ABSTRACT
Triple helix-forming oligonucleotides covalently linked to psoralen can be specifically
cross-linked to both strands of DNA at the triplex-duplex junction following UV irradiation. We have previously shown
that a 15mer psoralen-oligonucleotide conjugate forming a triple helix on the promoter of the
[alpha]
subunit gene of the interleukin-2 receptor inhibits transcription of reporter plasmids transfected into
living cells after irradiation. In the present work, we directly demonstrate
covalent triple helix formation at the target site inside cells. A primer
extension assay using Taq polymerase was developed to quantitate the DNA which
had reacted with the psoralen of the triple helix-forming oligonucleotide. Photoaddition of the psoralen at the DNA target
site was demonstrated, not only when the preformed triplex was electroporated
inside cells, but also when the oligonucleotide was added to the culture medium
after plasmid electroporation and before irradiation of the cells.
Oligopyrimidine and oligopurine oligonucleotides have been shown to bind to the
major groove of duplex DNA at polypurine[middot]polypyrimidine sequences forming a triple helical structure (
1
-
5
). Intermolecular triplex-forming oligonucleotides have been shown to inhibit transcription in
several systems at the level of either elongation (
6
-
10
) or initiation through inhibition of transcription factor binding (
3
,
11
,
12
). Several reports have described the effect of oligonucleotides in eukaryotic
cells (
9
,
12
-
17
). However, in all these experiments there was no direct demonstration of triple
helix formation inside cells.
Psoralen-oligonucleotide conjugates can be used to cross-link both strands of the target in a sequence-specific manner (
18
,
19
). Covalent triple helix can efficiently inhibit transcription of reporter
plasmids transfected into living cells, as previously demonstrated on the
promoter of the [alpha] subunit of the interleukin-2 receptor (IL-2R[alpha]) (
13
). Inhibition was observed when UV-induced cross-linking occurred both
in vitro
and
in vivo
. In these experiments a mutant of the target sequence was used to demonstrate
that the inhibitory effect was due to triple helix formation (
13
,
16
).
In the present work, we have used a 15mer psoralen-oligonucleotide conjugate directed to the IL-2R[alpha] promoter carried by a plasmid vector to directly detect
triple helix formation inside human cells. An amplified primer extension assay
has been developed to quantitate the site-specific psoralen photoadducts on DNA.
DNA polymerases are efficiently stopped at covalently modified bases in DNA (
20
,
21
). Recently it was shown that triple helix-forming oligonucleotide clamps coupled to a psoralen derivative induced,
after irradiation, a stop of elongation at the photoaddition site (
22
). In the present work, the use of Taq polymerase which is stopped at psoralen-induced mono- and bi-adducts (
20
) allowed detection of photoproducts on an electroporated plasmid and to
demonstrate triple helix formation inside cells.
The plasmid constructs containing wild-type and mutant IL-2R[alpha] promoter regions (positions -352 to +110) were described previously (
1
,
13
; see Fig.
1
). The psoralen derivative of the 15mer oligonucleotide (Pso-15meC; see Fig.
1
) was synthesized as previously described (
18
). Unmodified oligonucleotides were purchased from Eurogentec (Belgium). They
were purified on denaturing polyacrylamide gels.
Plasmids were incubated with various concentrations of Pso-15meC in the indicated buffer and irradiated using a Xenon lamp (150 W) in
a Cunow housing system. The light was filtered through a Pyrex filter to remove
radiation below 310 nm. A filter removing radiation below 390 nm (Type GG 395;
Schott) was added to obtain a majority of mono-adducts. The conditions of DNA concentration, buffer incubation, temperature and time of irradiation are
indicated in the figure legends.
The
Mbo
II endonuclease cleaves the template in the IL-2R[alpha] promoter sequence 24 nt downstream of the 3'-end of the oligopyrimidine strand of the triple helix
target sequence (see Fig.
1
). A primer (21pu) corresponding to a sequence beginning 114 nt upstream of the
Mbo
II site and 90 nt upstream of the oligopurine tract 5'-end (Fig.
1
) was used for extension reactions.This primer, 21pu (8 nM), was 5'-labeled with [gamma]-
32
P and mixed with DNA sample in 50 [mu]l extension reaction buffer [16 mM (NH
4
)
2
SO
4
, 67 mM Tris-HCl, 1.5 mM MgCl
2
, 0.01% Tween 20], 1 mM each dNTP and 2.5 U Taq polymerase (Eurobio) were added
and samples were overlaid with 50 [mu]l mineral oil. The primer extension reactions were performed in a thermal
cycling unit (Hybaid) under the following conditions: 95oC for 2 min, 55oC for 55 s, 72oC for 2 min; 25 cycles were performed. Samples were separated
after ethanol precipitation by electrophoresis on an 8% polyacrylamide
denaturing gel. The gel was dried and analyzed using a Molecular Dynamics
PhosphorImager to quantitate the primer extension stops at the photoaddition
sites.
