distribution of 2'-AE substitution. Accordingly we prepared a TFO with all but one position containing the
AE substitution (AE-01), or with 6 AE residues at the 3' end (AE-02), or with 4 AE residues at the 3' and 5' ends
(AE-03). The remainder of each oligonucleotide contained 2'-OMe sugars. In addition, we prepared a TFO uniformly modified with 2'-OMe sugars
(PS-01).
We constructed a shuttle vector plasmid, psupF12, with a
variant, but functional, supF mutation marker gene
containing the hamster HPRT target placed in the 5' pre-tRNA
region of the gene with the psoralen cross-link site embedded in the
first two bases of the mature tRNA gene (Fig. 1B) (see Levy
et al. (37), for the construction strategy). Psoralen
cross-links placed by the TFO in these positions cause mutations during
repair and replication of the plasmid in mammalian cells (17, 36). The
cross-link site was also positioned in a unique XbaI site
that allowed the presence or absence of a cross-link to be determined
by restriction enzyme protection.
Psoralen-conjugated TFOs--
Psoralen is a linear furocoumarin
which reacts via a [2 + 2] cycloaddition, in concert with long wave
UV light (UVA), to form photoadducts primarily with thymidines at
5'-TA-3' sites in double stranded DNA. This reaction is highly regio-
and stereospecific, forming the interstrand cross-link as the major
product under the conditions employed in our experiments. Conservation
of the lactone ring of psoralen is vital for preserving its
cross-linking activity. The unsaturated lactone ring of psoralen can be
susceptible to hydrolysis at elevated temperatures by harsh aqueous
bases like the ammonium hydroxide commonly used for deprotecting
oligonucleotides. Consequently it was important to identify
deprotection conditions that preserved the psoralen while removing the
trifluoroacetyl groups on the 2'-O-aminoethyl, the acetyl
group on N4 of the
2'-O-methyl-5-methylcytidine, and the
N-methylpyrrolidine amidine group on
N4 of the
2'-O-(2-aminoethyl)-5-methylcytidine.
Oligodeoxyribonucleotides with psoralen tethered to their 5' terminus
have been deprotected by concentrated ammonium hydroxide treatment
(55 °C, 16 h) (40). However, with AE oligos under these
deprotection conditions, even with reduced exposure times (55 °C,
1 h), we recovered quite heterogeneous products as analyzed by
HPLC (Fig. 2). Different
liquid phase deprotection conditions were tried
(tert-butylamine:methanol:water (1:1:2) (41), 10% 1,8-diazabicyclo[5.4.0.]undec-7-ene in ethanol (42), and sequential treatment with ammonia at room temperature, followed by ethylenediamine (43)) but none were satisfactory. Subsequently we tried a novel gas
phase deprotection protocol using anhydrous methylamine in an enclosed
steel chamber (44). The chromatogram (Fig. 2) of the oligonucleotide
following this treatment showed a homogeneous profile with a major
target peak. The oligonucleotide (AE-03 as an example in Fig. 2) was
purified by RP-HPLC and its capillary zone electrophoresis chromatogram
showed a single peak (Fig. 2). The matrix-assisted laser
desorption-time of flight spectroscopy data was consistent with the
theoretical value and an intact psoralen. Triplexes were formed with
the TFOs and duplex DNA containing the HPRT target sequence and an
XbaI site corresponding to the 5' end of the TFO. Psoralen
activity was measured by monitoring the resistance to XbaI
digestion of duplex DNA following triplex formation, photoactivation,
and removal of non-cross-linked TFO. TFOs prepared by gas phase
deprotection consistently displayed very high levels of DNA
cross-linking (Fig. 2).
TFO Affinity--
The affinity of each TFO for the HPRT target was
determined by a restriction enzyme protection assay ("Materials and
Methods") (35, 45). These assays were performed in buffers containing 10 mM MgCl2. Representative binding curves (for
PS-01 and AE-03) are shown in Fig.
3A, and the half-maximal
values for all the TFOs shown in the bar diagram (Fig. 3B).
The KD values ranged from 112 (PS-01) to 15 nM (AE-01).
Although commonly employed for these determinations, 10 mM
Mg2+ is unlikely to accurately reflect the concentration of
free Mg2+ in cells. Consequently we repeated the assays
with PS-01 and AE-03 in 1 mM MgCl2 (Fig.
