MolecularinteractionsbetweennuclearfactorkB(NF-kB) transcription
factors anda PNA–DNA chimeramimickingNF-kBbinding sites
Alessandra Romanelli
1
, Carlo Pedone
1
, Michele Saviano
1
, Nicoletta Bianchi
2
, Monica Borgatti
2
,
Carlo Mischiati
2
and Roberto Gambari
2,3
1
Biocrystallography Research Center, CNR, Napoli;
2
Department of Biochemistry andMolecular Biology, and
3
Biotechnology Centre,
Ferrara University, Ferrara, Italy
The decoy approach against nuclearfactorkB(NF-kB) is a
useful tool to alter NF-kB dependent gene expression using
synthetic oligonucleotides (ODNs) carrying NF-kB specific
cis-elements. Unfortunately, ODNs are not stable and need
to be extensively modified to be used in vivo or ex vivo.We
have previously evaluated the possible use of peptide
nucleic acids (PNAs) as decoy molecules. The backbone
of PNAs is composed of N-(2-aminoethyl)glycine units,
rendering these molecules resistant to both nucleases and
proteases. We found that the binding of NF-kB transcription
factors to PNAs was either very low (binding to PNA–PNA
hybrids) or exhibited low stability (binding to PNA–DNA
hybrids). The main consideration of the present paper was to
determine whether PNA–DNA chimeras mimicking NF-kB
binding sites are capable of stable interactions with proteins
belonging to the NF-kB family. Molecular modeling was
employed for the design of PNA–DNA chimeras; prediction
of molecularinteractionsbetween chimeras and NF-kB
nuclear proteins were investigated by molecular dynamics
simulations, andinteractionsbetween PNA–DNA chimeras
and NF-kB proteins were studied by gel shifts. We found
significant differences between the structure of duplex
NF-kB PNA–DNA chimeraand duplex NF-kBDNA–
DNA. However, it was found that these differences do not
prevent the duplex PNA–DNA chimera from binding to
NF-kB transcription factors, being able to suppress the
molecular interactionsbetween HIV-1 LTR and p50, p52
and nuclearfactors from B-lymphoid cells. Therefore, these
results demonstrate that the designed NF-kBDNA–PNA
chimeras could be used for a decoy approach in gene
therapy.
Keywords: peptide nucleic acids; PNA–DNA chimeras;
AIDS; NF-kB; transcription factors.
In vitro transfection of cis element decoys against nuclear
factors leads to the alteration of gene expression, and was
recently proposed as a novel molecular medicine tool for
possible use in therapy of a variety of well-characterized
disorders [1 –9]. Decoy molecules against HNF-1, RFX1,
NFYB, E2F, CRE and Sp1 were found to alter specific
functions in eukaryotic cells [7 –11]. One of the most
effective decoy approaches so far described involves nuclear
proteins belonging to the NF-kB superfamily. Decoy
molecules against NF-kB inhibit the expression of NF-kB
regulated genes (e.g. genes coding for MHC, IL2 receptor a,
Igk, IL6, d opioid receptor, preprogalanin and adhesion
molecule-1) [12–20]. More recently, dumbell DNA decoy
elements against NF-kB were demonstrated to be active in
inhibiting ex vivo transcription driven by the long-terminal
repeat (LTR) of human immunodeficiency type-1 virus
(HIV-1) [21]. A drawback of the decoy approach designed
for the modulation of gene expression is the presence of
intracellular DNases [1– 7]. Therefore, large amounts of
DNA must be internalized by target cells in order to obtain
biological responses leading to alteration of gene expression
[2]. In contrast, modified oligonucleotides (either methyl-
phosphonate or phosphorothioate) have been used by virtue
of their resistance to DNase cleavage, but these molecules
are highly toxic [22]. A further problem of the decoy
approach is the recently reported nonspecific activity of
these molecules. For example, dumbbell oligonucleotides to
RFX1, in addition to blocking activation of RFX1 regulated
genes, cause additional nonspecific effects most likely via
an interaction with the general transcription machinery [2].
In a recent paper, we investigated the possible use of
peptide nucleic acids (PNAs) [23–27] as alternative reagents
in experiments aimed at the control of gene expression
involving the decoy approach [28]. In PNAs, the pseudo-
peptide backbone is composed of N-(2-aminoethyl)glycine
units [23]. PNAs hybridize with high affinity to comple-
mentary sequences of single-stranded RNA and DNA,
forming Watson–Crick double helices [23,24] and are
resistant to both nucleases and proteases [29]. We demon-
strated that NF-kB p52 is able to bind to both NF-kBDNA–
DNA and DNA– PNA hybrids mimicking the NF-kB target
sites present in the HIV-1 LTR. Low binding of NF-kB p52
to PNA–PNA hybrids was found [28]. We have also
reported a conformational study to explain these binding
Correspondence to R. Gambari, Department of Biochemistry and
Molecular Biology, Via L. Borsari n.46, 44100 Ferrara, Italy.
Fax: 1 39 532 202723, Tel.: 1 39 532 291448,
E-mail: gam@dns.unife.it
(Received 25 May 2001, revised 19 September 2001, accepted
23 September 2001)
Abbreviations: NF-kB, nuclearfactor kB; Sp1, promoter-specific
transcription factor Sp1; AIDS, acquired immunodeficiency syndrome;
HIV-1, human immunodeficiency virus type 1; LTR, long-terminal
repeat; PNA, peptide nucleic acids; PDP, PNA–DNA–PNA chimera;
HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate; DIPEA, N,N
0
-diisopropylethylamine; MD,
molecular dynamics; ODN, synthetic oligonucleotides; cvff, consistent
valence force field.
