Targeting DNA G-quadruplex structures with peptide nucleic acids.
ABSTRACT Regulation of genetic functions based on targeting DNA or RNA sequences with complementary oligonucleotides is especially attractive in the post-genome era. Oligonucleotides can be rationally designed to bind their targets based on simple nucleic acid base pairing rules. However, the use of natural DNA and RNA oligonucleotides as targeting probes can cause numerous off-target effects. In addition, natural nucleic acids are prone to degradation in vivo by various nucleases. To address these problems, nucleic acid mimics such as peptide nucleic acids (PNA) have been developed. They are more stable, show less off-target effects, and, in general, have better binding affinity to their targets. However, their high affinity to DNA can reduce their sequence-specificity. The formation of alternative DNA secondary structures, such as the G-quadruplex, provides an extra level of specificity as targets for PNA oligomers. PNA probes can target the loops of G-quadruplex, invade the core by forming PNA-DNA guanine-tetrads, or bind to the open bases on the complementary cytosine-rich strand. Not only could the development of such G-quadruplex-specific probes allow regulation of gene expression, but it will also provide a means to clarify the biological roles G-quadruplex structures may possess.
[show abstract] [hide abstract]
ABSTRACT: We report here the results of a systematic search for the existence and prevalence of potential intramolecular G-quadruplex forming sequences in the human genome. We have also examined the tendency for particular sequences of 'loop' regions to occur in particular positions with respect to the G-tracts in a quadruplex. Using arithmetic ratio and probability techniques we have discovered frequent and systematic occurrence of certain sequence types, the most prominent being a potential quadruplex containing CCTGT in the first 'loop' position. Being able to highlight types of potential quadruplex sequences in G-rich regions is an important step in searching for biologically relevant sequences and finding their function.Nucleic Acids Research 02/2005; 33(9):2901-7. · 8.03 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Guanine-rich DNA sequences of a particular form have the ability to fold into four-stranded structures called G-quadruplexes. In this paper, we present a working rule to predict which primary sequences can form this structure, and describe a search algorithm to identify such sequences in genomic DNA. We count the number of quadruplexes found in the human genome and compare that with the figure predicted by modelling DNA as a Bernoulli stream or as a Markov chain, using windows of various sizes. We demonstrate that the distribution of loop lengths is significantly different from what would be expected in a random case, providing an indication of the number of potentially relevant quadruplex-forming sequences. In particular, we show that there is a significant repression of quadruplexes in the coding strand of exonic regions, which suggests that quadruplex-forming patterns are disfavoured in sequences that will form RNA.Nucleic Acids Research 02/2005; 33(9):2908-16. · 8.03 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: G-quartets are square planar arrangements of four guanine bases, which can form extraordinarily stable stacks when present in nucleic acid sequences. Such G-quadruplex structures were long regarded as an in vitro phenomenon, but the widespread presence of suitable sequences in genomes and the identification of proteins that stabilize, modify or resolve these nucleic acid structures have provided circumstantial evidence for their physiological relevance. The therapeutic potential of small molecules that can stabilize or disrupt G-quadruplex structures has invigorated the field in recent years. Here we review some of the key observations that support biological functions for G-quadruplex DNA as well as the techniques and tools that have enabled researchers to probe these structures and their interactions with proteins and small molecules.Molecular Biotechnology 03/2011; 49(2):198-208. · 2.17 Impact Factor
1984 Current Pharmaceutical Design, 2012, 18, 1984-1991
© 2012 Bentham Science Publishers
Targeting DNA G-Quadruplex Structures with Peptide Nucleic Acids
Igor G. Panyutin1,*, Mykola I. Onyshchenko1,3, Ethan A. Englund2, Daniel H. Appella2 and Ronald D.