3C). The results demonstrated the anticipated decline in
affinity with PS-01 (KD = 319 nM).
However, the affinity of AE-03 was essentially unchanged relative to
the measurement in 10 mM MgCl2.
Triplex Stability in Vitro--
In earlier biochemical studies
triplexes formed by TFOs with AE modifications were shown to be more
stable than those formed by unmodified equivalents (32). We measured
the stability, in vitro, of the triplexes formed on the
HPRT target by preparing them with each of the TFOs in
incubations in which the TFOs were at saturating concentrations
("Materials and Methods"). Under these conditions essentially all
plasmid molecules contained triplexes. Following removal of unbound
oligonucleotide, the plasmid-triplex complexes were incubated at
37 °C in a physiological buffer (140 mM KCl, 1 mM MgCl2, 10 mM Hepes, pH 7.2) that
included a 10-fold excess of a duplex trap oligonucleotide containing
the target sequence. At various times after the start of the
incubation, samples were exposed to UVA and then the extent of triplex
formation in each sample determined by restriction protection. The
results (Fig. 4) showed that all the
triplexes formed by the AE TFOs were extremely stable over the time
course, with 99% of the AE-01 triplex present after 8 h. The
AE-02 and AE-03 triplexes were also quite stable with 93 and 94%
maintained after 8 h. The least stable was the PS-01 triplex with
a decline to 83% in the same time period. These data were consistent
with previous results (32), and encouraged the expectation that
triplexes formed by the AE oligonucleotides would be more stable
in vivo than those formed by the PS-01 TFO with only the
2'-OMe modification.
Triplex Stability in Vivo--
We have described an approach to
the measurement of triplex stability in vivo based on a
shuttle vector mutation assay (36). The assay reports the presence of a
triplex in the nuclear compartment that supports replication and
mutagenesis of DNA carrying psoralen cross-links. Triplexes were formed
by incubation of saturating levels of a TFO with the supF12
plasmid containing the embedded HPRT triplex target sequence
adjacent to a TA step appropriate for psoralen cross-linking (Fig.
1B) (see "Materials and Methods"). Unbound TFOs were
removed, and the TFO-plasmid triplex complexes were electroporated into
Cos-1 cells. At the indicated times the cells were exposed to UVA to
activate the psoralen. After an additional 48 h the progeny
plasmids were harvested and the frequency of plasmids with mutations in
the supF gene determined in a microbiological screen.
Mutagenesis was dependent on psoralen cross-linking, which in turn was
dependent on the bound pso-TFO. Thus the mutation frequency measurement
reflected the frequency of plasmids with pso-TFOs bound in triplexes at
the time of photoactivation. The plot of mutation frequency (normalized
to the 0 time value) versus time of photoactivation gives a
picture of the decay of the triplexes in the replication compartment.
We found that the triplexes formed by AE-02 and AE-03 (both with
t1/2 = 105 min) were about 1.5 times as stable
in vivo as the PS-01 triplex (t1/2 = 72 min). In contrast, the stability of the AE-01 triplex was
substantially less than the other TFOs (t1/2 = 25 min) (Fig. 5). These results indicated
that there was not a simple correlation between the stability of a
triplex formed by a particular TFO, as measured in vitro,
and the stability in vivo of the triplex formed by the same
TFO.
Activity of AE TFOs in HPRT Knockout Assay in Vivo--
The
activity of the Pso TFOs was then measured in an HPRT gene
knockout assay in Chinese hamster ovary cells. The TFOs were introduced
into the cells by electroporation and the psoralen activated by
exposure to UVA ("Materials and Methods"). Control experiments
showed that no mutagenesis occurred without UVA treatment. The cells
were carried in culture for 8-10 days to allow pre-existing enzyme to
decay, and then were placed in selective medium. Each TFO was tested in
at least three independent experiments. The Ps-01 TFO showed only
marginal activity relative to the mock-transfected control cultures
(Fig. 6A), similar to our
previous experience with this oligonucleotide (18). However, both the
AE-02 and AE-03 TFOs were active, with AE-03 (0.04%) consistently
showing greater activity. Somewhat surprisingly, the AE-01 TFO was no more active than PS-01. A scrambled version of the AE-03 TFO with the
same distribution of AE substitution (Sc AE-03, Fig. 1A), but with 8 mismatches, was inactive.
