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Imagination is more important than

knowledge.

Albert Einstein

Peptide Nucleic Acid

characterization and applications

Advisor master :

Dr Majid Kheirollahi

By Aref Farrokhi Fard

aarreff@ymail.com3

OUTLINE

Introduction : nucleic acid analogues and PNA

PNA history

Syntesis procedures

Structural properties and its consequences

Binding properties and hybridization patterns

Aplications

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Introduction

The development of nucleic acid analogues has become an important feature due to the potential use of this newbiomolecular tool in genetic diagnostics and investigations:

XNA ( GNA / TNA / HNA )

LNA: locked nucleic acid

PNA

Morpholino

Among all the synthetic oligonucleotides designed, the peptide nucleic acids (PNA) constitute a remarkable class of nucleic acid mimics.

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PNA was invented by Drs. Nielsen,

Egholm, Berg, and Buchardt in

1991

PNA synthesis chemistry commercialized in 1993.

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.

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PNA is synthetic DNA analogue

• Peptide Nucleic Acid (PNA) is a powerful new

biomolecular tool with a wide range of important

applications.PNA mimics the behaviour of DNA.

• The phosphodiester backbone is replaced by

repetitive units of N-(2-aminoethyl) glycine to which

the purine and pyrimidine bases are attached via a

methyl carbonyl linker.

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PNA isn’t a nucleic acid and isn’t

a peptide

The various purine and pyrimidine bases are linked to

the backbone by a methylene bridge (-CH2-) and a

carbonyl group (-(C=O)-).

PNAs are depicted like peptides, with the N-terminus at

the first (left) position and the C-terminus at the last

(right) position.

PNA is not known to occur naturally but N-(2-

aminoethyl)-glycine (AEG), the backbone of PNA, has

been found produced by cyanobacteria.10

Synthesis procedures

standard solid-phase manual or automated synthesis.

The general principle of SPPS is one of repeated

cycles of coupling-wash-deprotection-wash

Coupling the carboxyl group or C-terminus of one

amino acid to the amino group or N-terminus of

another.

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SPPS allows the synthesis of

natural peptides which are difficult

to express in bacteria.

Solid-phase peptide synthesis (SPPS), pioneered by

Robert Bruce Merrifield.

SPPS is now the accepted method for creating

peptides and proteins in the lab in a synthetic manner.

Unlike ribosome protein synthesis, solid-phase peptide

synthesis proceeds in a C-terminal to N-terminal

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Modifications

The PNA molecules can routinely be labeled with biotin

or fluorophores.

Chimeric architecture developed in order to improve the

solubility and the cellular uptake of PNAs or to exhibit

new biological properties:

PNA–peptide chimeras

PNA–DNA chimeras

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Modifications

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Bis-PNA is prepared in a continuous synthesis

process by connecting two PNA segments via a

flexible linker.

PNA hetero-oligomers comprising normal

aminoethylglycine and aminoproline building blocks

are prepared using Boc strategy.

PNA advantages and

disadvantages

PNA advantages PNA disadvantages

Strong binding to NAs and

high Tm

Long halflife and high stability

Very specificity and mismatch

sensitivity

PNA solubility

PNA delivery

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Unlike DNA and RNA, the PNA

backbone is not charged

PNA is set apart from DNA in that the backbone of

PNA is acyclic, achiral and neutral. There is no

electrostatic repulsion when PNA hybridizes to its

target, giving a higher stability to PNA hybrids.

This greater stability results in higher thermal melting

temperature (Tm) values than is observed for natural

duplexes.

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PNAs have high Tm compared

with DNA

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Generally Tm of 10 mer PNA and 15mer PNA shows

around 50°and 70°respectively.

Early experiments with homopyrimidine strands have

shown that the Tm of a 6-base thymine PNA/adenine

DNA double helix was 31 °C in comparison to an

equivalent 6-base DNA/DNA duplex that denatures at

a temperature less than 10 °C.

PNAs have high Tm compared

with DNA

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Unlike DNA and RNA, the PNA

backbone is not charged

Hybridization independently of the salt concentration.

Tm of PNA–DNA duplex is barely affected by low ionic

strength.

Destabilizing intramolecular hybridization.

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Ionic strength dependence of thermal stability (Tm) of

PNA/DNA (open symbols) and DNA/

DNA duplex (closed symbols)

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Ionic strength dependence of thermal stability (Tm) of

PNA/DNA (open symbols) and DNA/DNA duplex

(closed symbols)

PNAs are resistant to enzymes

The lifetime of PNA is extended both in vivo and in vitro.

