γ-Hydroxymethyl PNAs: Synthesis, interaction with DNA and inhibition of protein/DNA interactions

Article abstract of DOI:10.1016/j.bioorg.2010.06.002

The ability of PNA to interact with DNA double stranded has been recently investigated. In a decoy approach these interactions are of great importance as may lead to inhibition of interactions of DNA sequences to specific transcription factors and may be employed as a strategy for the inhibition of gene transcription alternative to the antisense strategy (targeting transcription factors mRNAs) and the transcription factor decoy approach (targeting transcription factors). We explored the ability of PNA and PNAs with modified monomers to bind to DNA and to interfere in the formation of DNA/transcription factor complex. We report a procedure for the synthesis of Fmoc-γ-hydroxymetyl PNA, the synthesis and CD analysis of PNA oligomers containing the modified monomer in different positions and EMSA assays to test the: (a) binding to double stranded DNA and (b) inhibition of DNA-protein interactions.

Full text of DOI:10.1016/j.bioorg.2010.06.002

Bioorganic Chemistry 38 (2010) 196–201
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
c-Hydroxymethyl PNAs: Synthesis, interaction with DNA and inhibition
of protein/DNA interactions
Soccorsa Pensato a, Michele Saviano a, Nicoletta Bianchi b, Monica Borgatti b, Enrica Fabbri b,
Roberto Gambari b, Alessandra Romanelli a,c,
*
a Institute of Biostructures and Bioimaging, CNR, via Mezzocannone 16, 80134 Naples, Italy
b BioPharmaNet, Department of Biochemistry and Molecular Biology, Section of Molecular Biology, University of Ferrara, via Fossato di Mortara 74, 44100 Ferrara, Italy
c University of Naples ‘‘Federico II”, School of Biotechnological Sciences, via Mezzocannone 16, 80134 Naples, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 February 2010
The ability of PNA to interact with DNA double stranded has been recently investigated. In a decoy
approach these interactions are of great importance as may lead to inhibition of interactions of DNA
sequences to specific transcription factors and may be employed as a strategy for the inhibition of gene
transcription alternative to the antisense strategy (targeting transcription factors mRNAs) and the tran-
scription factor decoy approach (targeting transcription factors). We explored the ability of PNA and PNAs
with modified monomers to bind to DNA and to interfere in the formation of DNA/transcription factor
Keywords:
PNA
Solid phase synthesis
EMSA
Biological activity
complex. We report a procedure for the synthesis of Fmoc–c-hydroxymetyl PNA, the synthesis and CD
analysis of PNA oligomers containing the modified monomer in different positions and EMSA assays to
test the: (a) binding to double stranded DNA and (b) inhibition of DNA–protein interactions.
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
modified PNAs revealed that the introduction of a side chain in this
position does not hamper formation of duplexes with DNA and
PNAs (aeg PNA) are oligonucleotide mimics with an uncharged
backbone, composed by amino-ethyl glycine units [1]. Due to their
chemical and enzymatic stability and to their high affinity towards
complementary DNA and RNA, they are widely used for regulating
gene expression with basically two mechanisms of action, the first
being antisense against target mRNAs, the second being antigene
for the targeting of DNA stretches [2]. Other applications of PNAs
include the identification of single nucleotide polymorphism, and
isolation and purification of nucleic acids [3,4]. The research on
PNAs focused on improving those properties which are considered
the Achilles heel of PNAs, as solubility and ability to cross cell
membranes. Conjugation of PNAs to peptides, oligonucleotides
and small molecules strongly improved the PNA cellular uptake
[5]. On the other side PNAs with modified backbone have been ob-
tained to improve cellular uptake and selectivity towards DNA and
or RNA. Chiral PNAs bearing amino acid side chain in position alpha
and gamma of the backbone are widely investigated [6]. Append-
ing guanidine groups in the gamma position yields PNAs with an
increased ability to be taken up by cells, as compared to aeg PNAs.
The presence of chiral centers contributes to increase the selectiv-
ity in the recognition of complementary DNA. Studies on gamma
RNA; furthermore, insertion of such modified PNAs into a standard
aegPNA oligomer induces structural organization and allows for an
improvement in the binding affinity [7,8].
