Pandey VN. Mechanism of RNA cleavage catalyzed by
sequence specific polyamide nucleic acid-neamine
conjugate. Oligonucleotides 2007 17(3):302-13.
10. Wilks SA, Keevil CW. Targeting species-specific lowaffinity 16S rRNA binding sites by using peptide nucleic
acids for detection of Legionellae in biofilms. Applied
and Environmental Microbiology 2006 72:5453-62.
11. Marciniak RA, Cavazos D, Montellano R, Chen Q,
Guarente L, Johnson FB. A Novel Telomere Structure in
a Human Alternative Lengthening of Telomeres Cell
Line. Cancer Research 2005 65:2730-7.
12. Forrest GN. PNA FISH: present and future impact on
patient management. Expert Review of Molecular
Diagnostics 2007 7:231-6.
13. Heckl S, Pipkorn R, Waldeck W, Spring H, Jenne J, von
der Lieth CW, Corban-Wilhelm H, Debus J, Braun K.
Intracellular visualization of prostate cancer using
magnetic resonance imaging. Cancer Research 2003
63:4766-72.
14. Chakrabarti A, Zhang K, Aruva MR, Cardi CA, Opitz
AW, Wagner NJ, Thakur ML, Wickstrom E.
Radiohybridization PET imaging of KRAS G12D
mRNA expression in human pancreas cancer xenografts
with [ 64 Cu]DO3A-peptide nucleic acid-peptide
nanoparticles. Cancer Biology and Therapy 2007 6:94856.
15. Nagai Y, Miyazawa H, Huqun , Tanaka T, Udagawa K,
Kato M, Fukuyama S, Yokote A, Kobayashi K,
Kanazawa M, Hagiwara K. Genetic heterogeneity of the
epidermal growth factor receptor in non-small cell lung
cancer cell lines revealed by a rapid and sensitive
detection system, the peptide nucleic acid-locked nucleic
acid PCR clamp. Cancer Research 2005 65:7276-82.
16. Soh J, Toyooka S, Aoe K, Asano H, Ichihara S,
Katayama H, Hiraki A, Kiura K, Aoe M, Sano Y, Sugi
K, Shimizu N, Date H. Usefulness of EGFR mutation
screening in pleural fluid to predict the clinical outcome
of gefitinib treated patients with lung cancer.
International Journal of Cancer 2006 119:2353-8.
17. Miyazawa H, Tanaka T, Nagai Y, Matsuoka M, Huqun,
Sutani A, Udagawa K, Zhang J, Hirama T, Murayama Y,
Koyama N, Ikebuchi K, Nagata M, Kanazawa M,
Nukiwa T, Takenoshita S, Kobayashi K, Hagiwara K.
Peptide nucleic acid-locked nucleic acid polymerase
chain reaction clamp-based detection test for gefitinibrefractory T790M epidermal growth factor receptor
mutation. Cancer Science 2008 99:595-600.
18. Affymetrix GeneChip Globin-Reduction Kit Handbook
https://www.affymetrix.com/support/downloads/manuals
/globin_reduction_protocol_manual.pdf
19. Komiyama M, Aiba Y, Yamamoto Y, Sumaoka J.
Artificial restriction DNA cutter for site-selective
scission of double-stranded DNA with tunable scission
site and specificity. Nature Protocols 2008 3:655-62.
20. Germini A, Rossi S, Zanetti A, Corradini R, Fogher C,
Marchelli R. Development of a peptide nucleic acid
array platform for the detection of genetically modified
organisms in food. Journal of Agricultural and Food
Chemistry 2005 53:3958-62.
21. Panagne Inc. Unpublished results.
22. Brandt O, Hoheisel JD. Peptide nucleic acids on
microarrays and other biosensors. Trends in
Biotechnology 2004 22:617-22.
23. Sun C, Gaylord BS, Hong JW, Liu B, Bazan GC.
Application of cationic conjugated polymers in
microarrays using label-free DNA targets. Nature
Protocol 2007 2:2148-51..
24. Lee H, Jeon JH, Lim JC, Choi H, Yoon Y, Kim SK.
Peptide nucleic acid synthesis by novel amide formation.