The activity of the AE-02 and AE-03 TFOs against the intended
HPRT target prompted us to analyze the activity against
another selectable marker, the gene for APRT. This gene also
has polypurine:polypyrimidine tracts, some linked to psoralen target
sites (46). We reasoned that if the activity of the AE-02 and AE-03
TFOs were simply a function of nonspecific interactions with these
kinds of sequence elements then they would show activity against the
APRT gene. However, we found that all the TFOs were inactive
in the assay (Fig. 6B). These results argued that the
activity of the AE-02 and AE-03 TFOs was specific to the
HPRT gene.
We then analyzed the sequences of some of the mutant HPRT
genes from the experiments with the AE TFOs. As shown in Fig.
7, the mutations were all relatively
small deletions in the triplex-psoralen target region. The smallest
deletion removed the two bases that would be involved in the psoralen
cross-link, while the largest was 48 bases extending through the
triplex target and the adjacent exon 5. Of the 24 unique mutants (some
appeared multiple times in independent experiments) 9 could be
described as deletions between sequence microhomologies in the target
region. These were consistent with cellular processing of the
TFO-psoralen cross-link through a double strand break (47).
Generally, pyrimidine motif TFOs with deoxyribose sugars have been
characterized by relatively weak affinity for their duplex targets at
physiological pH and Mg2+ concentration (2). The pH problem
has been somewhat ameliorated by the replacement of cytosine by
5-Me-cytosine (23). However, the requirement for non-physiological
levels of Mg2+ still requires a satisfactory solution. The
realization that RNA-like third strands (as defined by an oxygen at the
2' position) formed more stable triplexes than the 2'-deoxy equivalents
(25) led to the use of the 2'-OMe substitution in pyrimidine TFOS
(28-30). Although TFOs with this modification clearly show improvement in conventional assays in vitro, we found that the binding
of our PS-01 TFO was diminished in physiological Mg2+, and
was essentially inactive in our knockout experiments despite the
uniform presence of 2'-OMe modified sugars (18) (this report). Significant biological activity was observed, in our earlier study, only when an intercalator was introduced into the oligonucleotide (18).
Those results underscored the need to extend the activity of pyrimidine
TFOs containing 2'-OMe sugars, but also indicated that appropriate
chemical modifications could confer biological activity. Consequently
it was of considerable interest to ask if TFOs with 2'-AE sugars would
show enhanced biological activity relative to their counterparts
carrying only 2'-OMe sugars.
It was crucial for these analyses that the TFOs with AE substitutions
carry fully functional psoralen moieties. TFOs with inactive psoralen
would compete with fully active molecules and thus reduce the overall
efficacy of the reagents. We found that protocols used previously for
deprotection of AE oligonucleotides gave heterogeneous products and
appreciable loss of psoralen activity. We eventually determined that
gas phase deprotection with anhydrous methylamine produced a largely
homogeneous product with excellent psoralen activity (44). While
methylamine is a stronger base than the ammonia used in conventional
procedures, the deprotection reactions were performed at room
temperature, which reduced the probability of hydrolysis of the
psoralen lactone ring.
TFO Design--
The Pso TFOs described here contained 2'-AE sugars
in three different formats. AE-01 was uniformly substituted save for
the 3' residue (for synthetic reasons). This TFO was analogous to the
fully modified 15-mer characterized in the earlier study by Cuenoud
et al. (32). That TFO displayed a dramatic enhancement of
target affinity and stability of the resultant triplex. AE-02 contained
6 contiguous 2'-AE residues at the 3' end and was designed with the
expectation that this organization would confer enhanced binding as
well as resistance to 3'-exonuclease activity (48). The AE-03 TFO
retained the motif of a 3' patch of AE combined with a 5' patch. Based
on the previous work (32) we anticipated that the AE TFOs would have
greater affinity for their targets than the 2'-OMe equivalent and this
was substantiated in the in vitro experiments (Fig. 2).
Furthermore, the 2'-AE modification appeared to reduce the requirement
for Mg2+ for triplex formation, which characterized the TFO
with only the 2'-OMe substitution. In addition, the AE TFOs formed
triplexes that were very stable in physiological buffer at 37 °C.
Remarkably stable purine motif triplexes have been described (14, 15) and the triplexes formed by the AE TFOs appear to be comparably stable.
The results of these biochemical analyses suggested that, while the AE
TFOs as a group were clearly superior to PS-01, there was little to
distinguish one from the other, and we expected them to display similar
activity in the biological assays.
