(The FASEB Journal. 2000;14:1041-1060.)
© 2000 FASEB
Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future
ARGHYA RAY and
BENGT NORDÉN1
Department of Physical Chemistry, Chalmers University of Technology, S 412 96, Gothenburg, Sweden
1Correspondence: Department of Physical Chemistry, Chalmers University of Technology, S 412 96, Gothenburg, Sweden. E-mail: norden@phc.chalmers.se; ray{at}phc.chalmers.se
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ABSTRACT
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Synthetic molecules that can bind with high sequence specificity to a
chosen target in a gene sequence are of major interest in medicinal and
biotechnological contexts. They show promise for the development of
gene therapeutic agents, diagnostic devices for genetic analysis, and
as molecular tools for nucleic acid manipulations. Peptide nucleic acid
(PNA) is a nucleic acid analog in which the sugar phosphate backbone of
natural nucleic acid has been replaced by a synthetic peptide backbone
usually formed from N-(2-amino-ethyl)-glycine units, resulting in an
achiral and uncharged mimic. It is chemically stable and resistant to
hydrolytic (enzymatic) cleavage and thus not expected to be degraded
inside a living cell. PNA is capable of sequence-specific recognition
of DNA and RNA obeying the Watson-Crick hydrogen bonding scheme, and
the hybrid complexes exhibit extraordinary thermal stability and unique
ionic strength effects. It may also recognize duplex homopurine
sequences of DNA to which it binds by strand invasion, forming a stable
PNA-DNA–PNA triplex with a looped-out DNA strand. Since its discovery,
PNA has attracted major attention at the interface of chemistry and
biology because of its interesting chemical, physical, and biological
properties and its potential to act as an active component for
diagnostic as well as pharmaceutical applications. In
vitro studies indicate that PNA could inhibit both
transcription and translation of genes to which it has been targeted,
which holds promise for its use for antigene and antisense therapy.
However, as with other high molecular mass drugs, the delivery of PNA,
involving passage through the cell membrane, appears to be a general
problem.—Ray, A., Nordén, B. Peptide nucleic acid (PNA): its
medical and biotechnical applications and promise for the future.
Key Words: DNA • PNA-DNA hybridization • antigene • antisense • gene therapy • nucleic acid biosensor
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INTRODUCTION
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PEPTIDE NUCLEIC ACIDS (PNA) originated from efforts
during the 1980s in organic chemist Prof. Ole Buchardt’s laboratory in
Copenhagen together with biochemist Peter Nielsen to develop new
nucleic acid sequence-specific reagents. Based on the observation with
flow linear dichroism that 
-helical poly-
-benzylglutamate (PBG)
can form stacked complexes with aromatic chromophores, it was proposed
that PBG with alternating nucleobases and acridine moieties instead of
phenyls might bind sequence selectively to duplex DNA by combined
Hoogsteen base pair formation and intercalation with the helix backbone
in the major groove. The proposed peptide nucleobase compound was given
the ad interim name peptide nucleic acid, or PNA.
Today’s PNAs (Fig. 1
) are DNA analogs in which a 2-aminoethyl-glycine linkage generally
replaces the normal phosphodiester backbone (1
, 2)
. A
methyl carbonyl linker connects natural as well as unusual (in some
cases) nucleotide bases to this backbone at the amino nitrogens. This
simple and yet entirely new synthetic molecule has an interesting and
nonprototype chemistry. PNAs are non-ionic, achiral molecules and are
not susceptible to hydrolytic (enzymatic) cleavage. Despite all these
variations from natural nucleic acids, PNA is still capable of
sequence-specific binding to DNA as well as RNA obeying the
Watson-Crick hydrogen bonding rules (3
, 4)
. Its hybrid
complexes exhibit extraordinary thermal stability and display unique
ionic strength properties. Although PNA was earlier considered
primarily a potential drug candidate for gene therapy, today there are
three to four major groups of applications for this novel compound.
First, it can be used as a molecular tool in molecular biology and
biotechnology (5
6
7
8
9)
. Second is its role as lead compound
for the development of gene-targeted drugs applying antigene or
antisense strategy (10
11
12)
. Third is the use of PNA for
diagnostics purposes and development of biosensors
(13
14
15
16)
. Fourth, the study of basic chemistry is related
to PNA (17
18
19
20)
for the improvement of basic architecture,
e.g., for supramolecular constructs and to possibly generate a
subsequent generation of PNA molecules.
This review will focus on different mechanistic aspects in order to
understand the physicochemical and biological properties of PNA and
PNA–nucleic acid complexes. We will discuss its mode of action in
relation to potential use as an antigene or antisense agent and related
developments for gene therapeutic application, detection systems for
genetic analysis, and novel tools for nucleic acid manipulation.
Finally, the cellular delivery of peptide nucleic acids will be
discussed in light of recent progress in this area.