Eur. J. Biochem. 268, 6066–6075 (2001) q FEBS 2001
data using amolecular dynamics approach. These data have
underlined that the loss of charged phosphate groups and the
different shape of helices in PNA–DNA and PNA–PNA
hybrids drastically reduce binding efficiency to NF-kB
transcription factor [30,31].
In order to develop PNA-based molecules able to stably
interact with transcription factors, in the present paper we
investigated whether PNA–DNA chimeras mimicking the
NF-kB bindingsites are able to interact with both purified
NF-kB p52 and p50, as well as nuclearfactors from
B-lymphoid cells.
In should be noted that PNA–DNA chimeras are com-
pounds of great interest [32– 34], as they are more water-
soluble than PNAs [33] and are far more resistant to
enzymatic degradation than oligonucleotides [32–34]. In
addition, they were recently found to be suitable primers for
DNA polymerases [32].
Finally, PNA–DNA chimeras generated with comple-
mentary RNA hybrid molecules were recognized by RNase
H [33]. However, PNA–RNA hybrids are not recognized by
RNase H [33].
In spite of these very promising results, no data on the
possible recognition of double stranded PNA–DNA chimeras
by transcriptionfactors are currently available in the
literature.
In the present paper, we designed, synthesized and tested
complementary PNA–DNA–PNA (PDP) chimeras poten-
tially able to interact with nuclear proteins belonging to the
NF-kB family.
With respect to the choice of the target sequence, we
decided to perform experiments using the nonsymmetric
NF-kB binding site of HIV-1 LTR in order to maximize
solubility (PNAs extremely rich in GC should be avoided)
and minimize the possibility to generate self- or inter-strand
hybridization, possibly forming highly stable complexes
[23–27]. In this respect, palindromic DNA sequences (for
example the symmetric GGGGATTCCCCT NF-kB binding
site of human p-selectin, human IL2Ra, mouse H2K, mouse
MHC EA promoter regions) are not the first choice [28].
Molecular modeling was employed for the design of
the NF-kB PNA–DNA–PNA chimeras, prediction of the
molecular interactionsbetweena double-stranded PNA–
DNA–PNA chimeraandnuclear proteins belonging to the
NF-kB family was performed by energy minimization and
molecular dynamics simulations, andinteractions between
PNA–DNA chimeras andNF-kB proteins were studied by
electrophoretic mobility shift assays [28].
MATERIALS AND METHODS
Synthetic oligonucleotides and peptide nucleic acids
The synthetic oligonucleotides used in this study were
purchased from Pharmacia (Uppsala, Sweden). HPLC-
purified PNAs were purchased from ISOGEN Biosciences
(Maarssen).
Synthesis of NF-kBPNA–DNA chimeras
Tetrabutylammonium N-[2-[(4-methoxytrityl)amino]ethyl]
ethyl]-N-[thymin-1-yl-acetyl] glycinate, tetrabutyl ammo-
nium N-[(N
6
-benzoyladenin-9-yl)acetyl]-N-[2-[(4-meth-
oxytrityl) amino] ethyl]glycinate, tetrabutylammonium
N-[(N
2
-isobutyrylguanin-9-yl)acetyl]-N-[2-[(4-methoxy-
trityl) amino]ethyl] glycinate, tetrabutylammonium N-[(N
4
-
benzoylcytosine-1-yl)acetyl]-N-[2-[(4-methoxytrityl)ami-
no]ethyl]glycinate PNA monomers were synthesized in the
laboratories of J H. Van Boom [35,36] (Leiden Institute of
Chemistry, Gorlaeus Laboratories, Leiden University, the
Netherlands); DNA monomers were obtained from Persep-
tive Biosystems. Methanol (Rathburn, HPLC grade) was
stored over molecular sieves (3 A
˚
) and used without further
purification. All the other solvents (Biosolve, synthesis
grade DNA) were used as received. Automatized syntheses
of the chimeras were performed on a Pharmacia Gene
Assembler, using highly cross-linked polystyrene (loading
26–28 mmol
:
g
21
) as the solid support on a 1-mmol scale.
The support was functionalized with a Fmoc-glycine via a
4-hydroxymethylbenzoic acid linker. Assembly of the PNA
parts was realized using 0.3
M solutions of the monomers
in acetonitrile/dimethylformamide 1 : 1 (v/v) (containing
25% of dimethylsulfoxide in the case of pyrimidine build-
ing blocks), 0.3
M N,N
0
-diisopropylethylamine (DIPEA) in
acetonitrile/dimethylformamide (1 : 1, v/v) and 0.3
M
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate (HATU) in acetonitrile/dimethylform-
amide (1 : 1, v/v). PNA monomers (15 equivalents per
mmol of resin) were preactivated for 1 min by mixing with
equal amounts of DIPEA and HATU, before coupling.