TFO Bioactivity--
We tested the TFOs in two bioassays, one that
measured triplex stability, the other gene targeting activity. The
plasmid based stability assay measures the persistence of a preformed
triplex in the nuclear compartment that supports plasmid replication
and mutagenesis of the TFO-psoralen cross-link. The triplex is exposed to the ionic environment as well as to the proteins and enzymes of this
compartment. In our earlier study we found that triplex stability could
be extended by lowering the temperature of the cells prior to
photoactivation (36), and we suggested that temperature-sensitive factors could destabilize triplexes in vivo. These might
include helicases (49), the enzymology of RNA and DNA synthesis,
chromatin assembly and modulation factors, and the proteins and enzymes involved in DNA repair (50). This is speculative and it is clear that
we lack an understanding of the ionic and protein effectors of triplex
stability in the nucleus. However, two conclusions can be drawn from
our experiments. The first is that it is possible to extend triplex
stability in this compartment by modification of the TFO, as
demonstrated by AE-02 and AE-03. This is encouraging because it
suggests that, whatever the nature of the destabilizing factors, they
can be countered, at least partially, by changes in the oligonucleotide
chemistry. The second, somewhat unexpected given the data in Fig. 4,
was that too much of the AE modification was deleterious (see below).
The results of the knockout assay showed that TFOs AE-02 and AE-03 were
active, unlike PS-01. The PS-01 TFO had the weakest affinity for the
target sequence in 10 mM Mg2+ concentration,
and showed a marked decline in activity when the Mg2+ was
reduced to 1 mM. In contrast, TFOs with 2'-AE substitutions formed triplexes at low concentrations (this report), or even in the
absence, of Mg2+ (32, 33). It is generally believed that a
TFO must be effective at low concentrations of Mg2+ if it
is to be active in vivo (51). Thus among the explanations for the activity differences between PS-01 and AE-02 and AE-03, it is
likely that triplex formation in low Mg2+ is an key
distinction. The importance of Mg2+ for triplex formation
by conventional oligonucleotides is well established (22), and the
contribution of oligonucleotide modifications to improved TFO activity
in reduced Mg2+ has been emphasized in recent publications
(51, 52). TFOs with a phosphoramidate backbone have been extensively
characterized and shown to form very stable triplexes in 10 mM Mg2+. However, in contrast to the AE TFOs,
triplex formation in the absence of Mg2+ was diminished
(51), and these TFOs appear to show only modest bioactivity (19).
The activity in vitro, in low levels of Mg2+,
does not explain the striking biological failure of the AE-01 TFO,
which was almost completely substituted with 2'-AE sugars. This TFO is
an analogue of one described previously, which bound its target in the
absence of Mg2+ (32). Although AE-01 had the greatest
affinity, and formed very stable triplexes in
vitro, it performed poorly in both assays of activity in
vivo. We suggest that there are at least two explanations. The
first is based on our observations of this oligonucleotide during the
preparation and purification. We had difficulty recovering material and
found that it was adherent to glass, columns etc. Apparently the
extensive aminoethoxy substitution altered the properties of the
molecule and we think it likely that the oligonucleotide may have been
bound by cellular membranes, proteins, nonspecifically to DNA, etc.,
greatly reducing the effective TFO concentration in the nucleus.
In the case of the stability assay in vivo, the triplexes
were preformed and would have been carried into the nucleus by the transfection of the plasmid. If the action of cellular proteins and
enzymes underlies triplex instability then, apparently, the AE-01
triplex was a better substrate for these activities than the triplexes
formed by the other TFOs. The 2'-AE modification makes two
contributions to the biochemistry of a TFO. In addition to the
interaction between the amine and an oxygen in the purine strand of the
duplex, this modification also preorganizes the C3'-endo,2'-exo conformation of the sugar (33). This
structure has been shown to be preferred in pyrimidine triplexes (31). In studies with oligonucleotides containing sugars locked in this conformation it has been shown that the local conformation of a single
locked sugar can influence the conformation of unmodified sugars in the
adjacent four nucleotides (53). If this is also true for the AE sugars
then the combination of the conformational influence on the immediate
and adjacent residues may produce a TFO that is perhaps too
constrained, unable to make subtle structural adjustments that may be
required for triplex stability in vivo. Whatever the actual
reason(s) for the poor activity of the AE-01 TFO our data do indicate
that extensive modification is not productive. It will be of interest
to determine if the extent of other sugar analogues that improve TFO
binding in vitro (51, 54) must also be limited in TFOs
intended for biological applications.