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Chemical properties and related methodology
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This section will discuss, in brief, the synthesis and
purification of peptide nucleic acids. Then the physicochemical
properties of PNAs, as characterized from different biophysical
measurements, will be discussed.
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Synthesis and purification
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The backbone of PNA carries 2-aminoethyl glycine linkages in
place of the regular phosphodiester backbone of DNA (1
, 2)
, and the nucleotide bases are connected to this backbone at
the amino nitrogens through a methylene carbonyl linker (Fig. 1)
. Being
achiral, peptide nucleic acids can be synthesized without need of any
stereoselective pathway. PNA oligomers can be prepared following
standard solid-phase synthesis protocols for peptides (21
, 22)
using, for example, a (methylbenzhydryl)amine polystyrene
resin as the solid support (23
24
25
26
27)
. The scheme for
protecting the amino groups of PNA monomers is based on either Boc or
Fmoc chemistry (23
24
25)
. The postsynthetic modification of
PNA uses coupling of a desired group to an introduced lysine or
cysteine residue in the PNA (23
, 27)
. Amino acids can be
coupled during solid-phase synthesis or compounds containing a
carboxylic acid group can be attached to the exposed amino-terminal
amine group to modify PNA oligomers. A bis-PNA is prepared in a
continuous synthesis process by connecting two PNA segments via a
flexible linker composed of multiple units of either
8-amino-3,6-dioxaoctanoic acid or 6-aminohexanoic acid
(28)
. It has been found that changing cytosine to
pseudoisocytosine in the Hoogsteen strand abolishes the pH dependence
of a DNA·PNA-DNA triplex without affecting the stability of the
triplex (28)
.
A subsequent generation of PNAs might contain an unusual
modification of the backbone or a chimeric architecture. The latter is
commonly known as a PNA/DNA chimera, where a PNA oligomer is fused to a
DNA oligomer to give rise to new architecture. The synthesis of PNAs
with modified backbones is carried out in a similar way. PNA
hetero-oligomers comprising normal aminoethylglycine and
aminoproline building blocks are prepared using Boc strategy (for
details, see ref 29
). Two principal strategies have been
adopted to facilitate the synthesis of PNA/DNA chimeras: block
condensation of presynthesized PNA and DNA oligomers in solution, and
stepwise, online solid-phase synthesis with suitably protected
monomeric building blocks. The second strategy has been successful for
the synthesis of chimera. Development of special protecting group
strategies is also going on. Koch et al. (30)
have
reported the synthesis of a PNA pentadecamer carrying a heptapeptide
attached to the NH2 terminus via a
tris(8-amino-3,6,-dioxaoctanoic acid) using Boc peptide synthesis. The
method made use of traditional PNA synthesis with Boc-benzyloxycarbonyl
strategy. The presence of the peptide does not significantly affect the
structure of the PNA–DNA duplex. Betts et al. (31)
have
reported the synthesis of a PNA-peptide-PNA chimera where two
pyrimidine PNA nanomers have been linked by a his-gly-ser-ser-gly-his
peptide. This construct is capable of forming a stable triple-helical
complex with an oligopurine complex (31)
.
Postsynthesis procedures involve cleaving PNA oligomers from the
solid support by treatment with either anhydrous hydrogenfluoride or
trifluoromethanesulfonic acid, followed by high-performance liquid
chromatography (HPLC) purification (23
, 28
, 32
, 33)
. The
crude PNA products can also be cleaved from the solid matrix by
treatment with trifluoroacetic acid and m-cresol (4:1) mixture. Then
PNA is precipitated by ice-cold diethyl ether and dissolved in 0.1%
trifluoroacetic acid solution (23
, 32
, 33)
. Further
purification and subsequent characterization are done using
reverse-phase HPLC, followed by matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) mass spectrometry (23
, 28
, 32
, 33)
.
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Physicochemical properties
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Since peptide nucleic acids and DNA have no functional groups in
common except for the nucleobases, the chemical stability of PNA
differs significantly from its DNA counterpart. Unlike DNA, which
depurinates on treatment with strong acids, peptide nucleic acids are
fairly acid stable. However, some chemical instability can derive from
the free amino functionality at the NH2 terminus,
where a slow N-acyl transfer of the nucleobase acetic acid or a
cleavage of the amino-terminal PNA unit by ring closure can take place,
particularly under basic conditions (34)
. PNAs are
charge-neutral compounds and hence have poor water solubility compared
to DNA. Neutral PNA molecules have a tendency to aggregate to a degree
that is dependent on the sequence of the oligomer. PNA solubility is
also related to the length of the oligomer and purine:pyrimidine ratio
(35)
. Some recent modifications, including the
incorporation of positively charged lysine residues (carboxyl-terminal
or backbone modification in place of glycine), have shown improvement
as to solubility. Negative charges may also be introduced, especially
for PNA–DNA chimeras, which will enhance the water solubility. The
extinction coefficients for PNA monomers are not as well established as
they are for DNA or RNA monomers. It is expected that PNA oligomers
should have extinction coefficients different from their DNA or RNA
counterparts, as dissimilar backbones would variously perturb the 
system of the nucleobases. Since the extinction coefficients of
different PNA monomers are not well characterized and contributions of
the peptide backbone to the perturbation in the system of the
nucleobases are not known, for all practical purposes the concentration
of a PNA oligomer is determined by measuring the absorption at 260 nm
at 80°C (36
, 37)
. At that temperature, the nucleobases
are considered completely destacked and contributions from an ordered
backbone can be assumed to be less important.