The protocol for the PNA oligomer synthesis on a 1 mmol
scale consisted of a cycle of the following steps. (a) Washing
with 2.5 mL of acetonitrile/dimethylformamide (1 : 1, v/v);
(b) coupling with the preactivated solutions of PNA, DIPEA
and HATU for 15 min in acetonitrile/dimethylformamide
(1 : 1, v/v); (c) washing with 2.5 mL of acetonitrile/dimethyl-
formamide (1 : 1, v/v); (d) capping with Ac
2
O/2,6-lutidine/
N-methylimidazole/tetrahydrofuran (1 : 1 : 1 : 7, v/v/v/v),
2.0 mL; (e) washing with 2.5 mL of acetonitrile and then
3.5 mL of dichloromethane; (f) detritylation with a solution
of trichloroacetic acid (2%) in dichloromethane for 3 min;
(g) washing with 2.5 mL of dichloromethane and 5 mL of
acetonitrile. DNA tract chain elongation was carried out
using 2-cyanoethyl-phosphoramidite-2
0
-ribonucleoside
building blocks (15 equivalents). Two successive couplings
were used to assure a high yield when obtaining the PNA-3
0
-
DNA junction via a phosphoramidate bond. 5-(O-nitrophe-
nyl)-tetrazole was used as activator. Standard DNA capping,
oxidation and detritylation cycles were used. Coupling
yields were gauged spectrophotometrically at 254 nm by the
absorption of the released trityl cation after each deprotec-
tion step. Finally, aDNA elongation step was performed
using a monomethoxytrityl protected 5
0
-amino-5
0
-deoxythy-
midine (T) phosphoramidite as the linker between the DNA
and PNA sections. The amidic bond between the 5
0
-amino-
5
0
-deoxythymydine phosphoramidite was realized using two
successive coupling cycles for the first PNA unit. PNA chain
synthesis was carried out following the above described
procedure. The yield of each PNA coupling was in the range
95–99 %, and the DNA couplings were quantitative.
After the last elongation step, the oligomers were cleaved
from the solid support and deprotected by treatment with
1.5 mL methanolic ammonia at 50 8C for 16 h [34].
The samples were filtered and then purified by RP-HPLC
on a LiChrosphere 100 RP-18 endcapped column
(4 Â 250 mm) on a Jasco HPLC system. Gradient elution
was performed at 40 8C, building up gradient starting with
q FEBS 2001 Binding of NF-kB proteins to PNA–DNA chimeras (Eur. J. Biochem. 268) 6067
buffer A (50 mM triethylammonium acetate in water) and
applying buffer B (50 m
M triethylammonium acetate in
acetonitrile/water, 75 : 25, v/v, with a flow rate of
1mL
:
min
21
. Chimera 1: HPLC purity 100%, t
R
¼ 18 min
(gradient 3–20% B in 25 min); chimera 2: HPLC purity
100%, t
R
¼ 16 min (gradient 5–25% B in 25 min). HPLC-
MS analysis was carried out on a Jasco LCMS system
equipped with a LiChrosphere 100 RP-18 endcapped
column (4 Â 250 mm) using a gradient of acetonitrile in
10 m
M ammonium acetate buffer with mass detection on a
Perkin Elmer Sciex API 165 equipped with an electrospray
interface (ESI). Chimera 1: t
R
¼ 7 min (gradient 5–20%
acetonitrile in 20 min); ESI-MS: [M 1 4H]
41
¼ 1438.2,
[M 1 5H]
51
¼ 1150.5, calculated. for C
193
H
245
N
90
O
95
P
13
5748.26. Chimera 2: t
R
¼ 8 min (gradient 0–20% aceto-
nitrile in 20 min); ESI-MS: [M 1 4H]
41
¼ 1443.6,
[M 1 5H]
51
¼ 1154.9, calculated for C
194
H
247
N
86
O
99
P
13
5770.26.
The chimera sequences were Gly-ccg-5
0
-TGGAAAGTCC
CCA-3
0
-gcg-Ac (1) and Gly-cgc-5
0
-TGGGGACTTTCCA-3
0
-
cgg-Ac (2).
Molecular dynamics simulations
All calculations and graphical analyses were run on a
Silicon Graphics O2 R10000 workstation. The package
INSIGHTDISCOVER (Biosym Technologies) was used to per-
form energy minimization andmolecular dynamics simu-
lations (MD) in vacuo at 300 K, with the consistent valence
force field (cvff), setting a pH 7 for all simulations.
In all simulations, the Arg, Glu, Gln, His, Lys, Asp and
Asn side chains carry a full charge, in agreement with the pH
value. The starting structures used in structural analysis and
simulations were those obtained from the Protein Data Bank
(http://www.rcsb.org/pdb/). Computational conditions were
chosen to avoid boundary effects [37].
The preparation of the starting models was performed in
agreement with other MD studies on this class of com-
pounds [38]. Using the solid state coordinates of the com-
plexes NF-kB p50/p65 homodimer with DNA, the PNA
duplex was generated by replacing the backbones of the
DNA strands of the DNA–DNA duplex with the PNA
backbone atoms. In all models, the coordinates of PNA
backbone atoms were generated by geometrical calculations
on their local topology and coordinates of their nearest
connected atoms and literature structural data. The coordi-
nates were then minimized, keeping the bases in a fixed
position. Then, the restraints were removed and further
energy minimization was performed. These resulting struc-
tures were then used for subsequent MD simulations.
The simulation was performed with a time step of 1.0 fs
at 300 K and the system was equilibrated for 80 ps. After
this first step, an additional 80 ps of simulation without
rescaling were carried out, as energy conservation was
observed and the average temperature remained essentially
constant around the target values. Coordinates and velocities
for the four simulations were dumped to a disk every 10
steps during the last 80 ps of the simulation.
Circular dichroism spectra
CD spectra were recorded at 20 8C on a Jasco model J-700
spectropolarimeter. The data were collected at 0.2 nm
intervals, with a 20-nm
:
min
21
scan rate, 1 nm band with a
16-s response, from 400 to 200 nm. Five scans were
performed for each sample, the CD spectra were obtained
as an average of the scans. The solutions were prepared
with concentration of 3.55 Â 10
27
M for PDP–PDP,
1.52 Â 10
26
M for DNA–DNA in 10 mM phosphate buffer
at pH 7. CD spectra are reported in molar ellipticity vs
wavelength. Single strand concentrations were determined
by UV. Double strands were annealed by warming up at
80 8C and cooling down at 4 8C before recording CD
spectra. Melting experiments were also performed on the
duplexes in 10 m
M phosphate buffer at pH 7.