1Department of Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA;
2Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA; 3Imaging Sciences Training
Program, Clinical Center and National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda,
MD 20892, USA
Abstract: Regulation of genetic functions based on targeting DNA or RNA sequences with complementary oligonucleotides is especially
attractive in the post-genome era. Oligonucleotides can be rationally designed to bind their targets based on simple nucleic acid base pair-
ing rules. However, the use of natural DNA and RNA oligonucleotides as targeting probes can cause numerous off-target effects. In addi-
tion, natural nucleic acids are prone to degradation in vivo by various nucleases. To address these problems, nucleic acid mimics such as
peptide nucleic acids (PNA) have been developed. They are more stable, show less off-target effects, and, in general, have better binding
affinity to their targets. However, their high affinity to DNA can reduce their sequence-specificity. The formation of alternative DNA
secondary structures, such as the G-quadruplex, provides an extra level of specificity as targets for PNA oligomers. PNA probes can tar-
get the loops of G-quadruplex, invade the core by forming PNA-DNA guanine-tetrads, or bind to the open bases on the complementary
cytosine-rich strand. Not only could the development of such G-quadruplex-specific probes allow regulation of gene expression, but it
will also provide a means to clarify the biological roles G-quadruplex structures may possess.
Keywords: Peptide Nucleic Acids, G-quadruplex, gene expression regulation.
tially form G-quadruplex structures [1, 2]. Usually, such sequences
consist of four runs of three or more guanines separated by short
linker sequences. The runs of guanines form the core of the quadru-
plex stabilized by guanine tetrads while the linker sequences form
loops of various conformations . Human telomeric repeats con-
tain the greatest number of such sequences . In addition, numer-
ous genes possess sequences capable of forming G-quadruplex
structures in their regulatory regions [5, 6]. Proto-oncogenes are
highly enriched with such G-quadruplex-forming sequences com-
pared to tumor suppressor genes . Furthermore, there is mount-
ing evidence that G-quadruplex structures may play roles not only
in telomere maintenance but in regulation of gene expression as
well . Numerous small molecules with higher affinity to G-
quadruplex versus duplex DNA have been synthesized [9, 10];
some of these are potent inhibitors of the telomere-extending en-
zyme, telomerase, which is overexpressed in cancer cells, and are
being extensively tested as anticancer drugs . Targeting G-
quadruplex structures in the intrachromosomal regulatory regions is
more challenging because of the wide variety of G-quadruplex
structures that can form in different genes based on their unique
sequences . Even though molecules have been designed to spe-
cifically bind to the G-quadruplex structures formed in particular
genes, the task remains very challenging.
The human genome is enriched with sequences that can poten-
structures is based on using short oligonucleotides that bind to the
complementary DNA bases that become accessible after G-
quadruplex formation. This approach is similar to antisense ,
antigene , and RNAi-based  approaches, and is based on
simple nucleic acids complementarity rules. While developing an-
tisense, antigene and RNAi-based therapies, it became clear that for
this approach to be effective, chemically modified analogs or nu-
cleic acid mimics should be used. One such class of molecules,
An alternative approach to targeting specific G-quadruplex
*Address correspondence to this author at the NIH/CC/RAD&IS, Bldg. 10,
Rm. 1C401, Bethesda, MD 20892-1180 USA; Tel: (301) 496-8308;
Fax: (301) 480-9712; Email: email@example.com
peptide nucleic acids (PNAs), are nucleic acid mimics in which the
natural nucleobases are connected to an achiral, uncharged polyam-
ide backbone  Fig. (1). PNA oligomers form very stable du-
plexes with complementary nucleic acids; they can even invade
double-stranded DNA under certain conditions. For example, when
the DNA is negatively supercoiled, PNA and DNA will complex
and form a P-loop [15, 16] Fig. (2). In addition, guanine-rich PNA
are able to form G-quadruplexes by themselves or DNA-PNA hy-
brid G-quadruplex structures .
Fig. (1). PNA (blue) and DNA (red) backbones. Shown PNA-DNA hybrid.
G-Quadruplex Targeting with Peptide Nucleic Acids Current Pharmaceutical Design, 2012, Vol. 18, No. 14 1985
Fig. (2). Various modes of PNA (blue) binding to duplex DNA (red).