Organic Letters 2007 9:3291-3.
25. http://www.advandx.com/about/news/detail.php?news_id=43.
Peptide Nucleic Acid
(PNA) and Its
Applications
Wonyong Koh, Ph.D.
[email protected]
Panagene Inc. www.panagene.com
816 Tamnip-dong, Yuseong-gu, Daejeon 305-510, Korea
Introduction
Peptide nucleic acid (PNA) shows remarkable hybridization
properties and has many exciting applications. PNA is a
DNA mimic, in which the entire negatively-charged sugarphosphate backbone is replaced with a neutral one consisting
of repeated N-(2-aminoethyl) glycine units linked by peptide
bonds. It is stable chemically and biologically. Peptide
bonds can make PNA readily conjugated with peptides,
fluorescent dyes, and other useful molecules. PNA was first
reported in 1991 by Nielsen et al. from the University of
Copenhagen in Denmark [1]. Among hundreds of DNA
mimics, the original PNA is one of the best performers. Over
2,000 papers on PNA were published on in the fields of
chemistry, molecular biology, antisense/antigene therapy,
molecular diagnostics, biosensors, and nanotechnology. This
article will describe recent notable examples of PNA
applications. More comprehensive information on properties
and applications of PNA can be found in a book published in
2004 [2].
such as DNA/RNA microarrays, single nucleotide
polymorphism (SNP) assays, polymerase chain reactions
(PCR), and fluorescent in situ hybridization (FISH).
Fig 1. PNA structure and Watson-Crick base paring with DNA
High affinity and sequence selectivity
Owing to proper intra-molecular spacing, PNA can
recognize specific sequence of DNA and RNA through
Watson-Click base pairing (Figure 1). Due to its uncharged
polyamide backbone, PNA can hybridize to negatively
charged DNA or RNA without electrostatic repulsion. PNA
can also hybridize under low salt concentration. Greater
affinity allows use of short PNA oligomers of 13-18 bases
typically. The stability of the PNA/DNA duplexes is
strongly affected by the presence of imperfect matches. A
mismatch in a PNA/DNA duplex is much more destabilizing
than a mismatch in a DNA/DNA duplex. For 15-mer
PNA/DNA duplex the average Tm of single mismatch was
found 15 C compared to 11 C for DNA/DNA duplex [2].
The high affinity and sequence selectivity of PNA can be
exploited in therapeutic and diagnostic applications based on
hybridization between complementary nucleic acid strands
Antisense and antigene drugs
PNA can bind to complementary sequence of mRNA and
can modulate its function. PNA can also act as antigene
agent because it can break up DNA duplex and form
PNA/DNA triplex or double duplexes without denaturing the
DNA duplex. PNA is typically conjugated with cellpenetrating peptides or lipophilic molecules for
antisense/antigene applications to enhance cellular delivery
because uncharged PNA diffuses very slowly through
cellular membrane. A recent review summarized biological
activity of PNA [3]. A few recent examples of these
applications are listed below:
PNA blocks liver-specific microRNA (miR-122) activity
in human and rat liver cells [4, 5]. Inhibition of miRNA122 in liver cells is achieved without transfection agent
[5].
PNA conjugated with oligophosphonates shows
antisense activity at nanomolar concentration range [6].
It indicates dramatically improved cellular delivery of
PNA and EC50 values down to 1 nM.
Splicing correction of mRNA by PNA was demonstrated
in a mouse model of Duchenne muscular dystrophy [7].
A peptide-conjugated 16-mer PNA suppresses cancer by
blocking transcription of the MYCN oncogene. Its DNA:
PNA duplex shows exceptionally stability (Tm 90 C)
[8].
Neamine-conjugated PNA inhibits HIV-1 replication by
blocking the initiation of reverse transcription and
sequence-specific cleavage of the target RNA [9].