Also, PNA are not recognized by polymerases and

therefore cannot be directly used as primers or be

copied.

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PNAs are resistant to enzymes

Inside cells, the half-life for most of the unmodified

DNA and RNA oligonucleotides is approximately 15

min or shorter.

PNAs are stable inside cells for at least 48 h.

This extreme stability makes PNAs an ideal

candidate for the antisense and antigene application.

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PNA hybridize to complementary

nucleic acids

PNA hybridize to complementary DNA or RNA in a

sequence-dependent manner, according to the Watson–

Crick hydrogen bonding scheme.

In contrast to DNA, PNA can bind in either parallel or

anti-parallel fashion.

PNA probes can bind to either single-stranded DNA or

RNA, or to double-stranded DNA.

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PNA–DNA hybridization is

significantly affected by base

mismatches

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In the PNA/DNA duplexes the average ΔTm was 15ºC,

whereas the average ΔTm for the corresponding

DNA/DNA duplexes was 11ºC.

More important than the average is that the lowest

measured ΔTm for the DNA/DNA duplex was 4ºC vs.

8ºC for the PNA/DNA duplex, i.e., the PNA is “twice” as

discriminating in the case of the least discriminating

base pair.

A single mismatch in 15mer PNA/DNA : 5°C decreased Tm

A single mismatch in 15mer DNA/DNA : 1°C decreased Tm

(1) Standard duplex invasion complex formed with some homopurine PNA.

(2) Double-duplex invasion complex, very stable but only possible with PNA

containing modified nucleo-bases (diaminopurine-thiouracil ‘base pairs’ that

sterically destabilize the competing PNA-PNA duplex)

(3) Conventional triple helical structure (triplex) formed with cytosine-rich

homopyrimidine PNA binding to complementary homopurine DNA targets.

(4) Stable triplex invasion complex, leading to the displacement of the second

DNA strand into a ‘D-loop’.25

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Ionic strength dependence of the formation of PNA triplex invasion complexes.

Homopyrimidine PNAs can form

triplex helixes with P-loop

(PNA)2–DNA triplex helixes displaying high Tm.

In these triplexes, one PNA strand hybridizes to DNA

through standard Watson–Crick base pairing rules,

while the other PNA strand binds to DNA through

Hoogsteen hydrogen bonds. The resulting structure

is called P-loops ..

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Bis-PNAs : tools for selectively

targeting short homo-purine sequence

in dsDNA with very high specificity and

efficacy

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More stable triplexes can be formed by bis-PNAs .

Watson-Crick PNA strand is connected to the

Hoogsteen PNA strand by continuous synthesis via

ethylene glycol type linkers.

Typical Tm for bis-PNAs are about 100ºC for a 10-mer

and about 65ºC for a 7-mer.

PNAs 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 .

Introducing a few lysine-based monomers into a PNA

oligomer greatly increase the aqueous solubility.

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PNAs are poorly penetrated

through

the cell membrane

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This is partially due to its uncharged property.

To enhance the efficiency in cellular delivery of

unmodified PNAs

many strategies have been explored:

Electroporation

Microinjection

permeabilization of eukaryotic cells by streptolycin-O

Increasing PNA delivery by

modifications

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Incorporating positively charged residues such as lysine

and arginine to the PNA molecules.

Using ligands to enhance the attachment of PNAs to the

cell membrane.

peptide sequences

Antibodies

lipophilic moieties

cell-specific receptor ligands

Main features of PNA are as

follows

High binding affinity to its complementary DNA or RNA.

Differentiation of single-base mismatch by high

destabilizing effect

High chemical stability to temperature and pH

High biological stability to nuclease and protease

Salt independence during hybridization with DNA sequence.

Triplex formation with continuous homopurine DNA.

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PNA world hypothesis

It has been hypothesized that the earliest life on

Earth may have used PNA as a genetic material

due to :

Extreme robustness

Simpler formation

Possible spontaneous polymerization at 100°C

If this is so, life evolved to a DNA/RNA-based

system only at a later stage.