G-quadruplexes with PNA have been proposed. First, guanine-rich
PNA can invade a DNA G-quadruplex and simultaneously bind to
the complementary cytosine-rich strand, thus facilitating G-
quadruplex formation [18, 19]. Second, short PNA oliomgers can
be designed to bind to the single-stranded DNA (ssDNA) in the
exposed loops of G-quadruplex structures . Third, PNA can
bind exclusively to the complementary cytosine-strand, thus facili-
tating G-quadruplex extrusion in the G-rich strand without interfer-
ing with native DNA G-quadruplex conformation  Fig. (3).
Based on these unique properties, several approaches to target
also will provide background on PNA chemistry and design, and
speculate on possible mechanisms of PNA-based strategies to regu-
late gene expression by targeting a G-quadruplex.
In this short review, we will describe all these approaches. We
PNA AS DNA SEQUENCE-SPECIFIC TARGETING DRUGS
system that takes advantage of nucleobase hydrogen bond recogni-
tion would be of general use for any gene target Fig. (4). Nucleic
acid derivatives are attractive drug candidates over natural nucleic
acids. PNAs are non-natural nucleic acids in which the natural nu-
cleobases are preserved but appended to an uncharged, achiral
pseudo-peptide backbone in lieu of the natural sugar phosphate
There are many approaches to make gene-specific drugs, but a
backbone . Because the PNA oligomers are uncharged, they
tend to exhibit tighter binding to natural nucleic acids due to the
lack of poly anionic charge-charge interactions . Furthermore,
PNA oligomers are resistant to degradation in vivo because they are
not recognized by nucleases or proteases, and their stability makes
them attractive candidates for antigene, antisense, or nucleic acid
its inherent shortcomings, such as solubility, cell permeability, or
bioavailability Fig. (5). The use of modified PNA residues in PNA
oligomers can also affect the binding affinity and selectivity to
nucleic acids through backbone rigidification and preorganization
[25-27], increase solubility , provide a handle for further conju-
gation or ligand display  or increase cellular uptake . Re-
cent work with diethylene glycol ?-substituted PNA residues af-
forded oligomers that had both higher binding affinity to DNA and
increased aqueous solubility . However, the thermodynamic
data indicated that preorganization might not be the reason for the
increased binding affinity in this example.
Chemical modification of PNA oligomers can abrogate some of
manner has already seen remarkable progress [32-36]. When cell-
penetrating peptides are conjugated to an antigene PNA oligomer,
inhibition of gene expression has been demonstrated [37-39]. Using
pseudovirion delivery agents as another means of delivering PNA
oligomers into cells has similarly shown promise in suppressing
gene expression associated with drug resistant cancer cells .
Besides targeted gene inhibition, another application of PNA oli-
gomers involves promoting the repair and recovery of gene function
[41, 42]. All of these approaches utilize the nucleic acid sequence
specificity rather than recognition of nucleic acid secondary struc-
PNA oligomers targeting genomic DNA in a sequence specific
G-QUADRUPLEX AND OTHER ALTERNATIVE DNA
yond the well-known B-form double helix . Examples include
A-form DNA duplex , the left handed Z-DNA , triplexes
formed from binding the Hoogsteen face of purine bases in the
major groove Fig. (4) , cytosine-rich i-motif  and
DNA forms a remarkable variety of secondary structures be-
Fig. (3). Proposed modes of PNA binding to DNA G-quadruplex formed in duplex DNA. Quadruplex binding PNA replaces one or more DNA strands con-
taining quadruplex-core-forming runs of guanines.
Current Pharmaceutical Design, 2012, Vol. 18, No. 14 Panyutin et al.
Fig. (4). (i). The hydrogen bonding formation of triplex form nucleic acids.
The guanine:cytosine form a pair on the Watson-Crick face of the guanine
forming a duplex as third oligomer containing a protonated cytosine binds
the Hoogsteen face of guanine. The third strand would be stationed in the
major groove of normal nucleic acid duplex. (ii). The same type base-
pairing with complementary thymine residues across both Hoogsteen and
Watson-Crick faces of the adenine.