The end point of the HPRT knockout assay is a mutation
frequency. This reflects a summation of events, most with negative effect, that influence the probability of a targeted mutation in
HPRT. The frequency can be no greater than the number of
cells with triplexes in place at the time of photoactivation of the psoralen. This will be a function of the TFO transfection efficiency, the intrinsic accessibility of the target sequence at the time of TFO
entry, and the frequency and stability of triplexes that form on
accessible targets. It is likely that many binding events are
unregistered because they fail to persist to the time of UVA treatment.
Following photoactivation the oligonucleotide-psoralen cross-link can
be a substrate for the machinery of DNA repair, whose action may have
no mutational consequences. Thus the mutation frequency is an
underestimate, perhaps considerable, of the binding activity of a TFO
in vivo. We expect that the development of efficient and
robust gene knockout protocols will involve manipulation of the cell
biology to enhance target accessibility and mutagenic processing of the
targeted DNA damage. In addition, we believe that further optimization
of the AE format TFOs, perhaps in combination with other sugar and base
modifications (54-57), will produce TFOs with potent biological activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cyanoethyl-N,N-diisopropyl)phosphoramidite,
the
5'-O-(4,4'-dimethoxytrityl)-5-methyluridine-2'-O-methyl-3'-O-succinamido-N6-hexanamido-N3-propyl-controlled
pore glass support, and anhydrous acetonitrile (low water) were
purchased from Chemgenes, Ashland, MA. The
N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-5-methylcytidine-2'-O-methyl-3'-O-(-cyanoethyl-N,N-diisopropyl)phosphoramidite and
6-[4'-(hydroxymethyl)-4,5',8-trimethylpsoralen]hexyl-1-O-(-cyanoethyl-N,N-diisopropyl)phosphoramidite were purchased from Glen Research, Inc., Sterling, VA.
-cyanoethyl-N,N-diisopropyl)phosphoramidite followed the shorter route reported previously (33). Starting from
2,2'-anhydro-5-methyluridine the 2,2'-ring was opened by the complex
generated from titanium isopropoxide and
N-(2-hydroxyethyl)phthalimide. Subsequent
5'-dimethoxytritylation, removal of the phthalimide group by hydrazine,
and the protection of the liberated amine by trifluoroacetyl, followed
by phosphitylation on the 3'-hydroxyl gave the required
phosphoramidite. For the synthesis of
N4-(N-methylpyrrolidineamidine)-5'-O-(4,4'-dimethoxytrityl)-5-methylcytidine-2'-O-(2-aminoethyl)-3'-O-(-cyanoethyl-N,N-diisopropyl)phosphoramidite, an earlier procedure (33) was followed starting from
N3-benzyloxymethyl-5',3'-O-1,1,3,3-tetraisopropyl-1,3-disiloxyl-5-methyluridine. The synthesis involved generation of the 2'-azidoethyl group through standard procedures with the initial alkylation of the starting material. With the azido group as the intermediate protection, the
O-4 oxygen was replaced by an amino group by triazolation followed by ammonolysis. Protection of the
N4-amino group with
N-methylpyrrolidine amidine, reduction of the 2'-azidoethyl
group and its protection followed by 5'-dimethoxytritylation and
3'-phosphitylation gave the required phosphoramidite.
max for psoralen). The purified oligos were
characterized by capillary zone electrophoresis and matrix-assisted
laser desorption-time of flight.
-D-galactopyranoside and
5-bromo-4-chloro-3-indolyl--D-galactopyranoside and the
frequency of white or light blue colonies, which contained plasmids
with mutations in the supF gene, was determined.
Mutagenesis was dependent on cross-link formation, which in turn was
dependent on bound TFO at the time of UVA treatment. Consequently the
mutation frequency was a measure of the frequency of triplexes at the
time of UVA treatment. Each experiment was done at least three times.