The plasmid IL-2R[alpha] CAT (1 [mu]g) was incubated with 10 [mu]M Pso-15meC oligonucleotide, irradiated and digested at 65oC with
Tfi
I endonuclease liberating a 37 bp fragment containing the triple helix site
(Fig.
3
A). Samples were electrophoresed on a denaturing 20% polyacrylamide gel. After
migration the gel was blotted on a nylon membrane (Hybond N
+
; Amersham) in 10* SSC. Membrane was prehybridized for 2 h at 45oC in 5* SSC, 0.1% SDS, 200 [mu]g/ml sonicated and denaturated herring sperm DNA.
Hybridization of 5'-
32
P-labelled oligonucleotide 25pu (complementary to the purine-rich strand of the target) or 25py (complementary to the pyrimidine-rich strand of the target) (10 pmol) was performed in 5* SSC, 5% SDS, in the presence of 10 [mu]g/ml denatured and sonicated herring sperm DNA, for
12 h at 47oC. The membrane was washed twice in 2* SCC, 0.1% SDS for 45 min at room temperature and analyzed using a
Molecular Dynamics PhosphorImager. Hybridization of a control oligonucleotide
complementary to another fragment of the cleaved plasmid allowed us to
normalize the quantity of DNA.
For oligonucleotide uptake and stability studies, 5 * 10
6
C8166 cells were incubated at 37oC with 10 [mu]M oligonucleotide Pso-15 meC in 75 [mu]l RPMI 1640, 20 mM HEPES with 10% heat-inactivated fetal calf serum. After various times of
incubation cells were centrifuged. The oligonucleotide was extracted from the
culture medium with phenol/chloroform saturated with 0.3 M sodium acetate and
precipitated with ethanol. The cell pellet was washed three times in PBS, lysed
in 10 mM Tris-HCl, pH 7.2, 10 mM NaCl, 2 mM MgCl
2
, 0.1% SDS and treated with proteinase K (100 [mu]g/ml) for 1 h at 37oC. Oligonucleotide was extracted with phenol/chloroform saturated with
0.3 M sodium acetate and precipitated with ethanol. Samples were
electrophoresed in a denaturing 20% acrylamide gel. After migration the gel was
blotted on a nylon membrane and hybridized with 5'-
32
P-labelled 15mer AS (5'-AGAGGGAGAGGAAAA-3') complementary to Pso-15 meC.
A pyrimidine oligonucleotide-psoralen conjugate was previously shown to induce cross-linking of the two DNA strands after irradiation when it formed a triple helix, provided that there was a 5'-TpA-3' step at the triplex-duplex junction (
18
). Analysis of cross-linked products showed that the 4'-5' (furan) double bond mainly reacts with the thymine
adjacent to the oligopurine target sequence. The resulting mono-adduct can absorb light at wavelengths near 365 nm and form a bi-adduct (Fig.
1
). Mono-adducts involving the psoralen 3-4 (pyrone) double bond cannot be
converted into bi-adducts.
The efficiency and specificity of triple helix formation with psoralen-15mer conjugates were studied on plasmid IL-2R[alpha] CAT with a sensitive detection method using Taq DNA
polymerase primer extension. After triple helix formation and irradiation, the plasmid was
digested with the endonuclease
Mbo
II. Extension of the radiolabelled primer 21pu yielded a full-length product of 114 nt (Fig.
1
). An arrest of Taq DNA polymerase by a psoralen adduct should give rise to a
truncated product of 90 nt.
When irradiation was omitted, or in the absence of third strand, only a 114 nt
product was obtained (Fig.
2
, lanes 3 and 4). This fragment corresponded to termination of DNA synthesis at
the
Mbo
II site. In the presence of the triplex-forming oligonucleotide and after irradiation ([lambda] > 310 nm), a band appeared corresponding to termination of
synthesis at the adenine preceeding the thymine which had reacted with the
psoralen on the template strand. This result is in agreement with those
obtained with triple helix-forming oligonucleotide clamps using modified T7 DNA polymerase
(Sequenase) (
22
). A second minor band one base before the major stop appeared when irradiation
time was increased (Fig.
2
, lane 6). The same phenomenon has been previously observed by Sage and
Moustacchi (
21
) using T4 DNA polymerase and DNA reacted with free psoralen. In experiments
performed with the mutated plasmid IL-2R[alpha] CATm, which cannot form a triple helix (see
1
), no stop of polymerase was detected at the TpA site (Fig.