G-quadruplexes [49, 50]. Many of these types of nucleic acid struc-
tures depend on non-Watson-Crick hydrogen bonding such as wob-
ble base pairing or sheared-type base pairing . However, the
question whether many of these alternative DNA structures play a
biological role remains open to this day. Some alternative structures
were found under the conditions that are normally not present in
cells, e.g., low pH, extremely supercoiled DNA, crystallized DNA,
etc. . Some of these secondary structures could not form readily
under physiological conditions. Nevertheless, all of these structures
tend to be sequence specific and some are observed in situ and may
play a direct role in biological processes. For example, DNA tri-
plexes utilizing Hoogsteen hydrogen bond recognition have been
linked to several disease states . This type of secondary struc-
ture only forms in stretches of polypurine/polypyrimidine tracks of
DNA where another pyrimidine rich strand can bind the Hoogsteen
face of the polypurine strand Fig. (3). The tips of vertebrate chro-
mosomes, telomeres, contain multiple GGGTTA repeats of single
stranded DNA which can form G-quadruplexes and play an integral
role in preventing chromosomal deterioration or unwanted chromo-
somal fusion . The sequences potentially forming G-quadruplex
DNA structures are highly abundant along the human genome,
especially in regulatory regions . This observation might indi-
rectly indicate the possible epigenetic role of these DNA structures.
structures, either with itself or in combination with natural nucleic
acids. Nielsen and coworkers originally designed PNA to bind to
the major groove (i.e. the Hoogsteen hydrogen bonding mode) of
DNA duplexes . It was only afterwards they discovered that
PNA oligomers invade DNA duplexes and form stable (PNA)2:
DNA triplexes. Today, PNA:DNA triplexes are well-known, and
PNA oligomers also have the potential to form many secondary
Bis-PNAs (PNA oligomers designed to bind polypurine DNA in
both Watson-Crick and Hoogsteen hydrogen bonding modes con-
nected through a flexible linker) invade even long tracks of duplex
DNA to form very stable triplex structures [57-59] Fig. (2). PNA
oligomers also form stable quadruplexes in the presence of appro-
priate cations (sodium, potassium, ammonium, etc.) . Guanine-
rich PNA oliogmers form stable heterocomplexes with guanine-rich
DNA oligomers . The stability and selectivity of PNA quadru-
plexes can be modified by using PNA derivatives. The use of ?-
substituted PNA can bias PNA oligomers towards binding in the
guanine-quadruplex mode versus duplex binding . Furthermore,
the selective arrangement of trans-cyclopentane PNA residues can
increase the stability of PNA:DNA heterocomplexes while discour-
aging the competing PNA quadruplex formation . The range
and diversity of secondary structures that PNA forms or recognizes
shows that trying to increase selectivity through targeting non-
duplex complexes could be a worthwhile strategy.
MODES OF TARGETING DNA QUADRUPLEXES WITH
strands of a G-quadruplex. Initially, PNA probes containing gua-
nines were designed to invade homologous DNA G-quadruplex-
forming sequences and participate in the formation of heterogene-
ous DNA-PNA guanine tetrads. This idea was first put forward by
Armitage and co-workers who noticed that guanine-rich PNA
formed hetero-quadruplexes with homologous DNA oligomers [17,
61]. This approach is based on targeting the guanine-rich quadru-
plex-forming DNA strand [17-19, 61, 63-66]. Instead of G-
quadruplex invasion, guanine rich PNA probes can alternatively
bind to the complementary cytosine-rich DNA strand, thus provid-
ing additional stabilization of the G-quadruplex-PNA complex.
DNA sequences with four guanine runs can also be targeted in this
mode via formation of two consecutive quadruplexes each consist-
ing of two PNA- and two DNA-strands  (Fig. (3), box, bottom).