PNAs hybridize to complementary DNA and RNA sequences in a
sequence-dependent manner, following the Watson-Crick hydrogen bonding
scheme A, multi-stranded triplex structure containing both Watson-Crick
and Hoogsteen base-pairing is also possible from a 2:1 PNA–DNA complex
(38
39
40
41)
(Fig. 2
). PNA-DNA hybridization is severely affected by base mismatches and PNA
can maintain sequence discrimination up to the level of a single
mismatch. On the other hand, base mismatches are less effective for
corresponding DNA-DNA hybridization. Different properties as well as
structures of different PNAs and PNA–DNA complexes have been well
characterized by a combination of various spectroscopic,
crystallographic, and computational methods.
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Figure 2. Schematic representation of different molecular complexes formed on
target-specific binding of PNA to DNA. A) Formation of a
prototype triple helical structure only possible with cytosine-rich
homopyrimidine PNAs. B) Stable triplex invasion complex
formed by homopyrimidine PNAs binding to a homopurine DNA target.
C) Double-duplex invasion complex only possible with
PNAs containing nonstandard nucleobases (116)
.
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PNA–DNA/RNA and PNA–PNA duplexes
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The properties of various duplexes of PNA, both pure and hybrid,
have been investigated extensively by absorption spectroscopy. The
thermal melting temperature (Tm), defined as the
temperature at which 50% of the complexes have been dissociated, gives
an idea of the stability of the PNA-DNA or PNA–RNA duplex. Moreover,
it provides information about different structural transitions. The PNA
oligomer H-TGTACGTCACAACTA-NH2 can form an
antiparallel duplex with complementary DNA sequence, which has a
Tm value of 70°C. But the corresponding
DNA–DNA complex melts at only 53°C. However, the corresponding
parallel PNA–DNA duplex shows a transition temperature of 56°C
(4)
. The thermal stability of a PNA–RNA duplex is even
higher than that of a PNA–DNA duplex (42)
. In contrast to
the pure DNA duplex, the stability of PNA–DNA hybrids is not affected
much by changes in ionic strength except in the limit of low ionic
strength, where the stability increases. The binding of PNA to a
corresponding complementary DNA oligomer takes place in a
sequence-specific manner, which means that the thermal stability of a
heteroduplex is considerably lowered by the presence of mismatches.
Corresponding DNA duplexes are not affected by mismatches in this way.
Owing to the higher sequence specificity of PNA on binding to nucleic
acids, incorporation of any mismatch in the duplex considerably affects
the thermal melting temperature of the heteroduplex. For the
heteroduplex comprising the PNA oligomer
H-egl-GGCAGTGCCTCACAA-NH2 (carboxyl-terminal) and
its full complementary DNA 5'-TTGTGAGGCACTGCC-3', a mismatched
incorporation of A in the in eighth position of the DNA sequence, in
place of G, reduces the Tm value from 72.3°C to
58.1°C (Table 1
; B. Nordén and co-workers, unpublished observations). PNA–DNA
chimeras also follow the basic Watson-Crick hydrogen bonding scheme on
hybridization with complementary DNA and RNA. The thermal melting
temperature of the duplex formed as a result of the interaction between
a PNA/DNA chimera and a DNA or RNA counterpart usually lies between
that of the corresponding PNA–DNA and DNA–DNA duplexes. The
Tm value of the chimera–DNA duplex also depends
on the ratio of PNA and DNA in the chimera strand and, above all, on
the nature and position of the linker molecule between PNA and DNA
bases (ref 29
and references therein).
Apart from these complexes with natural nucleic acids, peptide nucleic
acids can also bind to complementary sequences of PNA itself to form
extremely stable PNA–PNA duplexes (43)
. Preference is
found for antiparallel duplexes with high melting temperatures compared
to their hybrid (PNA–DNA duplex) counterparts. The PNA decamer
H-GTAGATCACT-NH2 forms both antiparallel and
parallel complexes with complementary PNA and DNA sequences. The
Tm values corresponding to antiparallel PNA–PNA,
PNA—DNA, and DNA–DNA sequences are 67°C, 51°C, and 33.5°C,
respectively. However, the parallel PNA–PNA complex with a
Tm value of ~47°C is still considerably more
stable than the corresponding DNA–DNA duplex (43)
. The
increased thermal stability of PNA–PNA duplexes relative to the
corresponding DNA–DNA duplexes is fundamentally due to the absence of
any significant electrostatic repulsion between the two strands in the
former complex.