The binding of PNAs to DNA duplexes has been investigated, pay-
ing attention to homopyrimidine sequences of single and bis-PNA
able to strand invade DNA and work as antigene. Binding of homo-
pyrimidine PNA to duplex DNA results in the formation of triple heli-
ces [9]. Strand invasion has also been reported by PNA targeting
regions of duplex DNA subject to breathing [10]. In this case the
resulting complex is a duplex PNA/DNA instead of a triplex. Exam-
ples of invasion by mixed sequence PNA and PNA-peptide conju-
gates have been described by Lundin and Corey respectively on
supercoiled DNA and chromosomal DNA [11,12]. Recently it was
foundthat mixed-sequence 15–20mer PNAs composed of only gam-
ma-methyl modified monomers can invade double-helical B-DNA
[13]. This last activity is of some interest, since binding of PNAs to
double stranded DNAs may lead to inhibition of interactions of
DNAsequencestospecifictranscriptionfactors(TFs)andmaybeem-
ployed as a strategy for the inhibition of gene transcription alterna-
tive to the antisense strategy (targeting transcription factors
mRNAs)and the transcription factordecoy approach(targetingtran-
scription factors). In this context antisense strategy against TFs is ex-
pected to be unefficient, due to the fact that usual regulation of gene
transcription is dependent from several TFs, belonging to the same
superfamily, but recognizing the same target gene sequence; on
the other hand PNA-based decoy molecules targeting TFs have been
* Corresponding author at: University of Naples ‘‘Federico II”, School of
Biotechnological Sciences, via Mezzocannone 16, 80134 Naples, Italy. Fax: +39
0812534574.
E-mail address: alessandra.romanelli@unina.it (A. Romanelli).
0045-2068/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2010.06.002

S. Pensato et al. / Bioorganic Chemistry 38 (2010) 196–201
197
reported to exhibit (with the exception of PNA–DNA chimeras) low
activity, as compared to ODN decoys.
In this work we explored the ability of gamma-hydroxymethyl
PNA to interfere in the binding of the transcription factor Sp1 to its
target DNA.
We describe a procedure for the synthesis of PNA monomers
with the backbone Fmoc–(O-benzyloxymethyl) amino-ethyl gly-
cine, and oligomers containing modified monomers. In order to
investigate the biological activity of the modified oligomers, we
and the product is precipitated in cold ether. The precipitate is sus-
pended in H2O, purified by preparative HPLC and lyophilized.
(Yield 100%.)
Rf: 0.48 (chloroform/methanol/water 14/6/1 v/v/v).
Mass analysis: m/z (ESI), [M+H]+ expected 404.3; found 404.2.
1H NMR (DMSO) (2 rotamers): 1.85 & 1.86 (3H, s, CH3 thymine),
4.10 (2H, s, –CO–CH2–thymine), 4.34 (2H, s, –N–CH2–COOH), 4.63
(2H, s, –OCH2–Ph), 7.37 & 7.41 (1H, s, H(6)-timina), 7.47 (5H, m, -
Ph-H), 11.41 & 11.45 (1H, s, COOH). Peaks corresponding to –NH–
CH–CH2–, –CH–CH2–N–e–CH–CH2–O– are covered by the signal of
water contained in DMSO in the range 3.2–3.6. 13C NMR (DMSO)
15.93 (CH3–thymine), 50.41 (N–CH2–COOH), 51.78 (CH–CH2–N–),
52.6 (NH–CH–CH2–), 52.91 (–CO–CH2–thymine), 71.61 (–CH–
CH2–O–), 76.50 (–O–CH2–Ph), 112.18 (C(5)–thymine), 131.70–
132.33 (–Ph), 141.52 (C(6)–thymine), 155.00 (C(2)–thymine),
168.31 (C(4)–thymine), 172.97 (–CO–CH2–thymine), 174.66
(COOH).
tested the ability of oligomers containing
c-hydroxymethyl PNA
in: (a) binding to double stranded DNA and (b) inhibiting DNA–
protein interactions. In both cases electrophoretic mobility shift as-
says have been performed.
2. Experimental
2.1. Synthesis of the PNA monomer and oligomers
2.1.3. Fmoc–NH (Bzl–OCH2)–T–OH (3)
Reagents for the synthesis of the PNA tOH monomer, Silica gel 60
(0.04–0.063 mm) for flash chromatography were purchased at Flu-
ka. TLC plates (Silica gel 60 F254, 5 ꢀ 20, 0.25 cm) are from Merk.