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PNA is a tool in molecular

biology, diagnosis and therapy

1. Solid-phase hybridization techniques : DNA

capturing(DNA purification), microarray and other

Biosensors

2. PNA–PCR strategies

3. Pre-gel hybridization : rapid alternative to Southern

blotting

4. PNA-assisted rare cleavage and artificial restriction

enzymes

5. In vivo imaging

6. PNA as probes for chromosomal analysis

7. Antisense technology: antiviral,

antibacterial,antiparasitic and anticancer agent37

PNA as antigene and antisense

agent

PNA molecules were first used in antigene and

antisense assays.

In theory PNA oligomers would be very strong

candidates for effective antisense agents with their

high affinity, high specificity, and their high stability in

vivo.

little or no binding to serum proteins

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PNA-mediated inhibition of gene

transcription : formation of strand-

invasion or strand displacement

PNA targeted against the promoter region of a gene

can form stable PNA–DNA complexes that restrict the

DNA access of the polymerase.

PNA as competitors with endogenous cis-element(s)

present in the target gene for trans-acting factors.

whereas PNA complexes located far from the

promoter can block the polymerase progression and

lead to the production of truncated RNA transcripts. 39

Applications of strand invasion by

PNAs

(A) Inhibition of gene expression.

(B) Activation of gene expression by PNA-peptide conjugates.

An artificial activation domain could be a peptide, a small molecule, or any other

synthetic construct capable of selectively recruiting transcription factors.40

PNA-mediated inhibition of mRNA

translation

PNA are able to interact with mRNA independently of

the RNA secondary structure.

PNA inhibits expression differently from anti-sense

oligonucleotides, acting through RNase-H-mediated

degradation of the mRNA– oligonucleotide hybrid.

Anti-sense effect acts through steric interference of

either RNA processing, transport into cytoplasm or

translation, caused by binding to the mRNA.

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Inhibition of gene expression

through recognition and destruction

of mRNA

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(A) Inhibition of gene expression by preventing ribosome binding.

(B) Inhibition of gene expression by preventing translocation of the

ribosome.

(C) Alteration of splicing.

(D) Inhibition of ribonucleoprotein activity.

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Solid-phase hybridization

techniques

The neutral backbone of PNA significantly increases the

rate of hybridization in assays where either the target or

the probe is immobilized.

Thus PNA can be used for sequence-specific capture of

single-stranded nucleic acids, taking advantage of the

tight complex formation at low ionic strength which

destabilizes nucleic acid secondary structure.

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Oligonucleotide/PNA-assisted

affinity

capture (OPAC)

Capturing of dsDNA using (PNA)2–DNA openers:

creating a large single-stranded DNA loop to which a

biotinylated oligonucleotide could hybridize.

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Targeting duplex DNA through PD-loop

5' 3'

3' 5'

NH2COOH

PNA openers = 6-10

Two PNA openers are able to sequence-specifically

hybridize to complementary target sites in duplex DNA

DNA probe can hybridize to the displaced strand forming a

stable complex

5' 3'

PD-loop

PNAS 199846

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PNA–PCR strategies

PNA probes have no direct interaction with DNA

polymerase but PNA can terminate the elongation of

oligonucleotide primers by binding to the template or

competing with the primers.

Moreover, PNA–DNA chimeras can be recognized by

the DNA polymerase and can thus be used as primers

for PCR reactions.

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Detecting single base pair

mutations by PCR

PNA-directed PCR clamping, uses PNA to inhibit the

amplification of a specific target by direct competition of

the PNA targeted against one of the PCR primer sites

and the conventional PCR primer.

This PNA–DNA complex formed at one of the primer

sites ,effectively blocks the formation of the PCR

product.

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Novel automated real-time PCR

has been developed using PNA

In this method, named Q-PNA PCR, a generic

quencher labeled PNA (Q-PNA) is hybridized to the

tag sequence of a fluorescent dye-labeled DNA

primer in order to quench the fluorescence of the

primer.

During PCR, the Q-PNA is displaced by incorporation

of the primer into amplicons and the fluorescence of

the dye label is liberated .

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An alternative to southern

analysis is the PNA pre-gel

hybridization process

Labeled PNA are then used as probes, allowing

hybridization to a denatured dsDNA sample at low ionic

strength prior to loading on the gel.

The mixture is directly subjected to electrophoresis for

separation of bound and unbound PNA probes.