Moreover, a PNA-PNA-PNA triplex construct is also possible from one
purine and two pyrimidine decamers, as evidenced from circular dichroic
measurements (20
, 44)
.
In general, PNA contains an achiral backbone and does not show any
circular dichroism (CD). However, PNAs with certain modifications of
the backbone structure and its conjugation with complementary DNA and
RNA sequences to form hybrids are capable of exhibiting circular
dichroic characteristics (Fig. 3
). A PNA duplex containing L-lysinyl amide attached to the carboxyl
terminal of the PNAs can demonstrate a handedness resulting from the
helicity of the structure. The chiral orientation of the base pairs
relative to each other is regarded as the source of the CD signal, and
the measured induced helicity is dependent on the nucleobase sequence
proximal center (45)
. The CD spectra of DNA–DNA and
antiparallel PNA–DNA and PNA–RNA duplexes are more or less similar.
This suggests that the base pair geometry of antiparallel right-handed
PNA-containing helices is not much different from that found in a B- or
an A-form DNA helix. In contrast, the spectra of parallel PNA–DNA and
PNA–RNA duplexes deviate more from the DNA-DNA spectrum, which
suggests a different kind of base stacking. It has been concluded that
incorporation of a PNA strand/strands drastically reduces the binding
capability of both minor groove binders and intercalators
(46)
.
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Figure 3. CD spectra of GTAGATCACT·AGTGATCTAC duplexes: DNA–DNA (curve 1) and
PNA–PNA (curve 2) duplexes. Base pair concentration, 50 µM; path
length of the cuvette, 1 cm. Adapted by permission from Dr. Pernilla
Wittung.
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In solution, the PNA monomer exists as both the cis
and trans rotamers about the tertiary amide bond, slightly
favoring the trans conformation (20)
as
revealed from the 1H-NMR (nuclear magnetic
resonance) spectra. The two rotamers interconvert on the time scale of
~1 s at room temperature. For longer sequences, such as the PNA
octamer H-GCTATGTC-NH2, the
1H-NMR spectrum reveals the existence of a
multitude of structural species. For a total of N number of residues,
there can be 2N possible structural species. The
solution structures of the antiparallel PNA-DNA
(H-GCTATGTC-NH25'd[GACATCGC]) duplex have been
determined by 1H-NMR (20
, 47
, 48)
.
The duplex shows a helical conformation with a Watson Crick
base-pairing scheme. The helical rise is ~42 Å with ~13 base pairs
per turn, whereas the helical diameter is close to 23 Å. The major
groove is wide and deep but the minor groove is quite narrow and
shallow. The carbonyl groups on the backbone base linkers of this
structure are oriented along the backbone, pointing in the
carboxyl-terminal direction. However, the sugar puckering is
predominantly in the C2'-endo conformation with all antiglycosidic
torsion angles and trans primary amide bond conformations.
The PNA–RNA duplex molecule (GAACTC-5'r[GAGUUC]), as revealed from
proton NMR spectrum, has features similar to a right-handed A helix
(20
, 49)
. The carbonyl groups of the PNA backbone linkers
are directed toward the carboxyl terminus and primary amide bonds are
in the trans configuration. The sugar puckering in the
nucleic acid strand is C3'-endo with glycosidic torsional angles near
-160°, features similar to an A-type conformation.
Rasmussen et al. (50)
reported the crystal structure of a
PNA duplex (H-CGTACG-NH2)2
at 1.7 Å resolution. The molecule crystallized in space group
P1bar, showing the presence of both a right- and
a left-handed helix in the unit cell (50)
. The helical
parameters estimated for this complex deviate significantly from the
canonical A and B conformations. The pitch is large (18 bp) and the
helix width is 28 Å, whereas the twist, rise, base tilt, and
displacement values are 19.8, 3.2, 1.0, and 8.3 Å, respectively. One
helical turn of the PNA duplex is ~58 Å long. From the results of
their crystallographic study, Nielsen and co-workers (20
, 50)
have confirmed that a sugar phosphate backbone is not a
prerequisite for the formation of a nucleobase double helix in
accordance with the inferences based on earlier circular dichroism
results when this species was discovered (43
, 45)
.
It is also pertinent to briefly describe here the thermodynamic
aspects of PNA binding to DNA and the binding motif. The need for a
detailed thermodynamic characterization of PNA-DNA interaction is
essential in order to improve our overall understanding of such a
reaction, particularly to accurately predict the thermodynamic
properties of relatively new complexes of sequences not studied before.