Fmoc (Bhoc) PNA monomers (Link Technologies), PAL–PEG
(0.19 mmol/g) (Applied Biosystem) were employed for the synthe-
sis of the PNA oligomers. 1He 13C NMR were recorded on a Varian
Innova 400 MHz spectrometer at 25 °C. LC–MS were carried out on
a Surveyor HPLC equipped with a mass spectrometer Thermo Finn-
46.4 mg of NH2–(Bzl–OCH2)–T–OH (0.11 mmol) are dissolved in
THF, 0.072 mL of a K2CO3aq 20% solution are added. The mixture is
stirred at 0 °C. 36.3 mg of Fmoc-OSu (0.11 mmol) are added and
the reaction is stirred at room temperature 1 h. The crude is ex-
tracted with ethyl acetate. The organic layer is washed with water
(2ꢀ) and dried over anhydrous Na2SO4. The solvent is evaporated
and the product is purified by silica gel chromatography. (Yield
84%.)
igan (electrospray source MSQ). Phenomenex Jupiter 5
l C18 300 Å
Rf: 0.58 (ethyl acetate/methanol/formic acid 95/3/2 v/v/v).
Mass analysis: m/z (ESI), [M+H]+ expected 627.1; found 627.2.
1H NMR (CDCl3) (2 rotamers): 1.76 (3H, s, CH3 thymine), 3.46–
3.70 (2H, m, –CH–CH2–O–), 3.99–4.05 (1H, m, –NH–CH–CH2–),
4.17–4.11 (2H, m, –CO–CH2–thymine), 4.25–4.27 (2H, m, –CH–
CH2–N–), 4.36 (2H, br s, –O–CH2–Ph), 4.44 (1H, br s, CH–Fmoc),
4.51 (2H, s, –N–CH2–COOH), 4.60 (2H, br s, CH2–Fmoc), 6.80 &
6.98 (1H, s, H(6)–thymine), 7.19–7.34 (5H, m, OCH2Ph),7.46–7.92
(8H, m, fluorenyl), 10.19 (1H, br s, COOH). 13C NMR (CDCl3) 11.98
(CH3–thymine), 47.04 (CH–Fmoc), 48.71 (–CH–CH2–N–), 48.91
(CH–CH2–N–), 49.60 ((N–CH2–COOH), 49.89 (–CO–CH2–thymine),
67.04 (CH2–Fmoc), 69.99 (–CH–CH2–O–), 73.26 (–O–CH2–Ph),
111.04 (C(5)–thymine), 125.01, 127.09, 127.76, 127.97, 128.08,
128.41, 128.58, 137.62 (Ph), 137.30 (C(6)–thymine), 141.22,
143.67 (Cq fluorenyl), 151.65 (C(2)–thymine), 156.48 (CO–Fmoc),
165.02 (C(4)–thymine), 168.68 (–CO–CH2–thymine), 173.06
(COOH).
and Phenomenex Jupiter 10 Proteo 90 Å columns were employed
l
respectively for analytical and semipreparative runs. Circular
dichroism spectra were recorded on a Jasco J-810 spectropolarim-
eter equipped with a Peltier thermal controller unity using a 1 cm
quartz cell at a temperature of 25 °C.
The modified monomer Fmoc–(BzlOCH2)–T–OH was synthes-
ised starting from the Boc (Bzl–OCH2)–T–OMe derivative obtained
following procedures reported in the literature [8].
2.1.1. Boc–(Bzl–OCH2)–T–OH (1)
56.0 mg of Boc (Bzl–OCH2)–T–OMe (0.11 mmol) are dissolved in
1.5 mL of dioxane and treated with 0.27 mL of NaOH 2 M solution.
The mixture is stirred 20 min, HCl 1 M is added until the pH of the
solution is 2. The crude is washed with ethyl acetate. The organic
layer is washed with H2O (2ꢀ) and dried with Na2SO4. The solvent
is evaporated and the resulting oil is dried under reduced pressure,
to give 55 mg of Boc–(Bzl–OCH2)–T–OH. (Yield 100%).
Rf: 0.48 (chloroform/methanol/water 14/6/1 v/v/v).
2.2. Synthesis of the oligomers
Mass analysis: m/z (ESI), [M+H]+ expected 504.3; found 504.2.