To increase the efficiency and specificity of quadruplex invasion,
Lusvarghi et al. utilized modified PNA probes. They incorporated
abasic sites as well as chiral modifications to the backbone of PNA
and showed an improvement in the selectivity of quadruplex versus
duplex formation . Paul and coauthors  proposed another
model of G-quadruplex targeting with PNAs. They designed a gua-
nine-rich PNA probe that combines with three guanine runs of a
human telomere sequence to form an intermolecular PNA-DNA G-
quadruplex in a “3+1” mode. The resulting complex mimics the
biologically relevant pure DNA telomeric quadruplex. However,
the PNA probes described above could not invade duplex DNA
even if the latter contained the motif complementary to the PNA
sequence. Therefore, further efforts are required to increase the
ability of PNA of invade duplex DNA to make these types of
PNAs were designed to target G-rich, C-rich, and/or both DNA
DNA nucleobases in the loops of G-quadruplex structure and ex-
clude disruption of guanine tetrads within the quadruplex. They
screened a small library of short PNAs complementary to a part of
quadruplex-forming DNA sequence that does not contain guanines
involved in G-quadruplex formation. Depending on PNA length
and ionic conditions, PNAs are able to bind to the loops of the G-
quadruplex and either stabilize or disrupt the quadruplex structure.
In their earlier studies, Amato et al. used short cytosine-rich PNA
probes to show that they could form novel G-quadruplex-PNA
complexes in addition to the expected DNA-PNA heteroduplexes,
depending on ionic conditions [65, 66].
An alternative approach was used by Amato et al.  to target
ing only the cytosine-rich strand complementary to the quadruplex-
forming DNA strand [21, 67]. In this case, a PNA probe invades a
double helix and binds the cytosine-rich strand while allowing the
guanine-rich strand to form native G-quadruplex. We studied this
Our team proposed a different approach that is based on target-
G-Quadruplex Targeting with Peptide Nucleic Acids Current Pharmaceutical Design, 2012, Vol. 18, No. 14 1987
mode of targeting using a double-stranded DNA plasmid model
containing a quadruplex-forming sequence from the human BCL2
gene promoter. Our studies on PNA invasion of duplex DNA en-
abled us to examine the role of quadruplex formation in PNA inva-
sion. In addition to the plasmid with the original BCL2 sequence,
we also used a plasmid with a mutant sequence incapable of quad-
ruplex formation but which still retained the PNA binding se-
quences. Chemical probing revealed that PNA oligomers were able
to invade and form a heteroduplex with the cytosine-rich strand
only in plasmid DNA with the original BCL2 sequence. We also
tested the effect of total PNA charge on the efficiency of the inva-
sion. PNAs that are positively charged or zwitterionic (which con-
tain both positive and negative charges but are charge neutral over-
all), invade the naturally supercoiled plasmid, but negatively
charged PNA does not invade. Zwitterionic PNA also showed the
highest specificity to the targeted sequence. Moreover, if triplex-
forming bis-PNAs targeting flanking sequences were used in com-
bination with central binding PNA Fig. (3) then invasion appeared
even more favorable. Overall, we demonstrated that the potential to
form a G-quadruplex facilitated PNA invasion. Our results provide
strong evidence that PNA probes can be designed that are not only
sequence-specific, but also quadruplex-dependent.
and binding facilitated by non-duplex secondary structures. Zhang
et al.  demonstrated that formation of cruciform structures in
palindromic regions of DNA facilitated PNA invasion. Amiard et
al.  showed that multiple t-loops (lariat-like structures that form
The aforementioned results are consistent with PNA invasion
when 3’ telemeric overhang DNA invades the double-stranded
telomeric repeat array) increase the ability of single-stranded DNA
to invade plasmid DNA. Furthermore, invasion took place only in
supercoiled DNA, implying that targeting may occur only in ac-
tively transcribed genes. As all these examples demonstrate, target-
ing double-stranded DNA with the potential to form alternative
secondary structures has the potential to affect transcription or rep-
POSSIBLE MECHANISMS TO REGULATE GENE EX-
PRESSION WITH G-QUADRUPLEX-SPECIFIC PNA
and other regulatory segments. Although guanine-rich regions
might directly interact with DNA binding proteins, it is likely that
formation of G-quadruplexes also directs interaction with proteins.