2
, lane 2). This showed that both stops observed in the wild-type plasmid were specific and due to triple helix formation. Since two
other TpA sites were present on the DNA fragment, the absence of stop at these
sites for long irradiation times provided evidence for the specificity of
psoralen reaction at the duplex-triplex junction directed by oligonucleotide binding to its target
sequence.
Oligonucleotide stability and uptake were studied by incubating cells or nuclei
with the psoralen 15mer conjugate. After various times of incubation,
oligonucleotide extracted from the cells was purified and analysed on a
polyacrylamide gel (see Materials and Methods). The gel was subsequently
blotted and hybridized to a
32
P-labelled oligonucleotide complementary to the 15mer sequence of Pso-15meC (15AS). This method avoided the problem of
32
P release due to phosphatase activity in the cell and allowed us to study cell
uptake and degradation of 5'- and 3'-modified oligonucleotides.
The Pso-15meC degradation profile in the cell line C8166 is presented in Figure
4
. After 2 h incubation, 50% of Pso-15meC remained intact in the medium. This oligonucleotide was 5'-protected by psoralen and was probably degraded by 3'-exonucleases and also by endonucleases.
Degradation products (shorter than 10 nt) could not be detected by
hybridization of the labelled complementary oligonucleotide. The
oligonucleotide was extracted from cells at different times of incubation and
analysed by gel electrophoresis (Fig.
4
). The intact Pso-15meC was recovered from C8166 cells after 30 min incubation. The
intracellular oligonucleotide concentration was maximal after 2 h incubation.
This result, in accordance with several reports, showed that full-length oligonucleotide persisted for several hours within cells (
14
,
23
-
25
). When incubated with isolated nuclei, the 3'-unmodified Pso-15meC was quickly degraded in the medium (data not shown).
After 1 h incubation, the full-length oligonucleotide was almost undetectable in the medium.
The plasmid IL-2R[alpha] CAT was transfected together with Pso-15meC into C8166 cells by electroporation. This cell line
constitutively expresses the Tax protein, which transactivates IL-2R[alpha] gene transcription (
26
-
29
).
Transfected cells were irradiated 15 min after electroporation. To avoid any
contamination by external plasmid or plasmid associated with the cell membrane,
plasmid DNA was extracted from nuclei and photoproducts were detected by primer
extension (Fig.
5
A). When cells were irradiated for 30 min (15 min after transfection), a
significant fraction of the plasmid was detected as cross-linked to the oligonucleotide, as shown by the stop of polymerase at the
photoaddition site (Fig.
5
A). Of the purine-rich strand, 30% had reacted with the psoralen linked to the
oligonucleotide after 30 min irradiation. To confirm that the signal was due to
intracellular plasmid, the same experiment was performed after treatment of the
cells with DNase I to degrade membrane-bound plasmids before isolating the nuclei. The fraction of the plasmid
template that had reacted with psoralen and the signal intensity were similar
to those obtained when cells were not treated with DNase I (data not shown).
These experiments showed that the triple helical complex preformed
in vitro
was stable, at least to a certain extent, within the cells. However, the
fraction of plasmid which was covalently modified by the psoralen was lower
(50%) than that obtained
in vitro
(~85%). Several mechanisms can account for this difference. Thirty minutes
irradiation might not be sufficient within cells to reach the plateau; the
triplex may partially dissociate during electroporation or inside the cells.
Previous studies have shown that CAT gene expression directed by the IL-2R[alpha] promoter was inhibited after irradiation of HSB2 cells transfected
with plasmid in the presence of Pso-15meC (
13
). The extent of inhibition was 70% when cells were irradiated 15 min after
electroporation.
Several reports have described specific inhibition of expression of endogenous
genes inside cells, but in all these experiments direct evidence for triplex
formation was not provided (
10
,
14
,
15
,
17
,
33
,
34
). Interpretation of the results can therefore be controversial. For example,
Michelotti
et al
. (
35
) recently proposed that the previously reported inhibition of c-
myc
transcription (
15
) involved oligonucleotide interaction with
trans
-acting factors rather than triplex formation. The complexity of potential
interactions of oligonucleotides with cellular components other than the
targeted nucleic acid sequence stresses the importance of correlating
inhibition of gene expression to triple helix formation on the target DNA.
As shown previously in transient assays, the oligonucleotide Pso-15meC targeted to the IL-2R[alpha] promoter leads after irradiation to a 70% decrease in reporter activity (
13
). In these experiments the plasmid and the oligonucleotide were co-transfected into the cells before irradiation. The results described above
provide direct evidence that the oligonucleotide Pso-15meC does form a triple helix inside cells.