Electrophoretic mobility shift assay
The electrophoretic mobility shift assay (EMSA) [39] was
performed by using the double-stranded synthetic oligo-
nucleotides mimicking the NF-kB (the nucleotide sequences
have been reported above and are shown in Fig. 1). The
synthetic oligonucleotides were 5
0
end-labelled using
[g-
32
P]ATP and T4 polynucleotide kinase (MBI Fermentas).
Binding reactions were set up as described elsewhere [39] in
a total volume of 25 mL containing buffer TF plus 5%
glycerol, 1 m
M dithiothreitol, 10 ng of human NF-kB p52
protein (Promega Corporation, Madison, WI) and 0.25 ng of
32
P-labelled oligonucleotides. When 2 mg of crude nuclear
extracts isolated from human cell lines were used instead of
purified NF-kB p50 and p52 factors, the binding reaction
was carried out in the presence of 1 mg of the nonspecific
competitor poly(dI-dC)
:
poly(dI-dC) [40]. After 20 min
binding at room temperature, the samples were electro-
phoresed at constant voltage (200 V) under low ionic
strength conditions (0.25 Â Tris/borate/EDTA buffer; 22 m
M
Tris/borate, 0.4 mM EDTA) on 6% polyacrylamide gels.
Gels were dried and subjected to standard autoradiographic
procedures [39]. In competition experiments, the competitor
Fig. 1. Structure of the HIV-1 genome, location of NF-kBand Sp1
binding sites, sequences of the ODNs, PNAs and PNA–DNA–PNA
(PDP) chimeras used.
6068 A. Romanelli et al. (Eur. J. Biochem. 268) q FEBS 2001
molecules carrying HIV-1 NF-kBbindingsites (DNA–
DNA, PDP–PDP and PNA–PNA) were preincubated for
20 min with purified NF-kB p52 protein, purified NF-kB
p50 factor or nuclear extracts, before the addition of labelled
target DNA. Nuclear extracts were prepared according to
Dignam et al. [40]. The nucleotide sequences of competitor
double stranded target DNAs used as controls were 5
0
-
TAATATGT AAAAACATT-3
0
(sense strand, NF-IL2A), 5
0
-
CACTTGAT AACAGAAAGTGATAACTCT-3
0
(sense
strand, GATA-1) and 5
0
-CATGTTATGCATATTCCTGTA-
AGTG-3
0
(sense strand, STAT-1).
Stability of decoy molecules
The stability of decoy molecules was evaluated after incu-
bation of DNAand PNA–DNA–PNA based decoys with
3
0
!5
0
exonuclease III, 5
0
!3
0
lambda exonuclease and
DNase I. ExoIII and lambda exonuclease were purchased
from MBI Fermentas and DNase I from Promega
Corporation, Madison, WI, USA. After incubation with
increasing amounts of the enzymes (for 10 min in the case
of ExoIII, for 30 min in the case of lambda exonuclease and
DNase I), the decoy molecules were layered on the top of a
2% agarose gel and detected by ethidium bromide staining.
Disappearence of the decoy molecule was considered as an
evidence of degradation by the employed enzymes. Results
were presented as percentage of recovery with respect to
control untreated reaction mixtures.
RESULTS
Design of synthetic oligonucleotides, PNAs and
PNA–DNA chimeras
The nucleotide sequence corresponding to a single asym-
metric NF-kBbinding site of the HIV-1 LTR was chosen in
order to maximize solubility of synthetized PNAs and
PNA–DNA chimeras. In addition, unlike symmetric NF-kB
binding sites, possible problems related to self and/or inter-
strand hybridization are expected to be minimal in the case
of asymmetric NF-kBbinding sites. For these reasons, the
experiments were performed with synthetic molecules
carrying the HIV-1 LTR asymmetric NF-kBbinding site
in both sense and antisense orientations (see Fig. 1).
The rational design of the NF-kB DNA–PNA chimera
was carried out, taking into account previous computational
analysis reported by us [30,31] and the solid state NF-kB
p50/p65 complex structure [41]. To preserve all protein–
DNA contacts the chimera was designed keeping the
nonsymmetric HIV-1 NF-kBbinding site (5
0
-GGGGACT
TTCC-3
0
) and linking to this DNA core a T base in the 5
0
position for synthetic reasons and an A at the 3
0
end for the
Watson–Crick base pair interactions. At both the 5
0
and 3
0
ends we have added three PNA monomers to ensure a highly
stable duplex.
Computational analysis
The MD simulation was performed on the complex between
nuclear factorsand the double stranded PNA–DNA–PNA
chimera mimicking the HIV-1 LTR NF-kBbinding sites, in
order to investigate the molecularinteractions between
nuclear factorsand the PDP– PDP hybrid molecule. The
comparison between the complex structure between NF-kB
p50/p65 bound to DNA–DNA, previously reported [41],
and the complex structure betweenNF-kB p50/p65 bound to
PDP–PDP, as obtained by MD simulation, demonstrates
that in both cases the central DNA duplex cores show a
comparable conformation (Fig. 2). In fact, the rmsd of all
atoms DNA of 5
0
-GGGACTTTC-3
0
/5
0
-GAAAGTCC-3
0
duplex from the canonical B-DNA is 2.2 A
˚
. This fragment
is slightly unwound with an overall twist of 11.2 bp per turn
(canonic DNA in B form has 10.0 bp per turn). On the
contrary, a large distortion of DNA due to PNA duplex is
present in the first (T) and last (CA) two DNA bp. In fact the
PNA duplexes have a conformation similar to that observed
in the solid state structure of PNA–PNA duplex [42]. The
PNA base pairs present a wide helix with an average calcu-
lated pitch of < 14 base pairs. The PNA helix conformation
could be described as comparable to a P-form duplex, and
this P-form would appear to be the natural conformation for
PNA [31]. The DNA bp near the PNA duplex in 5
0
and 3
0
show a distortion from the canonic DNA B-form. In par-
ticular, the sugar ring dihedral angles have values com-
parable to those observed in B-form DNA, but an average
overall twist of 12.5 bp per turn.