So far there are only a few reports concerning the determination of the
thermodynamic parameters 
H° and S° (4
, 36
, 51)
.
Two major techniques used to determine thermodynamic parameters are the
thermal melting of peptide nucleic acid complexes based on absorption
hypochromicity of bases measured at 260 nm and the isothermal
titration calorimetry (36
, 51
, 52)
. The thermal melting
profile or so-called equilibrium melting curve reflects a structural
transition from an ordered to a disordered state or vice
versa, and thermal melting temperature Tm,
H°, and S° are calculated from a van’t Hoff analysis of the
curve. In the simplest case, a bimolecular reaction (single strand
[PNA] + single strand [PNA/DNA/RNA] 
duplex) representing a
two-state model is assumed for analysis of the melting curves (for a
detailed discussion see refs 36
, 51
, 53
54
55
). The model
assumes that each single strand exists in one of two possible states:
completely based paired and stacked or completely denatured and
unstacked; partial base pairing is not considered. This model holds
well for short duplexes (5–20 base pairs). It is important to note
that single-stranded peptide nucleic acids, particularly at low
temperature, can also exhibit an ordered structure represented by a
self-melting profile of the oligomer (36
, 51)
.
In contrast to the thermal melting method based on the van’t Hoff
principle, calorimetric methods measure the heat of reaction and change
heat capacity, Cp, during a reaction; the
thermodynamic parameters are determined in a model-independent manner
(51
, 52)
. Isothermal titration calorimetry (ITC) measures
the released or absorbed heat of reaction, at a particular temperature,
as a ligand is titrated into a solution containing the reactant (for
details, see ref 52
). Another method, known as
differential scanning calorimetry, can also be used to complement the
ITC technique. It measures the change in heat capacity directly and the
reaction enthalpy, and entropy can be evaluated independent of any
model for binding. However, the requirement of a high sample
concentration (~100 µM) restricts the use of certain PNAs with low
solubility.
The overall free energy (G) can be divided into enthalpic and
entropic contributions. As discussed earlier, both PNA–PNA and
PNA–DNA complexes are thermally more stable than the corresponding
DNA–DNA duplexes. The additional stability of hybrid duplex over a
pure DNA duplex can be attributed to a more favorable entropic
contribution, whereas the higher stability of a PNA–PNA duplex over
its DNA–DNA counterpart is mainly due to the enthalpic contribution
(36
, 51)
. It is important to mention that for PNA–DNA and
DNA–DNA duplexes, enthalpic changes H are rather equal. This is
also reflected from the hypochromicity values obtained for both
complexes. It has been suggested that the entropic contribution is
possibly related to the ion release associated with the formation of a
hybrid duplex (36
, 51
, 52)
. On the other hand, a higher
H value for the PNA–PNA duplex compared to its DNA–DNA counterpart
signifies a larger structural rearrangements during the phase
transition (from native duplex to coiled state) of the duplex
(36
, 51
, 53
, 54
, 55)
. A higher hypochromicity value was
observed for PNA–PNA duplexes compared to their DNA–DNA counterparts,
which is consistent with the above interpretation. At low ionic
strength conditions, because PNA backbone is devoid of electrostatic
charges, a favorable enthalpy of hybridization is generally observed in
this order: PNA-PNA>PNA-DNA>DNA-DNA (36
, 51)
. It has
been suggested that hydrophobic interactions play an important role for
the stabilization of PNA-containing duplexes. The charge-neutral
backbone of PNA interacts with the surrounding water molecules, the
so-called spine of hydration. The water is probably organized
differently in PNA-containing duplexes. The interaction with
surrounding water molecules is not as favorable as with a DNA–DNA
complex containing charged sugar phosphate backbones, which could
possibly explain the relative importance of enthalpic contributions to
the free energy of hybridization for the PNA-containing duplexes over
DNA–DNA duplexes. It has been observed that the presence of terminal
lysine residues contributes to the solubility of a PNA oligomer and
stabilizes PNA–DNA duplexes. The H and S values for such
lys-PNA–DNA duplexes are less negative than the values determined for
corresponding hybrid duplexes containing PNAs without terminal lysine
residues. The thermal stability of nucleic acids is strongly dependent
on the sequence and so is the thermodynamic information derived from
the thermal melting data. The thermal stability range of PNA-containing
duplexes has been found to be larger than that for pure DNA duplexes
(36
, 51
, 52
, 56)
.
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(PNA)2–DNA triplexes
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Homopyrimidine PNAs bind to complementary DNA sequences to form
(PNA)2–DNA triplexes (Tm
> 70°C). The stability of the complexes depends on the length of the
oligomers in a regular manner with an increase in
Tm of ~10°C per base pair. A
PNA-(DNA)2 triplex is observed from an
interaction between a C-rich PNA and GC-rich DNA duplex. Since a
protonated cytosine is involved in the base triplet formation, this
particular triple helical structure involving
CGC+ triplets shows a pH dependence. The
structure is most stable in the pH range of 5.0–5.5. This problem
could be circumvented by incorporating pseudo-isocytosines (‘J
bases’) in place of normal cytosine, e.g., in bis-PNAs
(28)
(Fig. 4
), which can form a C.G*J base triplet instead of C.G*C+, which is
essentially independent of pH.