Imaging probes and FISH
PNA is especially good for FISH because it can bind to
DNA or RNA quickly even under low salt or other
unfavorable conditions for DNA [2]. Combined with
remarkable stability against enzymatic degradation,
fluorescent dye-conjugated PNA is used as a specific FISH
probe. PNA may be used to block repetitive sequences in
different FISH protocols. PNA s specificity was utilized to
discriminate 16S rRNA of bacteria species in drinking water
[10]. Secondary structure of 16S rRNA makes it difficult for
DNA FISH probes to detect the sequences. However, PNA
probes perfectly discriminated 47 different strains of
Legionella species. PNA FISH probes have been used to
analyze telomeres of chromosomes, which consist of
hundreds of repeats of a short sequence (5 -TTAGGG-3 in
human and primates). Telomere PNA FISH probes also used
for cancer diagnosis [11].
Notably AdvanDx s PNA FISH products for in vitro
diagnosis of blood infection were approved by Food and
Drug Administration (FDA) of United States in 2006 (Figure
Fig 2. FDA approved PNA FISH products of AdvanDx
Image from www.advandx.com
2). AdvanDx received Frost & Sullivan's 2008 Technology
Innovation of the Year Award in the field of rapid in vitro
molecular diagnostic technology. The products quickly
identify bloodstream pathogens in within 3 hours after blood
cultures turn positive, thereby ensure optimal therapy and
help reduce mortality rates for patients [12].
PNA probes also have been used for in vivo imaging of
mRNA for cancer research [13, 14]. In these applications
PNA is conjugated with a magnetic resonance imaging
(MRI) contrast agent or positron-emitting nuclide for
positron emission tomography (PET) imaging as well as a
cell-penetrating peptide. Specific hybridization with the
target mRNA allowed MRI or PET imaging of gene
expression in live rats or mice.
PCR clamping and artificial restriction enzyme
PNA PCR clamping technique was developed very early
to detect SNP in low abundance. For example, a few mutant
cancerous cells may be among healthy cells in a tissue
biopsy. PNA clamp complementary to wild type sequence
hybridizes specifically with wild type and blocks its PCR
amplification while allowing amplification of mutant
sequence of imperfect match. A single base mismatch is
enough to discriminate amplification of mutant type from
wild type. Epidermal growth factor receptor (EGFR) gene
mutations are associated with resistant to Gefitinib therapy
of lung cancer. A method using PNA clamp and locked
nucleic acid (LNA) primers to detect these mutations was
developed at Saitama Medical University in Japan and has
been practiced at many other sites [15-17].
Affymetrix recommends the use of globin reduction PNA
to enhance sensitivity of expression profile of human whole
blood RNA samples [18]. Specific binding of PNA to globin
mRNA inhibits reverse transcription during cDNA synthesis.
Artificial restriction enzymes have been developed for siteselective double strand DNA cutting [19]. In this system two
strands of pseudo-complementary peptide nucleic acid
invade DNA duplex to form 'hot spots' for scission, and then
Ce(IV)/EDTA complex acts as catalytic molecular scissors.
The scission fragments can be connected with foreign DNA
using DNA ligase.
Microarrays and biosensors
PNA can be used on microarrays and other biosensors. PNA
microarray combined with PCR could detect genetically
modified organisms (GMOs) in food [20]. Every Roundup
Ready soybean and Bt11, Bt176, Mon810, and GA21 maize
was correctly identified in the tested samples. PANArray
microarray for human papillomavirus (HPV) genotyping was
developed and correctly indentified genotypes of 195 HPVpositive samples among 894 clinical samples tested [21].
Certain strains of the HPV virus are known to cause cervical
cancer. 32 probes of PANArray HPV microarray can
identify 32 types of HPV unambiguously. Specific
hybridization of PNA to complementary DNA or RNA was
detected by surface plasmon resonance (SPR), time-offlight-secondary ion mass spectrometry (TOF-SIMS),
electrochemical detection, or direct electronic detection as
well as convention fluorescence-based techniques [22]. Noncharged PNA is beneficial to detect label-free DNA targets.
Positively-charged fluorescent polymers adsorb only onto
negatively-charged DNA targets hybridized with
complementary PNA probes on a microarray, but not onto
PNA probes, and thus allow detection of unlabeled DNA
targets [23].
PNA Synthesis
PNA oligomers had been synthesized by well established
solid-phase peptide synthesis using Boc or Fmoc chemistry
[2]. Boc chemistry was originally used by PNA inventors
and can be used with an automatic peptide synthesizer.