These results underline that PNA prefers a helical
structure that is different from that of DNA–DNA helices,
indicating that PNA seems to have clear conformational
preferences that are the driving force that leads to the
modification of the less flexible DNA backbone during the
formation of the duplex. It is worth noting that the PNAs in
Fig. 2. Average structure of NF-kB PDP–PDP duplex as obtained
from MD simulation in vacuo at 300 K of NF-kB p50/p65
heterodimer-PDP–PDP complex.
q FEBS 2001 Binding of NF-kB proteins to PNA–DNA chimeras (Eur. J. Biochem. 268) 6069
chimeric strands are able to modify the DNA conformation
of 2 or 3 bp starting from the PNA–DNA junction without
influencing the conformation of the remaining base pairs.
Computational analysis of the interaction of p50 NF-kB
with the PDP-PDP hybrid molecule
In the complexes between NF-kBPDP–PDPhybrid
molecule, the structural analysis reveals that the chimera
duplex has all base–specific interactions. In addition, the
system presents similar energy stabilization with respect to
the DNAtranscriptionfactor complex. Finally, the compari-
son of the relative orientation of the two subunits, both in the
solid-state structures and in the MD average models, reveals
that the NF-kB residues of both subunits are in optimal
positions to bind to PNA. In particular, for p50/p65 hetero-
dimer, the base–specific interactions mediated by Arg54,
Arg56, Glu60 and His64 of the p50 subunit and mediated by
Arg34, Arg35 and Arg187 of the p65 subunit are present,
and are comparable to the NF-kB DNA– DNA hybrid
molecule [41].
CD analysis and melting experiments
CD spectra of the duplex PDP–PDP chimera were recorded
and compared to the full DNA duplex sequence (Fig. 3). All
the comparisons were made on spectra normalized with
respect to the concentration. The analysis of the chimera
spectrum as compared to the DNA double-strand spectrum
[43] suggested that PDP –PDP tends to adopt a RNA A like
conformation. A-form RNA usually has a maximum close to
260 nm, a minimum close to 210 nm anda small negative
CD between 290 and 300 nm [43]. One maximum was
found at 267 nm in the chimera, the value observed in the
DNA analogue is 274 nm. Also, a shallow negative band
was at 294 nm. According to the conformational results
from the chimera duplex models, the amount of RNA A
form in the chimera, calculated considering a shift from the
DNA B form to the RNA A form, is < 50%. The intensity of
the bands was higher in the full DNA duplex than in the
chimera. This can be attributed to a bigger contribution of
stacking in the full DNA duplex than in the chimera duplex.
It could be speculated that adding PNA units at both termini
of the DNA strands causes a distortion in the double strand.
The rigid junction between the PNA and the DNA at the 5
0
end of the DNA might play an important role in the
conformation of the chimeric oligomer.
UV melting experiments were carried out on the same
duplex samples. A comparison between the obtained data
revealed that duplexes have similar melting temperatures
within the experimental error range, the chimeric duplex
being the less stable. The melting temperatures, calculated
assuming a two state process, were 53 ^ 1 8C for PDP–
PDP and 55 ^ 1 8C for DNA duplex (DNA).
The double stranded PDP–PDP chimera inhibits the
interactions betweenNF-kBtranscriptionfactors and
target DNA–DNA molecules
When 10 ng of human NF-kB p52 and p50 proteins were
incubated for 20 min in the presence of the cold double-
stranded PDP–PDP chimera, it was found that the
32
P-labelled NF-kB DNA–DNA probe was not efficiently
recognized by the NF-kB proteins (Fig. 4A). The results
obtained strongly suggest that, under these experimental
conditions, the double stranded PDP–PDP chimera
efficiently binds to p52 and p50 NF-kB transcription
factors. In addition, Fig. 4A shows that, unlike NF-kB
PDP–PDP molecules, NF-kB PNA–PNA hybrids do not
affect the binding of p50 and p52 NF-kB transcription
factors to
32
P-labelled NF-kB DNA–DNA target. As expected
[28], competition performed with GATA-1 and NF-IL2A
control oligonucleotides was also uneffective in inhibiting
interactions of NF-kBfactors with the target DNA.
Figure 4B shows a detailed study of the relationship
between the amount of competitor added and the inhibitory
effects observed. In this experiment, increasing amounts of
NF-kB DNA–DNA or PDP–PDP molecules were incu-
bated for 20 min in the presence of 10 ng of human NF-kB
p50 (Fig. 4B) and p52 (Fig. 4C) proteins; after this binding
period, a further 20 min incubation was performed in the
presence of the
32
P-labelled NF-kB DNA–DNA probe and
the samples were analysed by electrophoresis on native
6% polyacrylamide gels. These results demonstrate that the
NF-kB PDP –PDP hybrid does act as a competitive inhibi-
tor, despite having a lower efficiency than NF-kBDNA–
DNA hybrid in binding to purified p52 and p50 NF-kB
proteins.