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Figure 4. Hoogsteen-bonded base triplets (involving
pyrimidine*purine·pyrimidine). A) Different base
triplets involving Watson-Crick and Hoogsteen hydrogen bonding.
B) Schematic representation of the formation of a strand
invasion complex involving bis-PNA. Bis-PNA can bind to specific target
sequence to form an internal, triple-stranded invaded complex and a
looped-out single strand.
|
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From measurements of linear dichroism, defined as the anisotropic
absorption of linearly polarized light (57)
, it was early
demonstrated that PNA-T8 can form a right-handed
PNA2/DNA triple helix with poly(dA). The base
triplets are arranged in such a fashion that their planes are
approximately perpendicular to the helix axis, similar to a standard
poly(dA)-(poly(dT))2 triplex (58)
.
This PNA2/DNA triplex structure is remarkably
rigid, which indicates an efficient stacking interaction; segments of
PNA2–DNA triplexes, formed at subsaturated
stoichiometric strand ratios, are distributed evenly on the poly(dA)
strand, separated by flexible single-stranded regions. The formation of
(PNA)2–RNA triple helical structure has been
investigated by biological experiments. The Tm
values are more or less the same as those of their
(PNA)2–DNA counterparts (59)
. It
was observed that the translation of CAT-mRNA can be successfully
inhibited by the action of a triple helix-forming PNA.
Homopyrimidine PNAs can bind to complementary homopurine target sites
in double-stranded DNA by the mechanism of strand invasion (Fig. 2B
). The homopyrimidine PNA recognizes its complementary
site in the dsDNA and displaces the pyrimidine strand of dsDNA, forming
a stable internal PNA–DNA–PNA triplex involving the homopurine strand
and a looped-out, single-stranded structure containing pyrimidine
bases. The strand invasion is favored by the presence of internal
A-rich regions in a duplex DNA and by moderately low ionic strength (<
50 mM NaCl). Gel retardation assay using DNA restriction fragments
containing single PNA targets (1
, 38)
showed that strand
invasion occurs in a highly sequence-specific manner. The incorporation
of a single mismatch in a 10-mer PNA at 20°C reduces the binding
constant by more than a factor of 100. The presence of one further
mismatch could make the strand invasion virtually impossible.
The formation of a right-handed PNA2–DNA triplex
structure was first concluded from circular and linear dichroism
spectroscopic measurements (58)
. However, the crystal
structure of the PNA2–DNA triplex reveals
considerable deviations from its nucleic acid counterpart
(31)
. The purine DNA strand forms a Watson-Crick base pair
with the homopyrimidine PNA strand in an antiparallel orientation and
deploys a second set of base-pairing hydrogen bonds, obeying a
Hoogsteen base pairing scheme, to the second PNA strand of identical
sequence running parallel with the DNA. There is also another H-bond
set between the backbones of those two strands. The Watson-Crick paired
PNA–DNA strands in the triplex structure resemble the PNA–DNA duplex.
The PNA2–DNA triplex is wide (diameter ~26 Å)
and the pitch is ~16 (triplet) bases. The sugar puckering in the DNA
strand is C3'-endo and the backbone torsional angles are similar to
those of an A-form structure. The structure is further stabilized
through a network of water molecules specifically bound in the minor
groove.
Recently, another kind of invasion complex has been reported by Nielsen
(60)
that requires the presence of nonstandard nucleobases
in the PNAs. The complex formed after the binding of PNA molecules is
known as double invasion complex, since two PNA strands can
simultaneously bind (invade) the two strands (Fig. 2C
).
With the availability of NMR and X-ray crystallography data (20
, 31
, 47
48
49
50)
, a few computational studies have been made for
further characterization of peptide nucleic acid structures (61
, 62)
. The molecular dynamics simulation study of a PNA·DNA-PNA
triplex helix in aqueous solution by Shields et al. (62)
has been useful by concluding from energetic analysis that the hybrid
PNA-DNA-PNA triplex has a characteristic helicity that is quite
different from its actual DNA counterpart. The reason is not simply the
replacement of sugar phosphate by PNA backbones, but some definite
conformational preferences of the PNA strands that have effects on the
conformational flexibility. The structure of a PNA·DNA-PNA triple
helix is based on a total of 12 acyclic torsion angles, which determine
the conformation of adjacent bases in PNA vs. 9 variables, including
the sugar ring, in DNA (63)
. Moreover, a torsional
freedom, absent in the nucleic acid, is also introduced into the
structure involving PNA due to the replacement of the sugar ring by a
linear bond sequence. The 12 torsional angles are divided into two
groups. The first group involves the independent variables, including
torsional angles analogous to the glycosyl and ring torsions of the
nucleic acids. The second or the dependent set arises in the successful
closure of the PNA backbone (63)
. The results of these
computational (modeling) studies based on predicted structures and
cross-verified from the current spectroscopic, calorimetric, and
crystallographic data may explain why PNA never forms very B-like
structures.