However, it has to use extremely hazardous trifluoroacetic
acid repeatedly and cannot be used with an automatic DNA
synthesizer. Fmoc chemistry uses milder reaction condition
and can be used with a DNA synthesizer. However, it
produces PNA oligomers with more impurities, some of
which, such as generated by trans-acylation reaction, are
extremely difficult to separate because the impurities are of
the same molecular weight and other similar properties.
Recently Bts chemistry was developed by Panagene using
self-activated building blocks [24]. Bts chemistry produces
purer crude PNA oligomers (Figure 3). More importantly the
impurities are aborted oligomers because Bts chemistry does
not add building blocks to a misbehaved product. Aborted
oligomers are of smaller molecular weight and can be
readily removed by HPLC purification. This facile and near
perfect purification is especially beneficial for therapeutic
applications as well as diagnostic applications of PNA. For
Figure 3. Comparison of HPLC profiles of crude 15-mer PNAs
synthesized manually using Bts and Fmoc chemistries. Even though
the crude product can be purified by HPLC and appear as a single
peak after purification, PNA synthesized by Fmoc chemistry contains
impurities generated by side reactions whereas PNA synthesized Bts
chemistry is much purer.
example, AdvanDx announced that it would exclusively use
PNA produced by Bts chemistry for its FDA-approved
products [24].
PNA could have been more widely used if the former
licensed PNA supplier should not have had issues of long
delivery time and inability to deliver certain PNA sequences.
These problems were cleared when Panagene obtained the
world-wide exclusive right for custom PNA synthesis from
the PNA inventor group in 2006. High quality PNA
synthesized by Bts chemistry is commercially available in
large and small volume with great sequence flexibility.
Frequently requested PNA such as telomere probes are
readily available and typical quantity of custom PNA is
delivered in few weeks currently.
Conclusion
The strong and selective binding of PNA stimulated research
on the hybridization process and its therapeutic and
diagnostic applications in last 17 years. More novel
applications are coming and known applications such as
FISH and PCR clamping are becoming commercial
products. More diagnostic products will surely come and
therapeutic products are being actively sought. PNA has
very bright future for biotechnology.
References
1. Nielsen PE, Egholm M, Berg RH, Buchardt O. Sequenceselective recognition of DNA by strand displacement with
a thymine-substituted polyamide. Science 1991 254:14981500.
2. Nielsen PE, editor. Peptide Nucleic Acids-Protocol and
Applications. Norfolk: Horizon Bioscience; 2004.
3. Lundin KE, Good L, Stomberg R, Graslund A, Smith CIE.
Biological activity and biotechnological aspects of peptide
nucleic acid. Advances in Genetics 2006 56:1-51.
4. Fabani MM, Gait MJ. miR-122 targeting with LNA/2'-Omethyl oligonucleotide mixmers, peptide nucleic acids
(PNA), and PNA-peptide conjugates. RNA 2008 14:33646.
5. Ivanova GD, Fabani MM, Arzumanov AA, Abes R, Yin
Haifang, Lebleu B, Wood M, Gait MJ. PNA-peptide
conjugates as intracellular gene control agents. Nucleic
Acids Symposium Series 2008 52:31-32
6. Shiraishi T, Hamzavi R, Nielsen PE. Subnanomolar
antisense activity of phosphonate-peptide nucleic acid
(PNA) conjugates delivered by cationic lipids to HeLa
cells. Nucleic Acids Research 2008 36:4424-32.
7. Yin H, Lu Q, Wood M. Effective exon skipping and
restoration of dystrophin expression by peptide nucleic
acid antisense oligonucleotides in mdx mice. Molecular
Therapy 2008 16:38-45.
8. Faccini A, Tortori A, Tedeschi T, Sforza S, Tonelli R,
Pession A, Corradini R, Marchelli R. Circular dichroism
study of DNA binding by a potential anticancer peptide
nucleic acid targeted against the MYCN oncogene.
Chirality 2008 20:494-500.