The double stranded PDP–PDP chimeramimicking the
HIV-1 NF-kBbindingsites inhibits the interactions
between crude nuclear extracts and target NF-kB
DNA–DNA molecules
In order to determine the activity of the double stranded
PDP–PDP chimera carrying NF-kBbindingsites on a more
complex protein context, we repeated the experiments
reported in Fig. 4A by using, instead of purified NF-kB p50
and p52 proteins, crude nuclear extracts from B-lymphoid
Raij cells. In the experiment shown in Fig. 5, the double
stranded NF-kB PDP– PDP chimera was preincubated with
nuclear extracts from Raji cells, and processed as described
for the experiments shown in Fig. 4A. The obtained data
confirm that the double stranded PDP– PDP chimera inhibits
the binding of protein factors to the
32
P-end-labelled NF-kB
DNA–DNA target molecule. Control oligonucleotides
Fig. 3. CD spectra of NF-kB PDP–PDP (- - -) and of DNA–DNA
(– – –) duplexes.
6070 A. Romanelli et al. (Eur. J. Biochem. 268) q FEBS 2001
(NF-IL2A and GATA-1) were found to be inactive. In
agreement with data reported elsewhere by our research
group [28] the corresponding PNA–PNA hybrid molecule
was unable to inhibit the interactions between
32
P-labelled
DNA–DNA target and p52 or p50 NF-kB factors.
The double stranded PDP–PDP chimeramimicking the
HIV-1 NF-kBbindingsites does not inhibit the
interactions between NFIL2, GATA-1 and STAT-1
transcription factors to the relative target DNA–DNA
sequences
The experiment reported in Fig. 6 was performed using
32
P-end-labelled NF-IL2A, STAT-1 and GATA-1 DNA–
DNA target molecules andnuclearfactors isolated from Raji
and K562 cell lines. The results obtained firmly establish
that the effects of PDP– PDP chimera are sequence-specific.
In fact, while NF-IL2A, STAT-1 and GATA-1 cold oligo-
mers suppress the binding of nuclearfactors to the relative
32
P-end-labelled DNA–DNA target molecules, no inhibitory
activity was determined by addition of double stranded
PDP–PDP chimeramimicking the HIV-1 NF-kB binding
sites.
Differential effects of the HIV-1 NF-kB PDP–PDP chimera
on binding of NF-kB p52 and p50 to NF-kBbinding sites
of HIV-1 and IgK gene
The experiment reported in Fig. 7 demonstrates that DNA–
DNA and double stranded PDP –PDP chimeras mimicking
the HIV-1 NF-kBbinding sites, while effective inhibitors
of binding of NF-kB p52 and p50 to HIV-1 LTR sequences,
do not efficiently inhibit the binding of the same factors
to the palindromic GGGGATTCCCCT NF-kB IgK DNA
sequences. This result is of some relevance and is probably
due to the well-known differential affinity of NF-kB p52 and
p50 to HIV-1 or Igk NF-kBbindingsites [44]. The data
shown in Fig. 5 demonstrate that the NF-kB PDP–PDP
duplex chimera exhibits biological effects very similar to
those of NF-kBDNA–DNA.
Stability of the decoy molecules based on
PNA–DNA–PNA chimeras
The stability of the decoy molecules based on PNA– DNA–
PNA chimeras mimicking the NF-kBbindingsites was
evaluated after incubation with 3
0
!5
0
exonucleases, 5
0
!3
0
Fig. 4. Hybrid effects. (A) Effects of DNA–DNA, PNA–PNA and PDP–PDP hybrids, carrying the target sites of HIV-1 NF-kB, on the interaction
between purified NF-kB p50 (upper part of the panel) or p52 (lower part of the panel) and
32
P-labelled HIV-1 NF-kB DNA–DNA target molecules.
A total of 10 ng of NF-kBfactors were incubated for 20 min in binding buffer in the absence (–) or in the presence of 100 ng of DNA–DNA,
PNA–PNA and PDP–PDP molecules, as indicated. After this incubation period, a further 20 min incubation step was performed in the presence of
the
32
P-labelled HIV-1 NF-kB DNA–DNA target molecule. Protein – DNA complexes are indicated by an arrow. Asterisks indicate the free
32
P-labelled NF-kB DNA–DNA. Control DNA–DNA competitors carrying bindingsites for GATA-1 and NF-IL2A were used, as indicated. (B,C)
Effects of increasing amounts of DNA–DNA and PDP – PDP hybrids carrying the target sites of HIV-1 NF-kB, on the interaction between purified
NF-kB p50 (B) or p52 (C) and
32
P-labelled HIV-1 NF-kB DNA–DNA target molecules. A total of 10 ng of NF-kBfactors were incubated for 20 min
in binding buffer in the absence (–) or in the presence of the indicated concentrations of NF-kB DNA–DNA andNF-kB PDP–PDP molecules, as
indicated. After this incubation period, a further 20 min incubation step was performed in the presence of
32
P-labelled HIV-1 NF-kBDNA–DNA
target molecule. Protein–DNA complexes are marked with an arrow. Asterisks indicate the free
32
P-labelled NF-kB mer. Lane ‘b’ ¼ free
32
P-labelled
HIV-1 NF-kB DNA–DNA target molecule, no NF-kB protein added.
q FEBS 2001 Binding of NF-kB proteins to PNA–DNA chimeras (Eur. J. Biochem. 268) 6071
exonucleases, endonucleases, cellular extracts and serum.