1H NMR (CDCl3) (2 rotamers): 1.64 (9H, s, t-butyl–CH3–), 2.05 &
2.06 (3H, s, CH3 thymine), 3.51–3.68 (2H, m, –CH–CH2–O–), 3.74
(1H, m, –NHCH–CH2–), 3.85 (2H, s, –CO–CH2–thymine), 4.21–
4.50 (2H, m, –CH–CH2–N–), 4.70–4.78 (2H, m, –O–CH2–Ph),
4.95 (2H, dd, –N–CH2–CO), 5.10 (1H, br s, BocNH), 7.41 & 7.44
(1H, s, H(6)–thymine), 7.46–7.56 (5H, m, –Ph–H), 8.10 (1H, s,
NH–thymine). 13C NMR (CD3OD) 12.55 (CH3–thymine), 29.02
(t-butyl–CH3), 48.66 (N–CH2–CO), 49.65 (CH–CH2–N–), 49.79
(NH–CH–CH2–), 49.94 (–CO–CH2–thymine), 68.38 (–CH–CH2–O–),
74.52 (–O–CH2–Ph), 80.82 (t-butyl–C), 111.15 (C(5)–thymine),
128.98, 129.14, 129.66, 129.76, 139.74 (–Ph), 143.90 (C(6)–thy-
mine), 153.20 (C(2)–thymine), 158.10 (t-butyl–O–CO), 167.23
(C(4)–thymine), 170.50 (–CO–CH2–thymine), 172.53 (COOH).
The PNAs were synthesised by Fmoc chemistry on a PAL–PEG re-
sin (2 lmol scale) on a Expedite 8909 synthesiser. The modified
monomer Fmoc–(BzlOCH2)–T–OH was coupled following proce-
dures reported in the literature [8]. Cleavage and deprotection were
performed by treatment of the resin with a TFA/TFMSA/m-cresol/
thioanisole mixture (6:2:1:1); the oligomers were precipitated with
ethyl ether. The oligomers were purified by reverse-phase HPLC
using a gradient of acetonitrile (0.1% TFA) in water (0.1% TFA), from
5% to 40% in 30 min. Purified molecules were characterized by elec-
trospray mass analysis.
Sequences obtained:
PNA: tgaggcgtggcct
PNA-C: tgaggcgtggcctOH
PNA-M: tgaggcgtOHggcct
PNA-C+M: tgaggcgt OHggcctOH
PNA-N: OHtgaggcgtggcct
2.1.2. NH2-(Bzl-OCH2)-T-OH (2)
46.6 mg of Boc-(Bzl-OCH2)-T-OH are dissolved in 1 mL of TFA/
CH2Cl2/TIS 47/50/3 v/v/v and stirred 10 min. TFA is evaporated

198
S. Pensato et al. / Bioorganic Chemistry 38 (2010) 196–201
Mass analysis:
PNA: tgaggcgtggcct expected: 3590.4; 1197.79 [M+3H]3+
898.59 [M+4H]4+; found: 1198.10 [M+3H]3+; 898.35 [M+4H]4+
;
PNA-C: tgaggcgtggcctOH, PNA-M: tgaggcgtOHggcct and PNA-N:
OHtgaggcgtggcct: expected: 3622.08; 1208.36 [M+3H]3+
;
906.52 [M+4H]4+: found: 1208.95 [M+3H]3+; 906.20 [M+4H]4+
PNA-C+M: tgaggcgt OHggcctOH
: expected 3652.08; 1218.36
[M+3H]3+; 914.02 [M+4H]4+: found: 1217.75 [M+3H]3+; 914.7
[M+4H]4+
2.3. Electrophoretic mobility shift assay (EMSA)
Scheme 1. Synthesis of the Fmoc (Bzl–OCH2)–T–OH PNA monomer.
Electrophoretic mobility shift assay (EMSA) [14,15] was per-
formed by using double stranded 32P-labeled oligonucleotides as
target DNA. Binding reactions were set up as described elsewhere
in binding buffer (10% glycerol, 0.05% NP-40, 10 mM Tris–HCl pH
7.5, 50 mM NaCl, 0.5 mM DTT, 10 mM MgCl2), in the presence of
dium hydroxide solution, followed by a reaction with trifluoroacetic
acid, to free the amino terminus. The Fmoc group was installed and
theproductpurified(Scheme1). Oligomers were assembledona low
loading PAL–PEG resin using standard Fmoc–(Bhoc)–PNA mono-
mers and the modified Fmoc–(Bzl–OCH2)–T–OH. Fmoc (Bhoc)
monomers were coupled following standard procedures, while the
modified monomer was coupled using the mixture N-meth-
yldicyclohexylamine/pyridine as a base and HBTU as coupling re-
agent, as described in the literature [8]. Treatment of resin bound
oligomer with TFA/TFMSA/thioanisole/m-cresol afforded the crude
product, which was purified by HPLC and analyzed by mass spec-
trometry. The sequence of PNA oligomers is the DNA sequence rec-
ognized by Sp1, a transcription factor involved in the regulation of
the expression of several genes, as those encoding for the vascular
endothelial growth factor, plasminogen activator inhibitor type 1,
urokinase plasminogen activator and its receptor [16].