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Antigene and antisense applications of PNA
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Peptide nucleic acids have promise as candidates for gene
therapeutic drugs design. They require well-identified targets and a
well-characterized mechanism for their cellular delivery. In principle,
two general strategies can be adapted to design gene therapeutic drugs
(Fig. 5
). Oligonucleotides or their potential analogs are designed to recognize
and hybridize to complementary sequences in a particular gene whereby
they should interfere with the transcription of that particular gene
(antigene strategy). Alternatively, nucleic acid analogs can be
designed to recognize and hybridize to complementary sequences in mRNA
and thereby inhibit its translation (antisense strategy). PNAs are
chemically and biologically stable molecules and have significant
effects on replication, transcription, and translation processes, as
revealed from in vitro experiments. Moreover, no sign of any
general toxicity of PNA has so far been observed. As we shall see, PNA
can interfere with the translation process, and PNA-dsDNA strand
displacement complexes can inhibit protein binding and block RNA
polymerase elongation.
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Figure 5. Antigene and antisense strategy. An antigene oligomer (e.g., PNA)
could bind to a complementary sequence in the DNA and inhibit
transcription of the gene. On the other hand, cells can also be treated
with an antisense oligomer, and hybridization to a specific mRNA
sequence can inhibit the expression of a protein at the level of
translation.
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|
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Inhibition of transcription
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Peptide nucleic acids should be capable of arresting
transcriptional processes by virtue of their ability to form a stable
triplex structure or a strand-invaded or strand displacement complex
with DNA. Such complexes can create a structural hindrance to block the
stable function of RNA polymerase and thus are capable of working as
antigene agents (Fig. 4)
. Evidence from in vitro studies
supports the idea that such complexes are indeed capable of affecting
the process of transcription involving both prokaryotic and eukaryotic
RNA polymerases. PNA targeted against the promoter region can form a
stable PNA–DNA complex that restricts the DNA access of the
corresponding polymerase. PNA strand displacement complexes, located
far downstream from the promoter, can also efficiently block polymerase
progression and transcription elongation and thereby produce truncated
RNA transcripts; the PNA (DNA poly-purine) target must be present in
the gene of interest. Nielsen et al. (64)
have
demonstrated that even an 8-mer PNA (T8) is
capable of blocking phage T3 polymerase activity
(64)
. The presence of a PNA target within the promoter
region of IL-2R gene has been used to understand the effect of PNA
binding to its target on this gene expression (10
, 65)
.
The PNA2/DNA triplex arrests transcription
in vitro and is capable of acting as an antigene agent. But
one of the major obstacles to applying PNA as an antigene agent is that
the strand invasion or the formation of strand displacement complex is
rather slow at physiological salt concentrations (36
, 66)
.
Several modifications of PNA have shown improvement in terms of
binding. Modifications of PNA by chemically linking the ends of the
Watson-Crick and Hoogsteen PNA strands to each other (28)
,
introducing pH-independent pseudoisocytosines into the Hoogsteen strand
(28)
, incorporating intercalators (67)
, or
positively charged lysine residues (28
, 68)
in PNA strand
can drastically increase the association rates with dsDNA. Lee et al.
(69)
have demonstrated that PNA as well as the PNA–DNA
chimera complementary to the primary site of the HIV-I genome can
completely block priming by tRNA3Lys.
Consequently, in vitro initiation of the reverse
transcription by HIV-1 RT is blocked. Thus, oligomeric PNAs targeted to
various critical regions of the viral genome are likely to have a
strong therapeutic potential for interrupting multiple steps involved
in the replication of HIV-1 (69)
.
It has been found that under physiological salt conditions, binding of
PNA to supercoiled plasmid DNA is faster compared to linear DNA
(66
, 68)
. This result is relevant to the fact that the
transcriptionally active chromosomal DNA usually is negatively
supercoiled, which can act as a better target for PNA binding in
vivo. It has also been found that the binding of PNA to dsDNA is
enhanced when the DNA is being transcribed. This transcription-mediated
PNA binding occurs about threefold as efficiently when the PNA target
is situated on the nontemplate strand instead of the template strand.
As transcription mediates template strand-associated
(PNA)2/DNA complexes, which can arrest further
elongation, the action of RNA polymerase results in repression of its
own activity, i.e., suicide transcription (70)
. These
findings are highly relevant for the possible future use of PNA as an
antigene agent.