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PNA FISH for bacterial

detectionStaphylococcus aureus (green)

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PNA s specificity was utilized to

discriminate 16S rRNA of bacteria

species in drinking water

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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 are compatible with a

wide range of reporter

molecules and

fluorochromes including:

Additional benefits of

using PNA are:

Fluorescein

Rhodamine

Cyanine

Alex dyes

Lower background signals

Unlimited stability of the

probe mixture

Mild washing procedure

PNA probe for FISH

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PNA probes for chromosomal

analysis

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.

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PNA–FISH for telomers

Subsequently, telomere PNA probes were used in

several in situ studies of cancer and ageing.

The performance of PNA method for in situ detection

and sizing of telomeric repetitive sequences was

compared to the PRINS technique.

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PNA in biosensors

A single-stranded PNA probe is immobilized onto optical

or mass-sensitive transducers to detect the

complementary strand or corresponding mismatch in a

DNA sample solution.

The hybridization events are converted into electric

signals by the transducers.

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PANArray microarray for human papillomavirus

(HPV) genotyping was developed and correctly

indentified genotypes of 195 HPV positive

samples among 894 clinical samples tested

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PNA-assisted rare cleavage

(PARC)

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Peptide nucleic acids, in combination with

methylases and other restriction endonucleases,

can act as rare genome cutters.

It uses the strong sequence-selective binding of

PNAs, preferably bis-PNAs, to short

homopyrimidine sites on large DNA molecules.

Artificial restriction enzymes have

been developed for site selective

double strand DNA cutting

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In this system two strands of pseudo-complementary peptide nucleic acid invade DNA duplex to form 'hot spots' for scission, and thenCe(IV)/EDTA complex acts as catalytic molecular scissors.

PNA in combination with S1 nuclease can work as an ‘artificial restriction enzyme’ system.

References

Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991).

"Sequence-selective recognition of DNA by strand displacement with a

thymine-substituted polyamide". Science 254 (5037): 1497–500.

doi:10.1126/science.1962210. PMID 1962210.

Jump up ^ Cyanobacteria Produce N-(2-Aminoethyl)Glycine, a

Backbone for Peptide Nucleic Acids Which May Have Been the First

Genetic Molecules for Life on Earth

Jump up ^ Egholm M, Buchardt O, Christensen L, Behrens C, Freier

SM, Driver DA, Berg RH, Kim SK, Nordén B, and Nielsen PE (1993).

"PNA Hybridizes to Complementary Oligonucleotides Obeying the

Watson-Crick Hydrogen Bonding Rules". Nature 365 (6446): 566–8.

doi:10.1038/365566a0. PMID 7692304.

Wittung P, Nielsen PE, Buchardt Ole, Egholm M, and Nordén B (1994).

"DNA-like Double Helix formed by Peptide Nucleic Acid". Nature 368

(6471): 561–3. doi:10.1038/368561a0. PMID 8139692.

Jump up ^ Nelson KE, Levy M, and Miller SL (2000). "Peptide nucleic

acids rather than RNA may have been the first genetic molecule". Proc.

Natl. Acad. Sci. USA 97 (8): 3868–71. doi:10.1073/pnas.97.8.3868.

PMC 18108. PMID 10760258.

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References

Jump up ^ Zimmer C (January 2009). "On the Origin of Life on Earth".

Science 323 (5911): 198–9. doi:10.1126/science.323.5911.198.

PMID 19131603.

Jump up to: a b Markus Schmidt (9 May 2012). Synthetic Biology. John

Wiley & Sons. pp. 151–. ISBN 978-3-527-65926-5. Retrieved 9 May 2013.

Jump up ^ E. A. Jensen (27 November 2012). Manipulating the Last Pure

Godly Dna: The Genetic Search for God's Dna on Earth. Trafford

Publishing. pp. 257–. ISBN 978-1-4669-6106-7. Retrieved 9 May 2013.

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References

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http://www.blogsua.com/peptide-nucleic-acids.html

http://link.springer.com/article/10.1134%2FS0012500806050016

http://www.cell.com/chemistry-biology/abstract/S1074-

5521(11)00275-4?switch=standard

http://www.rsc.org/chemistryworld/2012/10/dna-directed-

synthesis

http://www.greiner-bio

one.co.jp/products/PNA/pcr_clamping.html

http://www.jbsdonline.com/Site-Specific-Nicking-of-Duplex-DNA-

using-PNAs-and-Restriction-Endonucleases-p11572.html

http://www.mdche.u-szeged.hu/~kovacs/PNA_Seili.pdf

Thank you for your attention

Any question?

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