Recently, Svinarchuk
et al
. (
36
) studied triplex formation inside cells on a plasmid gene with an oligopurine
third strand by
in vivo
DMS footprinting assay. They showed that a footprint was detectable only when
the triple helix was preformed
in vitro
before electroporation. The differences between these data and our results may
be explained by the use of different cell lines and because the methods of
detection were not the same. In our experiments, we formed a covalent triple
helix which is more favourable for detection than footprinting.
In the present study, we have shown that triple helix-directed cross-linking of the psoralen-oligonucleotide conjugate occurs not only when the plasmid
and oligonucleotide are preincubated before electroporation followed by
irradiation, but also when cells are incubated with the oligonucleotide after
electroporation of the plasmid and then irradiated. Control experiments showed
that the cross-linking reaction most likely occurred within the nucleus. Experiments with
nuclei isolated from cells electroporated with the plasmid revealed that the target sequence was accessible to the
oligonucleotide in the nuclear environment.
Several reports have described the repair of triple helix-induced photoproducts in cells (
37
-
40
). However, this mechanism seems to be inhibited under certain conditions (
41
,
42
). In our system we performed experiments indicating that the mono- and bi-adducts were not repaired either in HeLa nuclear extracts or in
C8166 cells after 72 h incubation (manuscript in preparation).
Our data demonstrate that an oligonucleotide can form a specific triple helix
inside cells on an exogenous target. The next step will be to demonstrate
covalent triplex formation on the endogenous gene. For this purpose, modified
oligonucleotides with increased stability in cell culture medium should be
used. The covalent attachment of a propylamino group at the 3'-terminus of Pso-15meC strongly reduced degradation in the medium, indicating
that the 3'-unmodified oligonucleotide was mainly degraded by 3'-exonucleases (data not shown). Further work with this
modified oligonucleotide will allow us to investigate triple helix formation in
a quantitative way on the endogenous gene. Oligonucleotide analogues in which
the phosphodiester linkages are replaced by N3' -> P5' phosphoramidate linkages form stable triple helices with
targeted sequences (
43
). These analogues constitute an interesting tool to investigate the
accessibility of DNA to triplex-forming oligonucleotides in cell nuclei.
This work was supported in part by a grant from Rhône-Poulenc-Rorer and the Agence Nationale de Recherche sur le SIDA
(ANRS). The Ligue Nationale Française contre le Cancer and the ANRS
are acknowledged for support to ALG.
*To whom correspondence should be addressed. Tel: +33 1 40 79 36 84; Fax: +33 1
40 79 37 05; Email: a-laure@laurel.mnhn.fr
+
Present address: Genetics and Biochemistry Branch, NIKDD, NIH, Building 10, Room
9, Bethesda, MD 20892, USA
Preformed triplex.
IL-2R[alpha] CAT plasmid (5 [mu]g) was preincubated for 2 h at 4oC with 20 [mu]M Pso-15-meC oligonucleotide in 10 [mu]l buffer containing 10 mM KH
2
PO
4
, pH 6, 0.1 M NaCl. Samples of 10
7
cells (tumor T cell line C8166) were transfected by electroporation as
previously described (
12
). After 15 min at 37oC, cells were irradiated at 4oC, washed three times with PBS and nuclei were isolated under mild
lysis conditions (5 min on ice in a buffer containing 0.4% Nonidet P-40, 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl
2
, centrifuged for 5 min at 500
g
at 4oC and washed three times in PBS with 10 mM MgCl
2
). This procedure was performed to eliminate extracellular plasmid (which could
interfere in the extension reaction). Nuclei were lysed with 0.5% SDS. After
proteinase K digestion (10 [mu]g/ml, 2 h at 37oC), total DNA was extracted in phenol/chloroform saturated with 0.3 M
sodium acetate, precipitated twice with 5 vol ethanol and digested with
Mbo
II endonuclease (0.2 U/[mu]l).
Triplex formation in isolated nuclei.
Samples of 10
7
cells were transfected with IL-2R[alpha] CAT plasmid (5 [mu]g). After 15 min at 37oC nuclei were isolated and incubated with 20 [mu]M Pso-15meC at 37oC in PBS with 10 mM MgCl
2
in a final volume of 100 [mu]l. After various incubation times nuclei were irradiated for 30 min and DNA
was extracted, precipitated and digested with
Mbo
II endonuclease as described.
Triplex formation in cells.
Samples of 10
7
cells transfected with IL-2R[alpha] CAT plasmid (5 [mu]g) were incubated at 37oC. After 15 min, 20 [mu]M Pso-15meC was added. Cells were irradiated after 1 h
incubation. Nuclei were isolated before DNA extraction and cleavage with
Mbo
II endonuclease.
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