We wish to refer to reports describing the ability of PNA to activate
transcription, although this is not actually related to its antigene
effect. Mollegaard et al. (71)
have efficiently
demonstrated that the looped-out single-stranded structure formed as a
result of strand invasion is also capable of acting as efficient
initiation sites for Escherichia coli and mammalian RNA
polymerases in which the polymerase might start transcription using the
single-stranded loop as a template (65)
. This is
consistent with the affinity of RNA polymerase for single-stranded DNA
and its ability to transcribe single-stranded DNA.
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Inhibition of translation
|
|---|
The basic mechanism of the antisense effects by
oligodeoxynucleotides is considered to be either a ribonuclease H
(RNase H) -mediated cleavage of the RNA strand in oligonucleotide-RNA
heteroduplex or a steric blockage in the oligonucleotide–RNA complex
of the translation machinery (59)
.
Oligodeoxynucleotide analogs such as phosphorothioates activate RNase H
and thus hold promise of working as antisense agents. However, they
also exhibit some nonspecificity in their action. PNA/RNA duplexes, on
the other hand, cannot act as substrates for RNase H. Normally, the
peptide nucleic acid antisense effect is based on the steric blocking
of either RNA processing, transport into cytoplasm, or translation. It
has been concluded from the results of in vitro translation
experiments involving rabbit reticulocyte lysates that both duplex-
(mixed sequence) and triplex-forming (pyrimidine-rich) PNAs are capable
of inhibiting translation at targets overlapping the AUG start codon
(59)
. Triplex-forming PNAs are able to hinder the
translation machinery at targets in the coding region of mRNA. However,
translation elongation arrest requires a
(PNA)2–RNA triplex and thus needs a homopurine
target of 10–15 bases. In contrast, duplex-forming PNAs are incapable
of this. Triplex-forming PNAs can inhibit translation at initiation
codon targets and ribosome elongation at codon region targets.
Mologni et al. (72)
showed effects of three different
types of antisense on the in vitro expression of PML/RAR
gene. The first one was complementary to the first AUG (initiation)
site. The second could bind to a sequence in the coding region that
includes the second AUG, the starting site for the synthesis of an
active protein. The third PNA was targeted against the 5'-untranslated
region (UTR) of the mRNA, the point of assembly of the translation
machinery. Together, these three PNAs could efficiently inhibit
translation even at a concentration much below the critical
concentration used for each individual. The result suggests that the
PNA targeting of RNA molecules like PML/RAR requires effective
blocking of different sequences on the 5' part of the messenger. A
5'-UTR PNA target can also be used as efficiently as an initiation
(AUG) target to achieve an antisense activity of PNA, and a more
effective translation inhibition can be achieved by combining PNA
directed toward 5'-UTR and AUG regions.
Triple helix-forming PNAs can also hinder the translation process.
Bis-PNA or clamp-PNA structures are capable of forming internal triple
helical constructs. In principle, if targeted against the coding region
of mRNA, PNA2/RNA triple helix-forming
derivatives can also cause a stop in translation, which can be easily
verified by the detection of a truncated protein. (59)
.
However, this methodology requires a sequence optimization for each new
target.
Recent studies show that E. coli cells are somewhat
permeable for PNA molecules. Good and Nielsen (73
, 74)
have shown that it is possible to achieve PNA antisense effects in the
‘leaky’ mutant strains of E. coli. PNAs targeted against
the AUG region of the mRNA corresponding to ß-galactosidase and
ß-lactamase genes were indeed capable of down-regulating the
expression of these two genes (73)
. Another study
(74)
demonstrated the effect of two bis-PNAs, targeted
against the homopurine stretches in rRNA, either in the peptidyl
transferase center or in the -sarcin loop, in inhibiting the
ribosome function in a cell-free system. The translation was arrested
at submicromolar range of PNA concentration. The growth of a mutant
strain of E. coli, namely, AS19, was also inhibited by using
the same PNAs at low micromolar concentration.
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Inhibition of replication
|
|---|
It is also possible by using PNA to inhibit the elongation of DNA
primers by DNA polymerase. Further, the inhibition of DNA replication
should be possible if the DNA duplex is subjected to strand invasion by
PNA under physiological conditions or if the DNA is single stranded
during the replication process. Efficient inhibition of
extrachromosomal mitochondrial DNA, which is largely single-stranded
during replication, has been demonstrated by Taylor et al.
(75)
. The PNA-mediated inhibition of the replication of
mutant human mitochondrial DNA is a novel (and also potential) approach
toward the treatment of patients suffering from ailments related to the
heteroplasmy of mitochondrial DNA. Here wild-type and mutated
DNA are both present in the same cell. Experiments have shown that PNA
is capable of inhibiting the replication of mutated DNA under
physiological conditions without affecting the wild-type DNA in
mitochondria.
TOP
ABSTRACT
INTRODUCTION