9. Chaubey B, Tripathi S, Desire J, Baussanne I, Decout JL,
cellular membrane. A recent review summarized biological
activity of PNA [3]. A few recent examples of these
applications are listed below:
PNA blocks liver-specific microRNA (miR-122) activity
in human and rat liver cells [4, 5]. Inhibition of miRNA122 in liver cells is achieved without transfection agent
[5].
PNA conjugated with oligophosphonates shows
antisense activity at nanomolar concentration range [6].
It indicates dramatically improved cellular delivery of
PNA and EC50 values down to 1 nM.
Splicing correction of mRNA by PNA was demonstrated
in a mouse model of Duchenne muscular dystrophy [7].
A peptide-conjugated 16-mer PNA suppresses cancer by
blocking transcription of the MYCN oncogene. Its DNA:
PNA duplex shows exceptionally stability (Tm 90 C)
[8].
Neamine-conjugated PNA inhibits HIV-1 replication by
blocking the initiation of reverse transcription and
sequence-specific cleavage of the target RNA [9].
Imaging probes and FISH
PNA is especially good for FISH because it can bind to
DNA or RNA quickly even under low salt or other
unfavorable conditions for DNA [2]. Combined with
remarkable stability against enzymatic degradation,
fluorescent dye-conjugated PNA is used as a specific FISH
probe. PNA may be used to block repetitive sequences in
different FISH protocols. PNA s specificity was utilized to
discriminate 16S rRNA of bacteria species in drinking water
[10]. Secondary structure of 16S rRNA makes it difficult for
DNA FISH probes to detect the sequences. However, PNA
probes perfectly discriminated 47 different strains of
Legionella species. PNA FISH probes have been used to
analyze telomeres of chromosomes, which consist of
hundreds of repeats of a short sequence (5 -TTAGGG-3 in
human and primates). Telomere PNA FISH probes also used
for cancer diagnosis [11].
Notably AdvanDx s PNA FISH products for in vitro
diagnosis of blood infection were approved by Food and
Drug Administration (FDA) of United States in 2006 (Figure
Fig 2. FDA approved PNA FISH products of AdvanDx
Image from www.advandx.com
2). AdvanDx received Frost & Sullivan's 2008 Technology
Innovation of the Year Award in the field of rapid in vitro
molecular diagnostic technology. The products quickly
identify bloodstream pathogens in within 3 hours after blood
cultures turn positive, thereby ensure optimal therapy and
help reduce mortality rates for patients [12].
PNA probes also have been used for in vivo imaging of
mRNA for cancer research [13, 14]. In these applications
PNA is conjugated with a magnetic resonance imaging
(MRI) contrast agent or positron-emitting nuclide for
positron emission tomography (PET) imaging as well as a
cell-penetrating peptide. Specific hybridization with the
target mRNA allowed MRI or PET imaging of gene
expression in live rats or mice.
PCR clamping and artificial restriction enzyme
PNA PCR clamping technique was developed very early
to detect SNP in low abundance. For example, a few mutant
cancerous cells may be among healthy cells in a tissue
biopsy. PNA clamp complementary to wild type sequence
hybridizes specifically with wild type and blocks its PCR
amplification while allowing amplification of mutant
sequence of imperfect match. A single base mismatch is
enough to discriminate amplification of mutant type from
wild type. Epidermal growth factor receptor (EGFR) gene
mutations are associated with resistant to Gefitinib therapy
of lung cancer. A method using PNA clamp and locked
nucleic acid (LNA) primers to detect these mutations was
developed at Saitama Medical University in Japan and has
been practiced at many other sites [15-17].
Affymetrix recommends the use of globin reduction PNA
to enhance sensitivity of expression profile of human whole
blood RNA samples [18]. Specific binding of PNA to globin
mRNA inhibits reverse transcription during cDNA synthesis.
Artificial restriction enzymes have been developed for siteselective double strand DNA cutting [19]. In this system two
strands of pseudo-complementary peptide nucleic acid
invade DNA duplex to form 'hot spots' for scission, and then
Ce(IV)/EDTA complex acts as catalytic molecular scissors.
The scission fragments can be connected with foreign DNA
using DNA ligase.
