peptide nucleic acid

Compiled by Dr. Taruna Anand, Scientist

VTC, NRC Equine, Hisar (Haryana)-India

& Dr. Dharmendra Kumar, Scientist

CIRB, Hisar (Haryana)- India

NH2

N

NH

O

N

NH

N

NH

O

B

O

O

B

O

N

OH

B

O

O

O

B

DNA PNA

O

O

O

P OO

O B

O

O

P OO

O B

O

O

P OO

O B

O

O

P OO

OH B

-

-

-

-

What is PNA ?

PNA is a DNA analog in which

2-aminoethyl-glycine linkage

generally replaces phosphodiester

backbone

A methyl carbonyl linker connects

Nucleotide bases to the backbone

at the amino nitrogen

(Nielsen et al,1992)

The Miller Experiment Primordial Soup

•Amino acids

•PNA units

Characteristics of PNA

• Higher affinity to complementary nucleic acid (DNA, RNA)

• Strong hybridization independent of salt concentration

• Greater specificity and sensitivity of interaction

• Thermal and chemical stability

• Resistance to nucleases and proteases

(Nielsen et al,1992)

PNA

G

T

A

N

NH

NH2

N

O

O

N

NH

NH

N

O

O

CONH2

O

O

OC

RNA

C

A

OH

O

OOPO

O

O

O

OH

PO

O O-

O

OPO

O O-

O-

HO

HO

HO

HO

T

G

Interaction of PNA with RNA

PNA Properties: Hybridization

Thermal stability

PNA-PNA > PNA-RNA > PNA-DNA > DNA-DNA

Tmpred = 20.79 + 0.83 x TmDNA -26.13 x fpyr + 0.44 x length

TM of a PNA-DNA-Duplex

(Nielsen et al,1993)

Triple Helix

ON

O

NH

CH3

T

CH3

HN N

OH H

N

N

N N

N

O

AT

Hoogsteen

Watson-Crick

C+C

G

N

N N

N

O

NH

H

H

H

H

N

N

O

N

+NN

N

H

H

HO

(Nielsen et al,1995)

A TRIPLEX

B TRIPLEX INVASION

D DOUBLE DUPLEX INVASION

Molecular complexes formed on binding of PNA to the target DNA

DUPLEX INVASIONC

(Pellestor et al,2004)

PNA vs. DNA

DNA PNA

Hybridization affinity with DNA At least 1 oC higher per base

Hybridization rate with DNA 100 - 5000 times

Salt concentration for hybridization Dependent Independent

Tm single mismatch Lowering 10 oC Lowering 15 oC

Chemical stability Unstable in acid and base Stable Water solubility Soluble Restricted solubility

Required base length for diagnosis 20 - 30 13 - 17

Biological stability Degradation by nuclease Stable

Thermal stability Moderate Good

PNA applications

1. Molecular tool in Molecular Biology and Biotechnology

2. Lead compound for gene-targeted drugs (antisense & antigene)

3. Diagnostic purpose and development of biosensor

More than 25 probes for clinical and industrial microbiology

4. The study of basic chemistry – improvement of basic architecture

• Self reporting PNALight Up probesMolecular Beacon

• PCR Tools PCR clamping

Q PNA

ANTIGENE and ANTISENSE ACTION of PNA

DNA

RNA

PROTEIN

NORMAL CELL

DNA

NO RNA and NO PROTEIN

ANTIGENE INHIBITION

DNA

RNA

NO PROTEIN

ANTISENSE INHIBITION

ANTIGENE and ANTISENSE STRATEGY

STERIC INTERFERENCE

RNase H

Chimeric PNA-DNA activates RNase-H

PNA-DNA chimera

(Nielsen,1996)

Molecular tool in Molecular Biology and Biotechnology

Molecular tool in Molecular Biology and Biotechnology

• Pre-gel hybridization – A rapid alternative to Southern blotting

• PNA-assisted rare cleavage

• Artificial restriction enzyme system

• Nucleic acid purification

•PNA-assisted topological labelling of duplex DNA

940C

ds DNA

Labelled PNA probe

ssDNA

PNA/DNA hybrid ssDNA

Excess PNA

-

ssDNA

PNA/DNA hybrid

Excess PNA

+

ELECTROPHORESIS

Pre-gel hybridization

A rapid alternative to Southern blotting(Egholm,1996)

Binding of PNA

Methylation

Methylase inactivation and disruption of PNA –DNA complex

Cleavage by Restriction enzyme

PNA-ASSISTED RARE CLEAVAGE SYSTEM(Veselkov,1996)

Sample DNA

SNP siteEnzymatic digestion

Staining dye

Full match Mismatch

PNA for SNP GENOTYPING ARTIFICIAL RESTRICTION ENZYME SYSTEM

(Komiyama et al,2004)

Enzyme mediated ligation

ccDNA

Linear ds-DNA

Oligonucleotide

Biotin

F

F

F

F

FMOLECULES FROM LIBRARY

INCUBATIO

N

SIZE-EXCLUSION

CHROMATOGRAPHY

PROTEINS

F

FF

HYBRIDIZATION

DNA microarray

Molecule sorting on Microarrays(Winssinger et al,2001)

PCR Clamping

Wild Type DNA

PNA BindsDNA Primer

PCR PNA Binds Amplification Blocked

Mutant DNA

PNA DNA Primer

PCR Amplification

Analysis of point mutation

(Orum,1993)

Q-PNA

DYE

Q(Fiandaca,2001)

PNA

Targeting peptide/Ligand

Desired gene

Targeting peptide Site of action

Importin Nuclear import

Antennapedia Cell membrane translocation

Folate Cancer cell

Transferrin Blood-brain barrier

access

Peptide-PNA conjugates as targeting reagents for plasmids

(Dean,2000)

CONCLUSION PNA has a unique position compared with

other nucleic acids PNA offer enormous potential for

improved assay performance It can make an impact in the

establishment of assay procedures for routine applications

PNA can be used in the same applications as DNA, or RNA with many added benefits