In particular, the high prevalence of guanine-rich sequences in
regulatory regions suggests possible regulatory roles for these quad-
ruplex DNA structures. For example, the number of guanine-rich
sequences with G-quadruplex forming potential in the human ge-
nome is almost 2 orders of magnitude higher than that in a random
DNA sequence of the same length .
Numerous genes possess guanine-rich sequences in promoter
enhancer regions of many genes, and particularly in oncogenes .
Recent studies show that these guanine-rich sequences could play a
role in the regulation of gene transcription. Quadruplex formation
has been studied in PDGF-A [70, 71], VEGF [71-73], c-myc [71,
74], KRAS , C-KIT [76, 77], BCL2 [78, 79] hTERT , Rb
Multiple runs of guanines can be found in the promoter and
Fig. (5). The composition of aminoethyl glycine PNA (aegPNA) and some of the common derivatives based on maintaining the basic form of the PNA back-
bone while augmenting it with ring structures or side chains.
aminoethyl glycyl PNA residue
(S,S) trans-cyclopentyl PNA residue
L-Lysine γ-PNA residue
L-Alanine γ-PNA residue
D-Lysine α-PNA residue
D-Arginine α-PNA residue
Current Pharmaceutical Design, 2012, Vol. 18, No. 14 Panyutin et al.
, PDGFR-? genes . This provides an appealing opportunity
for gene regulation by targeting guanine quadruplexes. While G-
quadruplex formation has been demonstrated in models of single-
stranded DNA oligonucleotides, several studies provide evidence of
quadruplex formation in supercoiled double-stranded DNA 
plex formation was implicated to affect gene expression. In this
respect the G-quadruplex forming sequence located in the promoter
region of human C-MYC gene  was one of the first described
and most extensively studied. Hurley and co-workers proposed the
following model of C-MYC expression regulation. Dynamic stress
(negative supercoiling) resulting from transcription converts the
duplex DNA to a G-quadruplex on the guanine-rich strand and an i-
motif (proposed secondary structure in cytosine-rich sequence) on
the pyrimidine-rich strand. This displaces activating transcription
factors and silence gene expression. Specific proteins, namely
NM23-H2 and nucleolin, that recognize the G-quadruplex are able
to fold or to resolve its structure and hence regulate C-MYC expres-
sion [8, 84]. Authors found that inhibition of NM23-H2 silences C-
MYC and redistribution of nucleolin from the nucleolus to the nu-
cleoplasm negatively affects expression of this gene.
Below, we describe examples of several studies where quadru-
during transcription in vitro was found using electron microscopy
. Authors developed a plasmid with guanine rich inserts and
observed a “G-loop”, i.e. DNA/RNA hybrid on the cytosine-rich
strand and a G-quadruplex on the complimentary strand during
transcription. They concluded that the RNA/DNA hybrid was criti-
cal for G-quadruplex stabilization in the post-transcriptional G-
In another example, quadruplex formation in plasmid DNA
quadruplex-forming sequence element within the promoter of hu-
man thymidine kinase 1 (TK1). Their data suggested that this se-
quence forms an intramolecular G-quadruplex with two G-tetrads.
A cell-based reporter assay revealed the role of this G-quadruplex
motif in TK1 transcription. Nucleotide substitutions designed to
destabilize G-quadruplex structure formation resulted in increased
promoter activity, therefore, pointing on direct involvement of the
G-quadruplex structure in transcription regulation of TK1 .
Another group of authors identified a characteristic potential G-
the maintenance of genome stability in yeast) in stabilizing DNA
sequences that could otherwise form G-quadruplex structures by
acting as a G-quadruplex resolvase. They found that for the human
Pif1 to resolve a G-quadruplex, an extended (>10 nucleotide) 5'
ssDNA tail is required. The authors suggest that human Pif1 could
therefore have a role in processing G4 structures that arise in the
single-stranded nucleic acid intermediates formed during DNA
replication and transcription .