poly(dI:dC)ꢁpoly(dI:dC) (1
den), 5.75 g/reaction of crude nuclear extracts from K562 cells
or 45 ng of purified nuclear factor human Sp1 (Promega, Milano)
and labeled oligonucleotide, in a total volume of 20 l. For analysis
lg/reaction) (Pharmacia, Uppsala, Swe-
l
l
of molecular interactions between PNA and double stranded DNA
(dsDNA), binding reactions were carried on for 40 min at room
temperature. In both cases, the samples were electrophoresed at
constant voltage (200 V for 1 h) through a low ionic strength
(0.25ꢀ TBE buffer) (1ꢀ TBE = 0.089 M Tris–borate, 0.002 M EDTA)
on 6% polyacrylamide gels until tracking dye (bromophenol blue)
reached the end of a 16 cm slab. Gels were dried and exposed for
autoradiography with intensifying screens at ꢂ80 °C. In studies
on the inhibition of protein/DNA interactions, addition of the re-
agents was as follows: (a) poly(dI:dC).poly(dI:dC); (b) labeled oli-
gonucleotides (c) competitor molecules (50 ng, 100 ng, 400 ng/
reaction); (d) binding buffer; (e) nuclear factors or purified Sp1,
for Fig. 4A and C, while for Fig. 4B and D nuclear extracts were
added before additions of PNAs. The nucleotide sequences of dou-
ble stranded target DNA utilized in these experiments were: 50-
TGA GGC GTG GCC T-30 (sense strand) and the complementary
50-AGG CCA CGC CTC A-30.
Sequences obtained are reported in Table 1. The chemical struc-
ture of the modified monomer as it is in the deprotected oligomers
is illustrated in Fig. 1 and is indicated as tOH
.
3.2. CD analysis
Single strand PNAs and their hybrids with the complementary
DNA were analyzed by Circular Dichorism, in Phosphate buffer pH
7 (Fig. 2). PNA bearing hydroxymethyl in position gamma are re-
ported to display optical activity, due to the presence of chiral cen-
ters [8]. The PNA obtained by us, bearing one or two chiral centers
exhibit very low optical activity; the PNA single strand with the
modification at the C-terminus (PNA C), in the middle (PNA M)
and at the C-terminus and in the middle (PNA C+M) have superim-
posable CD spectra, while the PNA modified at the N-terminus
hardly shows optical activity. The low optical activity may be due
2.4. CD analysis
CD spectra were recorded using concentration of PNA single
strand and PNA/DNA duplexes 2 lM in 10 mM Phosphate buffer
pH 7. The spectra are an average of 5 consecutive scans from 320
to 220 nm, recorded with a bandwidth of 0.5 nm, a time constant
of 4 s and a scan rate of 20 nm/min. Concentration of the PNAs
and DNA single strands was calculated measuring the absorbance
at 260 nm and using
a
molar extinction coefficient of
molꢂ1 cmꢂ1 for PNA C, PNA M, PNA C+M, PNA N and
Table 1
PNA sequences.
129.5 mL
124.4 mL
l
l
molꢂ1 cmꢂ1 for the complementary DNA strand
(AGGCCACGCCTCA).
