Molecular interactions between nuclear factor kB NF-kB transcription factors and a PNA – DNA chimera mimicking NF-kB binding sites Alessandra Romanelli1, Carlo Pedone1, Michele Saviano1,
Trang 1Molecular interactions between nuclear factor kB (NF-kB) transcription factors and a PNA – DNA chimera mimicking NF-kB binding sites
Alessandra Romanelli1, Carlo Pedone1, Michele Saviano1, Nicoletta Bianchi2, Monica Borgatti2,
Carlo Mischiati2and Roberto Gambari2,3
1 Biocrystallography Research Center, CNR, Napoli;2Department of Biochemistry and Molecular Biology, and3Biotechnology Centre, Ferrara University, Ferrara, Italy
The decoy approach against nuclear factor kB (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 molecular interactions between chimeras and NF-kB nuclear proteins were investigated by molecular dynamics simulations, and interactions between 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 chimera and duplex NF-kB DNA – 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 interactions between HIV-1 LTR and p50, p52 and nuclear factors from B-lymphoid cells Therefore, these results demonstrate that the designed NF-kB DNA – 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-kB DNA – 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, nuclear factor 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,N0-diisopropylethylamine; MD,
molecular dynamics; ODN, synthetic oligonucleotides; cvff, consistent
valence force field.
Trang 2data using a molecular 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 binding sites are able to interact with both purified
NF-kB p52 and p50, as well as nuclear factors 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 transcription factors 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 interactions between a double-stranded PNA –
DNA – PNA chimera and nuclear proteins belonging to the
NF-kB family was performed by energy minimization and
molecular dynamics simulations, and interactions between
PNA – DNA chimeras and NF-kB proteins were studied by
electrophoretic mobility shift assays [28]
M A T E R I A L S A N D M E T H O D S
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-kB PNA – DNA chimeras
Tetrabutylammonium N-[2-[(4-methoxytrityl)amino]ethyl]
ethyl]-N-[thymin-1-yl-acetyl] glycinate, tetrabutyl
ammo-nium N-[(N6
-benzoyladenin-9-yl)acetyl]-N-[2-[(4-meth-oxytrityl) amino] ethyl]glycinate, tetrabutylammonium
N-[(N2 -isobutyrylguanin-9-yl)acetyl]-N-[2-[(4-methoxy-trityl) amino]ethyl] glycinate, tetrabutylammonium N-[(N4 - 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:g21) 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 0.3M solutions of the monomers
in acetonitrile/dimethylformamide 1 : 1 (v/v) (containing 25% of dimethylsulfoxide in the case of pyrimidine build-ing blocks), 0.3MN,N0-diisopropylethylamine (DIPEA) in acetonitrile/dimethylformamide (1 : 1, v/v) and 0.3M 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 Ac2O/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-20-ribonucleoside building blocks (15 equivalents) Two successive couplings were used to assure a high yield when obtaining the PNA-30 -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, a DNA elongadeprotec-tion step was performed using a monomethoxytrityl protected 50-amino-50 -deoxythy-midine (T) phosphoramidite as the linker between the DNA and PNA sections The amidic bond between the 50
-amino-50-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 (4 250 mm) on a Jasco HPLC system Gradient elution was performed at 40 8C, building up gradient starting with
Trang 3buffer A (50 mM triethylammonium acetate in water) and
applying buffer B (50 mM triethylammonium acetate in
acetonitrile/water, 75 : 25, v/v, with a flow rate of
1 mL:min21 Chimera 1: HPLC purity 100%, tR¼ 18 min
(gradient 3 – 20% B in 25 min); chimera 2: HPLC purity
100%, tR¼ 16 min (gradient 5 – 25% B in 25 min)
HPLC-MS analysis was carried out on a Jasco LCHPLC-MS system
equipped with a LiChrosphere 100 RP-18 endcapped
column (4 250 mm) using a gradient of acetonitrile in
10 mMammonium acetate buffer with mass detection on a
Perkin Elmer Sciex API 165 equipped with an electrospray
interface (ESI) Chimera 1: tR¼ 7 min (gradient 5 – 20%
acetonitrile in 20 min); ESI-MS: [M 1 4H]41¼ 1438.2,
[M 1 5H]51¼ 1150.5, calculated for C193H245N90O95P13
5748.26 Chimera 2: tR¼ 8 min (gradient 0 – 20%
aceto-nitrile in 20 min); ESI-MS: [M 1 4H]41¼ 1443.6,
[M 1 5H]51¼ 1154.9, calculated for C194H247N86O99P13
5770.26
The chimera sequences were Gly-ccg-50-TGGAAAGTCC
CCA-30-gcg-Ac (1) and Gly-cgc-50-TGGGGACTTTCCA-30
-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 and molecular 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
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:min21scan 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 1027M for PDP – PDP, 1.52 1026Mfor DNA – DNA in 10 mMphosphate 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 mMphosphate 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 50 end-labelled using [g-32P]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 mM dithiothreitol, 10 ng of human NF-kB p52 protein (Promega Corporation, Madison, WI) and 0.25 ng of
32P-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 Tris/borate/EDTA buffer; 22 mM 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-kB and Sp1 binding sites, sequences of the ODNs, PNAs and PNA – DNA – PNA (PDP) chimeras used.