Sanders et al. described the role of Pif1 proteins (implicated in
interaction. They found that the quadruplex-forming GA-element in
the KRAS promoter responds to a Myc-associated zinc finger and
poly(ADP-ribose) polymerase 1 proteins. Through use of an im-
munoprecipitation assay, they discovered that the Myc-associated
zinc finger protein specifically binds to the duplex and quadruplex
conformations of the GA-element, whereas poly(ADP-ribose) po-
lymerase binds only to the G-quadruplex. Introduction of a point
mutation into the quadruplex-forming sequence showed down-
regulation of KRAS, while addition of phthalocyanines (G-
quadruplex stabilizing agents) up-regulated KRAS expression .
Cogoi et al. described another example of G-quadruplex-protein
indicate that formation of G-quadruplex in the regulatory gene se-
quences can play a dual role in gene expression; it can cause up- or
down-regulation of the gene expression . In this respect, PNA
probes can provide a powerful instrument to target guanine-rich
regulatory DNA sequences, stabilize or destabilize G-quadruplexes,
and ascertain the effect of G-quadruplex formation on the expres-
The above examples along with a recent genome-wide study
sion of a particular gene. Based on existing experimental data, sev-
eral modes of gene transcription regulation through quadruplex
targeting with PNA probes are possible. In one mode, PNA probes
are designed to interfere with G-quadruplex recognition by protein
factors. This can be achieved with PNA probes that invade G-
quadruplex [17-19, 61, 63-66], or that are complementary to the
loops of G-quadruplex structure  Fig. (6A). Alternatively, PNA
oligomers complementary to the cytosine-rich strand can be used to
invade DNA duplex, bind the cytosine rich strand forming a
DNA:PNA duplex and allow the guanine-rich strand to form a na-
tive quadruplex structure [21, 67]. In these cases, PNA-DNA inter-
action leads to stabilization of native G-quadruplex structures and
promotes binding of G-quadruplex specific protein factors. Once a
quadruplex is stable, it can become a target for quadruplex-binding
proteins. In turn, these proteins can switch on or off gene transcrip-
tion, depending on that particular protein’s function Fig. (6B).
Fig. (6). Possible effects of PNA binding to a quadruplex-forming sequence
on G-quadruplex recognition by a protein factor and gene transcription. A.
PNA bind G-quadruplex-forming sequence interfering with a protein factor
(green) recognition of the quadruplex; gene regulation by the protein factor
is disrupted. B. PNA bind cytosine-rich strand complementary to G-
quadruplex-forming sequence, thus, helping quadruplex formation. A pro-
tein factor recognizes the quadruplex, binds to it and accomplishes its tran-
scription regulation functions.
strated only in vitro thus far, and in many cases only models con-
taining single-stranded guanine rich DNA are used. The next step
would be to demonstrate the efficiency and specificity of this ap-
proach in cell culture models. To achieve this goal, both the affinity
and specificity of PNA binding to their targets must be improved,
along with methods of delivery of PNA to cells and, to DNA targets
within the cell nuclei. Nevertheless, recent developments in the
PNA field give us optimism that in the near future, an anti-G-
quadruplex PNA will be developed with good biological activity.
Targeting G-quadruplex structures with PNA has been demon-
Training Program supported in part by the Radiology and Imaging
Sciences Department, Clinical Center and Intramural Research
The study was partially sponsored by the Imaging Sciences
G-Quadruplex Targeting with Peptide Nucleic Acids Current Pharmaceutical Design, 2012, Vol. 18, No. 14 1989
Program at the National Institute of Biomedical Imaging and Bio-
engineering, and supported by Intramural Research Programs of
Clinical Center and National Institute of Diabetes and Digestive and
Kidney Diseases, NIH.
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Received: October 19, 2011 Accepted: November 28, 2011