Name
Sequence
PNA
tgaggcgtggcct
PNA C
3. Results and discussion
3.1. Synthesis of the Fmoc protected monomer and the oligomer
PNA M
PNA C+M
PNA N
We developed a procedure for the synthesis of the Fmoc–(Bzl–
OCH2)–T–OH monomer, to use on an automated PNA synthesiser
(Scheme 1). Yields in PNA synthesis increase when reactions are per-
formed under argon, as on a PNA synthesiser. The protocol requires
first the synthesis of the Boc–(Bzl–OCH2)–T–OH analog and then the
successive conversion of protecting groups. The Boc–(Bzl–OCH2)–T–
OCH3 monomer was obtained following procedures reported in the
literature [8]. The methyl ester was removed by treatment with a so-

S. Pensato et al. / Bioorganic Chemistry 38 (2010) 196–201
199
3.3. Binding studies: PNA analogs bind to target DNA and inhibit DNA–
protein interactions
In a first set of experiments the interactions of the modified PNA
and double strandedSp1 target DNA were analyzedand compared to
that of standard control PNA (Fig. 3). As evident from the Fig. 3, a
sharp decrease of free ds-DNA (panel A) and the appearance of
low-migrating dsDNA/PNA complexes (panel B) are clearly evident
with addition of 50 and 100 ng/reaction of the PNA molecules. In
these experimental conditions PNA molecules, when used at
50 ng/reaction, are about 10-fold in respect to target 32P-labeled
double stranded Sp1 DNA. In using DNA-based oligonucleotides,
specific competition is usually obtained in the range of 15–50 ng/
reaction using experimental conditions similar to those followed
in the present paper. Among the four PNAs analyzed, PNA-C and
the standard reference PNA appear to be slightly more efficient. This
is evident with two complementary observations: (a) the absence of
free probe only in the presence of PNA-C and the standard reference
PNA and (b) the high amount of blocked band evident when 50 ng of
PNA are added. The retarded bands may be attributed to a complex
formed after binding of the PNA on the DNA duplex, likely after
strand invasion. This phenomenon was reported for homopurine
PNAs [18] and in our case it may be attributed to the use of a PNA oli-
gomer with a high content of purines. The effect observed may be
further driven by the use of large excesses of PNA. The ability to
sequester the DNA is lowered when the modified monomer is in-
serted at in the center or when two such monomers are present.
PNA-N demonstrated an activity similar to standard PNA.
Fig. 1. Chemical structure of the
these studies.
c-hydroxymethyl PNA monomer employed in
In the second set of experiments, EMSA was performed using
two alternative approaches. In the first one (panels A and C of
Fig. 4), standard PNAs and PNA analogs were pre-incubated with
32P-labeled double stranded Sp1 DNA and then allowed to interact
with purified Sp1 nuclear factor (Fig. 4A) or nuclear extracts puri-
fied from K562 cells (Fig. 4C). In the second approach (panels B and
D of Fig. 4), purified Sp1 nuclear factor (Fig. 4B) or nuclear extracts
(Fig. 4D) were allowed to interact with target 32P-labeled double
stranded Sp1 DNA and then PNA molecules added.
Fig. 4A and C shows the data obtained performing the first ap-
proach, demonstrating that the synthesised PNA analogs inhibit
Sp1/DNA interactions when pre-incubated with the target double
stranded DNA. The efficiency of inhibition of the Sp1/DNA interac-
tion exhibited by the PNA analogs approached that of the standard
PNA. Again, PNA-C and the standard reference PNA appear to be
slightly more efficient.
Fig. 2. CD spectra of: duplexes PNA N/DNA (—); PNA M/DNA (ꢁ ꢁ ꢁ); PNA C/DNA (- - -
); PNA C+M/DNA (- ꢁꢁ -) and single strands PNA M (ꢁ ꢁ ꢁ); PNA C (- - -); PNA C+M (- ꢁꢁ -)
in Phosphate buffer 10 mM pH 7.
to the scarce incidence of one or two chiral monomer on a sequence
of 13 bases, a phenomenon particularly manifest for PNA N. With
the exception of the duplex formed by PNA N, all duplexes obtained
after annealing to complementary PNAs are very similar in second-
ary structure. The PNA N/DNA duplex shows one positive band
around 280 and one negative band, of comparable intensity around
245; this spectrum resembles to the spectrum of an antiparallel
PNA/DNA duplex. Hybrids formed by PNA C, PNA M and PNA
C+M, instead show a strong positive band around 265 and a mini-
mum around 240; the structure of these duplexes looks very similar
to that of PNA/RNA duplexes, suggesting that the presence of the
hydroxymethyl chain influences the structure of the duplex [17].
Overall CD data indicate that the position in which the chiral mono-
mer is inserted and the length of the oligomer dictates the second-
ary structure of the single strand and of the hybrids with DNA.
Fig. 4B and D, on the other hand, suggests that PNA-mediated
inhibition does not take place on pre-formed Sp1/DNA complexes;
even when (in the case of the use of nuclear extracts) PNA analogs
are added at 400 ng/reaction the inhibition does not take place.