Microarrays and biosensors
PNA can be used on microarrays and other biosensors. PNA
microarray combined with PCR could detect genetically
modified organisms (GMOs) in food [20]. Every Roundup
Ready soybean and Bt11, Bt176, Mon810, and GA21 maize
was correctly identified in the tested samples. PANArray
microarray for human papillomavirus (HPV) genotyping was
developed and correctly indentified genotypes of 195 HPVpositive samples among 894 clinical samples tested [21].
Certain strains of the HPV virus are known to cause cervical
cancer. 32 probes of PANArray HPV microarray can
identify 32 types of HPV unambiguously. Specific
hybridization of PNA to complementary DNA or RNA was
detected by surface plasmon resonance (SPR), time-offlight-secondary ion mass spectrometry (TOF-SIMS),
electrochemical detection, or direct electronic detection as
well as convention fluorescence-based techniques [22]. Noncharged PNA is beneficial to detect label-free DNA targets.
Positively-charged fluorescent polymers adsorb only onto
negatively-charged DNA targets hybridized with
complementary PNA probes on a microarray, but not onto
PNA probes, and thus allow detection of unlabeled DNA
targets [23].
PNA Synthesis
PNA oligomers had been synthesized by well established
solid-phase peptide synthesis using Boc or Fmoc chemistry
[2]. Boc chemistry was originally used by PNA inventors
and can be used with an automatic peptide synthesizer.
However, it has to use extremely hazardous trifluoroacetic
acid repeatedly and cannot be used with an automatic DNA
synthesizer. Fmoc chemistry uses milder reaction condition
and can be used with a DNA synthesizer. However, it
produces PNA oligomers with more impurities, some of
which, such as generated by trans-acylation reaction, are
extremely difficult to separate because the impurities are of
the same molecular weight and other similar properties.
Recently Bts chemistry was developed by Panagene using
self-activated building blocks [24]. Bts chemistry produces
purer crude PNA oligomers (Figure 3). More importantly the
impurities are aborted oligomers because Bts chemistry does
not add building blocks to a misbehaved product. Aborted
oligomers are of smaller molecular weight and can be
readily removed by HPLC purification. This facile and near
perfect purification is especially beneficial for therapeutic
applications as well as diagnostic applications of PNA. For
Figure 3. Comparison of HPLC profiles of crude 15-mer PNAs
synthesized manually using Bts and Fmoc chemistries. Even though
the crude product can be purified by HPLC and appear as a single
peak after purification, PNA synthesized by Fmoc chemistry contains
impurities generated by side reactions whereas PNA synthesized Bts
chemistry is much purer.
example, AdvanDx announced that it would exclusively use
PNA produced by Bts chemistry for its FDA-approved
products [24].
PNA could have been more widely used if the former
licensed PNA supplier should not have had issues of long
delivery time and inability to deliver certain PNA sequences.
These problems were cleared when Panagene obtained the
world-wide exclusive right for custom PNA synthesis from
the PNA inventor group in 2006. High quality PNA
synthesized by Bts chemistry is commercially available in
large and small volume with great sequence flexibility.
Frequently requested PNA such as telomere probes are
readily available and typical quantity of custom PNA is
delivered in few weeks currently.
Conclusion
The strong and selective binding of PNA stimulated research
on the hybridization process and its therapeutic and
diagnostic applications in last 17 years. More novel
applications are coming and known applications such as
FISH and PCR clamping are becoming commercial
products. More diagnostic products will surely come and
therapeutic products are being actively sought. PNA has
very bright future for biotechnology.
References
1. Nielsen PE, Egholm M, Berg RH, Buchardt O. Sequenceselective recognition of DNA by strand displacement with
a thymine-substituted polyamide. Science 1991 254:14981500.
2. Nielsen PE, editor. Peptide Nucleic Acids-Protocol and
Applications. Norfolk: Horizon Bioscience; 2004.
3. Lundin KE, Good L, Stomberg R, Graslund A, Smith CIE.
Biological activity and biotechnological aspects of peptide
nucleic acid. Advances in Genetics 2006 56:1-51.