After incubations, the decoy molecules were isolated,
layered on top of an agarose gel, electrophoresed and stained
with ethidium bromide (results of a typical experiment are
shown in Fig. 8A). Disappearance of the ethidium bromide
stained bands gives evidence for degradation of the decoy
molecules under the stated experimental conditions. The
observed stabilities of PDP –PDP chimeras were compared
to those of DNA–DNA decoy molecules. Examples of
the results obtained are depicted in Fig. 8B –D, which
clearly demonstrates that the decoy molecules based on
PNA–DNA–PNA chimeras are resistant to Exo III 3
0
!5
0
exonucleases (Fig. 8B) and 5
0
!3
0
lambda exonuclease
(Fig. 8C), unlike the corresponding DNA–DNA hybrid.
Interestingly, also when experiments were performed
employing DNase I, higher stability of the PNA–DNA–
PNA based decoys was obtained (Fig. 8D). Taken together,
these data suggest that PDP–PDP chimeras exhibit higher
levels of resistance to nucleases with respect to decoy
molecules based on DNA–DNA hybrids. When cytoplasmic
extracts from human leukemic K562 cells or human serum
was employed, we obtained results conferming the
increasing stabilities of PDP– PDP chimeras when com-
pared to DNA–DNA hybrid molecules (data not shown and
M. Borgatti, C. Mischiati, N. Bianchi and R. Gambari,
unpublished results).
DISCUSSION
The NF-kB/Rel family of transcriptionfactors is involved in
the control of the expression of a number of mammalian
genes, such as those encoding for major histocompatibility
complex (MHC) proteins, interferons and growth factors
[12– 20]. In addition, transcriptionfactors belonging to
the NF-kB/Rel family are involved in the transactivation
of viral genomes, such as HIV-1 [21]. In fact, it has
been demonstrated that HIV-1 transcription depends on
interactions between cellular transcriptionfactors of the
NF-kB/Rel family and two target sites (5
0
-GGGGACT-
TTCC-3
0
) present within the long terminal repeat [45].
Accordingly, biomolecular approaches able to inhibit
NF-kB activity could be of interest for the experimental
therapy of AIDS. For example, triple-helix-forming
oligonucleotides are able to inibit HIV-1 LTR-directed
transcription [46].
With respect to gene therapy, the decoy approach against
NF-kB has been proposed as a useful tool to alter NF-kB
dependent gene expression [12–20]. This was achieved by
using ODNs as decoy molecules, carrying NF-kB specific
cis-elements. Unfortunately, synthetic ODN are not stable
and therefore should be extensively modified in order to be
used in vivo or ex vivo [1 –7].
Fig. 5. Effects of NF-kB DNA–DNA, PNA–PNA and PDP–PDP
and GATA-1 and NF-IL2 DNA–DNA hybrids on the interaction
between crude nuclear extracts from B-lymphoid Raij cells and
32
P-labelled HIV-1 NF-kB DNA–DNA target molecules. A total of
1 mg of nuclearfactors were incubated for 20 min in binding buffer in
the absence (–) or in the presence of 50–200 ng of competitor molecules,
as indicated. After this incubation period, a further 20 min incubation step
was performed in the presence of
32
P-labelled HIV-1 NF-kBDNA – DNA
target molecule. Protein–DNA complexes are marked with an arrow.
Asterisks indicate the free
32
P-labelled NF-kBmer.
Fig. 6. Effects of DNA–DNA and PDP – PDP
hybrids carrying the target sites of HIV-1
NF-kB, on the interaction between crude
nuclear extracts from B-lymphoid Raij or
human leukemic K562 cells, as indicated, and
32
P-labelled NF-IL2A, STAT-1 and GATA-1
DNA–DNA target molecules. A total of 1 mg
of nuclearfactors were incubated for 20 min in
binding buffer in the absence (–) or in the presence
100 ng of DNA–DNA and PDP – PDP molecules,
as indicated. After this incubation period, a further
20 min incubation step was performed in the
presence of
32
P-labelled DNA–DNA target
molecules. Protein–DNA complexes are marked
with an arrow. Asterisks indicate the
32
P-labelled
NF-IL2, STAT-1 and GATA-1mers. Control
DNA–DNA competitors carrying bindingsites for
STAT-1, GATA-1 and NF-IL2Awere used, in order
to verify the specificity of protein–DNA
interactions observed.
6072 A. Romanelli et al. (Eur. J. Biochem. 268) q FEBS 2001
In a recent paper, we have proposed PNAs as alternative
reagents in experiments aimed at the control of gene
expression involving the decoy approach [28]. In PNAs, the
pseudopeptide backbone is composed of N-(2-aminoethyl)-
glycine units [23,24]. PNAs hybridize with high affinity to
complementary sequences of single-stranded RNA and
DNA, forming Watson– Crick double helices [23,24] and
are resistant to both nucleases and proteases [29]. We
demonstrated that NF-kB p52 is able to bind to both NF-kB
DNA–DNA and DNA–PNA hybrid mimicking the NF-kB
target sites present in the HIV-1 LTR. However, the binding
of the NF-kB DNA–PNA to NF-kBtranscription factors
was found to exhibit low stability, and therefore this reagent
is expected to be not suitable for a decoy approach [28].