4 M hypoxanthine, 5 × 106 M aminopterin, 105
M thymidine) for 1 week to remove pre-existing
HPRT-deficient cells. Cells were suspended at
107/ml in complete medium and Pso-TFOs added to 5 µM. The cell/TFO mixture was then electroporated
(Bio-Rad) at a setting of 110 volts, 960 microfarads, followed by
incubation at room temperature for 3 h, and exposure in the
Rayonet chamber to UVA light for 3 min at 1.8 J/cm2. The
cells were plated in complete medium for 8-10 days with 2-3 passages,
and then placed in selective medium depleted of hypoxanthine and
containing 20 µM thioguanine (200,000 cells/100-mm dish).
Cells were also plated in selective medium without thioguanine to
determine plating efficiency. After 10 days resistant colonies were
counted and picked for expansion and DNA analysis.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
A, schematic of the HPRT
gene, the triplex target sequence, and TFOs. The bases indicated in
small bold letters are those with 2'-aminoethoxy sugars.
B, schematic of the supF12 variant gene with the
HPRT target sequence embedded in the pre-tRNA region and
first bases of the acceptor stem. The 5' TA involved in the psoralen
cross-linking are boxed. The boxed nucleotides
are also part of a unique XbaI site (5'-TCTAGA).
View larger version (21K):
[in a new window]
Fig. 2.
Effect of deprotection protocol on
TFO-psoralen preparation. The HPLC profile following deprotection
in ammonium hydroxide, 55 °C, 1 h, upper left; or
after anhydrous methylamine, room temperature, 30 min, upper
right. The analysis by capillary electrophoresis of the peak from
the gas phase deprotection is shown in the lower left. The
activity of psoralen as demonstrated by XbaI protection is
shown in lower right (lane 1 is an undigested
marker; lane 2 is the digested marker; lane 3 the
plasmid was cycled through the precipitation protocol used to remove
noncross-linked TFO and then digested; lane 4, a triplex was
formed and the TFO was removed by multiple rounds of precipitation
followed by UVA treatment and digestion; lane 5 the triplex
was formed followed by photoactivation followed by multiple rounds of
precipitation to remove noncross-linked TFO and then digestion. The A
band is uncleaved, while the bands at B are the cleavage
products.
View larger version (10K):
[in a new window]
Fig. 3.
A, binding of PS-01 (filled
circles) and AE-03 (filled diamonds) to the HPRT target
sequence in 10 mM MgCl2; B,
KD of TFOs determined in 10 mM
MgCl2; C, KD of PS-01 and
AE-03 determined in 1 mM MgCl2.
View larger version (11K):
[in a new window]
Fig. 4.
Stability of triplexes in
vitro. Triplexes were formed on the HPRT
target in supF12 by PS-01 (filled circles), AE-01
(filled diamonds), AE-02 and AE-03 (open square
and cross, these are superimposed). The triplexes were
separated from unbound TFO and then suspended in physiological buffer
in the presence of excess competitor duplex oligonucleotide
("Materials and Methods"). At the indicated times the psoralen was
photoactivated, and the samples digested with XbaI. The
extent of resistant and sensitive target was determined by
electrophoresis on agarose gels.
View larger version (14K):
[in a new window]
Fig. 5.
Stability of triplexes in vivo.
Triplexes were formed on the supF12 plasmid and were
introduced into Cos-1 cells. At the indicated times the cells were
treated with UVA to activate the psoralen. After an additional 48 h the progeny plasmids were harvested and the frequency of plasmids
with mutations in the supF12 gene was determined by colony
color. AE-01, filled diamonds; PS-01, filled
circles; AE-02, cross; AE-03, filled
squares.
View larger version (13K):
[in a new window]
Fig. 6.
A, frequency of HPRT knockout
by Pso-TFOs. B, frequency of APRT knockout by
Pso-TFOs.
View larger version (14K):
[in a new window]
Fig. 7.
Mutations in HPRT induced by
the AE Pso-TFOs.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
|---|
We thank Dr. Serge Beaucage for helpful discussions and JiLan Liu and Juanita Thorpe for expert technical assistance.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: LMG/NIA National Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8565; Fax: 410-558-8157; E-mail: seidmanm@grc.nia.nih.gov.
Published, JBC Papers in Press, June 1, 2001, DOI 10.1074/jbc.M103409200
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ABBREVIATIONS |
|---|
The abbreviations used are: TFO, triplex forming oligonucleotides; OMe, 2'-O-methyl; AE, 2'-aminoethyl; RP-HPLC, reverse phase-high performance liquid chromatography; HPRT, hypoxanthine-guanine phosphoribosyltransferase.
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