4. Fabani MM, Gait MJ. miR-122 targeting with LNA/2'-Omethyl oligonucleotide mixmers, peptide nucleic acids
(PNA), and PNA-peptide conjugates. RNA 2008 14:33646.
5. Ivanova GD, Fabani MM, Arzumanov AA, Abes R, Yin
Haifang, Lebleu B, Wood M, Gait MJ. PNA-peptide
conjugates as intracellular gene control agents. Nucleic
Acids Symposium Series 2008 52:31-32
6. Shiraishi T, Hamzavi R, Nielsen PE. Subnanomolar
antisense activity of phosphonate-peptide nucleic acid
(PNA) conjugates delivered by cationic lipids to HeLa
cells. Nucleic Acids Research 2008 36:4424-32.
7. Yin H, Lu Q, Wood M. Effective exon skipping and
restoration of dystrophin expression by peptide nucleic
acid antisense oligonucleotides in mdx mice. Molecular
Therapy 2008 16:38-45.
8. Faccini A, Tortori A, Tedeschi T, Sforza S, Tonelli R,
Pession A, Corradini R, Marchelli R. Circular dichroism
study of DNA binding by a potential anticancer peptide
nucleic acid targeted against the MYCN oncogene.
Chirality 2008 20:494-500.
9. Chaubey B, Tripathi S, Desire J, Baussanne I, Decout JL,
Pandey VN. Mechanism of RNA cleavage catalyzed by
sequence specific polyamide nucleic acid-neamine
conjugate. Oligonucleotides 2007 17(3):302-13.
10. Wilks SA, Keevil CW. Targeting species-specific lowaffinity 16S rRNA binding sites by using peptide nucleic
acids for detection of Legionellae in biofilms. Applied
and Environmental Microbiology 2006 72:5453-62.
11. Marciniak RA, Cavazos D, Montellano R, Chen Q,
Guarente L, Johnson FB. A Novel Telomere Structure in
a Human Alternative Lengthening of Telomeres Cell
Line. Cancer Research 2005 65:2730-7.
12. Forrest GN. PNA FISH: present and future impact on
patient management. Expert Review of Molecular
Diagnostics 2007 7:231-6.
13. Heckl S, Pipkorn R, Waldeck W, Spring H, Jenne J, von
der Lieth CW, Corban-Wilhelm H, Debus J, Braun K.
Intracellular visualization of prostate cancer using
magnetic resonance imaging. Cancer Research 2003
63:4766-72.
14. Chakrabarti A, Zhang K, Aruva MR, Cardi CA, Opitz
AW, Wagner NJ, Thakur ML, Wickstrom E.
Radiohybridization PET imaging of KRAS G12D
mRNA expression in human pancreas cancer xenografts
with [ 64 Cu]DO3A-peptide nucleic acid-peptide
nanoparticles. Cancer Biology and Therapy 2007 6:94856.
15. Nagai Y, Miyazawa H, Huqun , Tanaka T, Udagawa K,
Kato M, Fukuyama S, Yokote A, Kobayashi K,
Kanazawa M, Hagiwara K. Genetic heterogeneity of the
epidermal growth factor receptor in non-small cell lung
cancer cell lines revealed by a rapid and sensitive
detection system, the peptide nucleic acid-locked nucleic
acid PCR clamp. Cancer Research 2005 65:7276-82.
16. Soh J, Toyooka S, Aoe K, Asano H, Ichihara S,
Katayama H, Hiraki A, Kiura K, Aoe M, Sano Y, Sugi
K, Shimizu N, Date H. Usefulness of EGFR mutation
screening in pleural fluid to predict the clinical outcome
of gefitinib treated patients with lung cancer.
International Journal of Cancer 2006 119:2353-8.
17. Miyazawa H, Tanaka T, Nagai Y, Matsuoka M, Huqun,
Sutani A, Udagawa K, Zhang J, Hirama T, Murayama Y,
Koyama N, Ikebuchi K, Nagata M, Kanazawa M,
Nukiwa T, Takenoshita S, Kobayashi K, Hagiwara K.
Peptide nucleic acid-locked nucleic acid polymerase
chain reaction clamp-based detection test for gefitinibrefractory T790M epidermal growth factor receptor
mutation. Cancer Science 2008 99:595-600.