Fig. 3. Efficiency of addition of PNA, PNA-C, PNA C+M, PNA M on the conversion of free-dsDNA (A) to dsDNA/PNA complexes. Free ds-DNA (panel A) and PNA complexed
dsDNA (panel B) were identified by polyacrylamide gel electrophoresis. Free ds-DNA migrated as a fast band, while dsDNA/PNA complexes exhibited a very low migration
efficiency.

200
S. Pensato et al. / Bioorganic Chemistry 38 (2010) 196–201
Fig. 4. Effects of PNA molecules on Sp1/DNA interactions. The effects of the indicated concentrations of standard PNA PNA-C, PNA C+M, PNA-M, and PNA-N were determined
on the de novo formation of Sp1/DNA complexes (A and C) and on pre-formed Sp1/DNA complexes (B and D). In the experiments described in panels A and C PNA molecules
were pre-incubated with 32P-labeled double stranded Sp1 DNA and the purified Sp1 nuclear factor (A) or K562 nuclear extracts (B) were added. In the experiment described in
panels B and D, purified Sp1 factor (B) or nuclear factors (D) were pre-incubated with 32P-labeled double stranded Sp1 DNA and then PNA molecules added. (–) = no addition
of purified Sp1 or nuclear extracts.
The complex formed by the transcription factor Sp1 and its target
DNA is thus very strong and, probably, the DNA is not easily accessed
by external molecules, therefore the PNA oligomer cannot reach its
target and move the equilibrium
us and here reported, the PNA analogs carrying the modified
monomer at the C-terminus appears to be the most promising
(see Fig. 3). On the other hand, our results suggest that the addition
of these PNA analogs has inhibitory effects on the generation of
Sp1/DNA complexes. In addition to the generation of PNA/DNA/
DNA complexes unable to interact with Sp1, our data are also com-
patible with the generation of PNA/DNA duplexes which, as pub-
lished by Borgatti et al. [19] are not able to interact efficiency
with Sp1. In any case, these (as well as the employed standard
PNA) are unable to disassemble pre-formed Sp1/DNA complexes.
In our opinion, this is very important, since we can hypothesize
these molecules as reagents able to interact and affect only ‘‘open”
chromatin structure interacting with transcription factors. What-
ever being the mechanism of action, our data sustain the concept
that this approach might interfere to the recruitment of transcrip-
tion factors to the promoter site, thereby inhibiting transcription,
allowing to sustain strategies alternative to the use of transcription
factor decoy oligonucleotides to control gene expression.
DNA þ protein ¢ complex DNA=protein
backwords; sequestering the DNA:
Overall, these data suggest that: (1) mixed sequences aegPNAs
form complexes with DNA double strand, but the introduction of
a modification in the middle of a sequence or at its C-terminus pre-
vents complex formation. (2) C-terminal modified PNAs keep the
ability to sequester DNA duplexes; (3) the complex PNA/DNA does
not interact with the transcription factor; (4) the capability of PNA
oligomers to sequester the Sp1 DNA duplex is strongly reduced
when the DNA is in complex with the transcription factor, probably
due either to the fact that the DNA duplex cannot be approached
by the PNA oligomer for steric reasons and to the lack of interac-
tions of the modified PNA with the transcription factor.
Acknowledgments
4. Conclusions
R.G. is granted by AIRC, by Fondazione Cariparo (Cassa di
Risparmio di Padova e Rovigo), by Cofin-2002, by Fondazione Itali-
ana per lo Studio della Fibrosi Cistica, by STAMINA Project (Univer-
sity of Ferrara) and by UE ITHANET Project. This research is also
supported by Regione Emilia-Romagna (Spinner Project) and by
Associazione Veneta per la Lotta alla Talassemia, Rovigo. The
authors are grateful to MIUR PRIN07 for funding.
In conclusion this work sustains the concept that mixed se-
quence PNAs can form complexes with duplex DNA. This feature
might be used in the future for approaches aimed at interfering
with transcription of targeted genes. This strategy for interfering
in the DNA transcription mediated by transcription factors was
proposed by several groups, based on the use of molecules able
to inhibit the recognition of specific DNA sequences by transcrip-
tion factors. In this context, the ability of PNA oligomers to bind
to DNA duplexes is a required pre-requisite for proposing these
molecules for further studies in this field. The introduction of one
or more modified PNA monomers in the oligomer influences the
ability to complex DNA. Among the novel molecules designed by
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