The main issue of the present paper was to determine
whether PNA–DNA chimeras mimicking the NF-kB bind-
ing sites are capable of stable interactions with both purified
NF-kB p52 and p50, as well as nuclearfactors from
Fig. 7. Effects of increasing amounts of
DNA–DNA and PDP–PDP hybrids carrying
the target sites of HIV-1 NF-kB or Igk NF-kB,
as indicated, on the interaction between purified
NF-kB p50 (A) or p52 (B) and
32
P-labelled
IgK NF-kBDNA – DNA target molecules. A
total of 10 ng of NF-kBfactors were incubated
for 20 min in binding buffer in the absence (–)
or in the presence of the indicated concentrations
of DNA–DNA and PDP–PDP molecules, as
indicated. After this incubation period, a further
20 min incubation step was performed in the
presence of
32
P-labelled IgK NF-kBDNA – DNA
target molecule. Protein–DNA complexes are
marked with an arrow. Asterisks indicate the free
32
P-labelled NF-kB IgK DNA. Lane ‘b’ ¼ free
32
P-labelled Igk DNA–DNA target molecule, no
NF-kB protein added.
Fig. 8. Stability of decoy molecules.
(A) Preliminary experiment showing the effects
of ExoIII on DNA–DNA and PDP–PDP decoy
molecules. A total of 250 ng of NF-kB PDP–PDP
and DNA–DNA decoys were incubated for 10 min
in the absence (a) or in the presence of 0.001 (b),
0.01 (c), 0.1 (d), 1 (e), 10 (f) and 100 (g) U of
ExoIII in 20 mL reaction mixture. After incubation
the decoy molecules were layered on the top of a
2% agarose gel and detected by ethidium bromide
staining. B–D. Differential effects of ExoIII (B),
lambda exonuclease (C) and DNase I (D) on DNA
(open symbols) and PNA–DNA–PNA (closed
symbols) based decoys. Length of incubation was
10 min with ExoIII, 30 min with lambda
exonuclease and 30 min with DNase I.
Disappearence of the decoy molecules was
considered as an evidence of degradation by the
employed enzymes. Results shown in panels B–D
are presented as percentage of recovery with
respect to control untreated reaction mixtures.
q FEBS 2001 Binding of NF-kB proteins to PNA–DNA chimeras (Eur. J. Biochem. 268) 6073
B-lymphoid cells. DNA– PNA chimeras were originally
designed to improve the poor cellular uptake and solubility
of PNAs. More recently, they were found to exhibit
biological properties typical of DNA, such as the ability to
stimulate RNaseH activation and to act as substrate for
cellular enzymes (for example DNA polymerases) [32,33].
No information is available in the literature on the possible
use of double stranded PNA–DNA chimeras as target
molecules of transcription factors. This is not an unexpected
result, as the PNA–PNA hybrid structure is considerably
different compared to the DNA–DNA double helix and
therefore could alter the molecular structure of double-
stranded PNA–DNA chimeras, perturbing the interactions
with specific transcription factors.
Therefore, molecular modeling was firstly employed for
the design of the NF-kB PNA–DNA chimera [30] and
prediction of molecularinteractionsbetween the PNA–DNA
chimera andnuclear proteins belonging to the NF-kB family
was performed by energy minimizations and molecular
dynamics simulations [30,31]. Furthermore, the conforma-
tional behaviour and the thermal stability of the PDP– PDP
duplex chimera were studied by circular dichroism analysis
and melting experiments, respectively. The results obtained
with these independent approaches convergently demon-
strated significant differences between the duplex NF-kB
PDP–PDP chimeraand the duplex NF-kBDNA–DNA.
However, when interactionsbetween the PNA– DNA
chimeras andNF-kB proteins were studied by electrophoretic
mobility shift assay, it was clearly demonstrated that the
differences in molecular structure and conformation do not
prevent the PDP–PDP chimera from binding to NF-kB
transcription factors. We found indeed that the double
stranded PDP –PDP chimeras mimicking the HIV-1 NF-kB
binding sites are able to suppress the molecular interactions
between HIV-1 LTR and p50, p52 andnuclearfactors from
B-lymphoid cells. Therefore, the results obtained conclu-
sively demonstrate that the designed NF-kBDNA–PNA
chimeras could be proposed as powerful decoy molecules.
To our knowledge, this is the first report indicating that
double stranded PNA–DNA chimeras are target molecules
for transcription factors.
In addition, we hope our results will have practical
implications. The finding that DNA–PNA chimeras stably
interact with NF-kBtranscriptionfactors encourages further
experiments focused on the possible use of these molecules
for the development of potential agents for a decoy approach
in gene therapy. In this respect, the finding that PDP-based
decoy molecules are more resistant than DNA–DNA hybrids
to enzymatic degradation (Fig. 8 and data not shown) appears
to be of great interest. Furthermore, their resistance can be
improved further after complexation with cationic lipo-
somes or microspheres (M. Borgatti, C. Mischiati, N.
Bianchi and R. Gambari, unpublished results) to which
PDP–PDP chimeras are able to bind in virtue of their
internal DNA structure.
ACKNOWLEDGEMENTS
This work was supported by Istituto Superiore di Sanita
`
(AIDS/1998–
99), CNR-PF Biotecnologie, MURST-PRIN-98 and Finalized Research
funds (year 2001) from the Italian Ministry of Health. Mr Giuseppe
Perretta is acknowledged for technical assistance. The authors thank
Prof. J. H. van Boom and J. C. Verhejien for giving the possibility of
synthesizing the chimeras in their laboratory.
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q FEBS 2001 Binding of NF-kB proteins to PNA–DNA chimeras (Eur. J. Biochem. 268) 6075
. Molecular interactions between nuclear factor kB (NF -kB) transcription
factors and a PNA DNA chimera mimicking NF -kB binding sites
Alessandra Romanelli
1
,. stranded target DNAs used as controls were 5
0
-
TAATATGT AAAAACATT-3
0
(sense strand, NF-IL 2A) , 5
0
-
CACTTGAT AACAGAAAGTGATAACTCT-3
0
(sense
strand, GATA-1)