18. Affymetrix GeneChip Globin-Reduction Kit Handbook
https://www.affymetrix.com/support/downloads/manuals
/globin_reduction_protocol_manual.pdf
19. Komiyama M, Aiba Y, Yamamoto Y, Sumaoka J.
Artificial restriction DNA cutter for site-selective
scission of double-stranded DNA with tunable scission
site and specificity. Nature Protocols 2008 3:655-62.
20. Germini A, Rossi S, Zanetti A, Corradini R, Fogher C,
Marchelli R. Development of a peptide nucleic acid
array platform for the detection of genetically modified
organisms in food. Journal of Agricultural and Food
Chemistry 2005 53:3958-62.
21. Panagne Inc. Unpublished results.
22. Brandt O, Hoheisel JD. Peptide nucleic acids on
microarrays and other biosensors. Trends in
Biotechnology 2004 22:617-22.
23. Sun C, Gaylord BS, Hong JW, Liu B, Bazan GC.
Application of cationic conjugated polymers in
microarrays using label-free DNA targets. Nature
Protocol 2007 2:2148-51..
24. Lee H, Jeon JH, Lim JC, Choi H, Yoon Y, Kim SK.
Peptide nucleic acid synthesis by novel amide formation.
Organic Letters 2007 9:3291-3.
25. http://www.advandx.com/about/news/detail.php?news_id=43.
Peptide Nucleic Acid
(PNA) and Its
Applications
Wonyong Koh, Ph.D.
[email protected]
Panagene Inc. www.panagene.com
816 Tamnip-dong, Yuseong-gu, Daejeon 305-510, Korea
Introduction
Peptide nucleic acid (PNA) shows remarkable hybridization
properties and has many exciting applications. PNA is a
DNA mimic, in which the entire negatively-charged sugarphosphate backbone is replaced with a neutral one consisting
of repeated N-(2-aminoethyl) glycine units linked by peptide
bonds. It is stable chemically and biologically. Peptide
bonds can make PNA readily conjugated with peptides,
fluorescent dyes, and other useful molecules. PNA was first
reported in 1991 by Nielsen et al. from the University of
Copenhagen in Denmark [1]. Among hundreds of DNA
mimics, the original PNA is one of the best performers. Over
2,000 papers on PNA were published on in the fields of
chemistry, molecular biology, antisense/antigene therapy,
molecular diagnostics, biosensors, and nanotechnology. This
article will describe recent notable examples of PNA
applications. More comprehensive information on properties
and applications of PNA can be found in a book published in
2004 [2].
such as DNA/RNA microarrays, single nucleotide
polymorphism (SNP) assays, polymerase chain reactions
(PCR), and fluorescent in situ hybridization (FISH).
Fig 1. PNA structure and Watson-Crick base paring with DNA
High affinity and sequence selectivity
Owing to proper intra-molecular spacing, PNA can
recognize specific sequence of DNA and RNA through
Watson-Click base pairing (Figure 1). Due to its uncharged
polyamide backbone, PNA can hybridize to negatively
charged DNA or RNA without electrostatic repulsion. PNA
can also hybridize under low salt concentration. Greater
affinity allows use of short PNA oligomers of 13-18 bases
typically. The stability of the PNA/DNA duplexes is
strongly affected by the presence of imperfect matches. A
mismatch in a PNA/DNA duplex is much more destabilizing
than a mismatch in a DNA/DNA duplex. For 15-mer
PNA/DNA duplex the average Tm of single mismatch was
found 15 C compared to 11 C for DNA/DNA duplex [2].
The high affinity and sequence selectivity of PNA can be
exploited in therapeutic and diagnostic applications based on
hybridization between complementary nucleic acid strands
Antisense and antigene drugs
PNA can bind to complementary sequence of mRNA and
can modulate its function. PNA can also act as antigene
agent because it can break up DNA duplex and form
PNA/DNA triplex or double duplexes without denaturing the
DNA duplex. PNA is typically conjugated with cellpenetrating peptides or lipophilic molecules for
antisense/antigene applications to enhance cellular delivery
because uncharged PNA diffuses very slowly through