Trang 4molecules carrying HIV-1 NF-kB binding sites (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 50
-TAATATGT AAAAACATT-30 (sense strand, NF-IL2A), 50
strand, GATA-1) and 50
-CATGTTATGCATATTCCTGTA-AGTG-30(sense strand, STAT-1)
Stability of decoy molecules
The stability of decoy molecules was evaluated after
incu-bation of DNA and PNA – DNA – PNA based decoys with
30!50 exonuclease III, 50!30 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
R E S U L T S
Design of synthetic oligonucleotides, PNAs and
PNA– DNA chimeras
The nucleotide sequence corresponding to a single
asym-metric NF-kB binding 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-kB binding sites For these reasons, the
experiments were performed with synthetic molecules
carrying the HIV-1 LTR asymmetric NF-kB binding 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-kB binding site (50-GGGGACT
TTCC-30) and linking to this DNA core a T base in the 50
position for synthetic reasons and an A at the 30end for the
Watson – Crick base pair interactions At both the 50 and 30
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 factors and the double stranded PNA – DNA – PNA
chimera mimicking the HIV-1 LTR NF-kB binding sites, in
order to investigate the molecular interactions between
nuclear factors and 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 between NF-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 50-GGGACTTTC-30/50-GAAAGTCC-30 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 50 and 30 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.
Trang 5chimeric 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-kB PDP – PDP hybrid
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 DNA transcription factor 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 and a 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 50 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 between NF-kB transcription factors 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
32P-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 to32P-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-kB factors 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 the32P-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-kB DNA – DNA hybrid in binding to purified p52 and p50 NF-kB proteins
The double stranded PDP – PDP chimera mimicking the HIV-1 NF-kB binding sites 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-kB binding sites 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 the32P-end-labelled NF-kB DNA – DNA target molecule Control oligonucleotides Fig 3 CD spectra of NF-kB PDP – PDP (- - -) and of DNA – DNA
( – – – ) duplexes.
Trang 6(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 between32P-labelled
DNA – DNA target and p52 or p50 NF-kB factors
The double stranded PDP – PDP chimera mimicking the
HIV-1 NF-kB binding sites 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
32P-end-labelled NF-IL2A, STAT-1 and GATA-1 DNA –
DNA target molecules and nuclear factors 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 nuclear factors to the relative
32P-end-labelled DNA –DNA target molecules, no inhibitory
activity was determined by addition of double stranded
PDP – PDP chimera mimicking 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-kB binding 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-kB binding 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-kB binding sites [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-kB DNA – 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-kB binding sites was evaluated after incubation with 30!50exonucleases, 50!30
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) and32P-labelled HIV-1 NF-kB DNA – DNA target molecules.
A total of 10 ng of NF-kB factors 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 binding sites 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-kB factors were incubated for 20 min
in binding buffer in the absence ( – ) or in the presence of the indicated concentrations of NF-kB DNA – DNA and NF-kB PDP – PDP molecules, as indicated After this incubation period, a further 20 min incubation step was performed in the presence of32P-labelled HIV-1 NF-kB DNA – 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.
Trang 7exonucleases, 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 30!50 exonucleases (Fig 8B) and 50!30 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)
D I S C U S S I O N
The NF-kB/Rel family of transcription factors 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, transcription factors 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 transcription factors of the NF-kB/Rel family and two target sites (50 -GGGGACT-TTCC-30) 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 nuclear factors 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-kB DNA–DNA
target molecule Protein–DNA complexes are marked with an arrow.
Asterisks indicate the free32P-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 nuclear factors 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 of32P-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 binding sites for STAT-1, GATA-1 and NF-IL2A were used, in order
to verify the specificity of protein – DNA interactions observed.
Trang 8In 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-kB transcription 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 nuclear factors 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) and32P-labelled
IgK NF-kB DNA – DNA target molecules A
total of 10 ng of NF-kB factors 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-kB DNA – 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.
Trang 9B-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 molecular interactions between the PNA– DNA
chimera and nuclear 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 chimera and the duplex NF-kB DNA – DNA
However, when interactions between the PNA – DNA
chimeras and NF-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 and nuclear factors from
B-lymphoid cells Therefore, the results obtained
conclu-sively demonstrate that the designed NF-kB DNA – 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-kB transcription factors 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
A C K N O W L E D G E M E N T S
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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