Compiled by Dr. Taruna Anand, Scientist VTC, NRC Equine, Hisar (Haryana)-India & Dr.

r. Dharmendra Kumar, Scientist CIRB, Hisar (Haryana)- India

What is PNA ?
H2N
HO O O -O P O O B

O N O HN O N B O HN B

PNA is a DNA analog in which 2-aminoethyl-glycine linkage generally replaces phosphodiester

backbone
O N B O

O -O P O O

A methyl carbonyl linker connects Nucleotide bases to the backbone

O

O -O P O O

HN N

B O OH

at the amino nitrogen

O -O P O O

DNA

PNA

(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)

Interaction of PNA with RNA

HO

OH O O O P O HO O O O O P O HO O

NH2 N O NH N O NH O

C
O

G
N

O O P O HO O

O NH N O C ON H 2

O OH

PNA

RNA

PNA Properties: Hybridization

Thermal stability
PNA-PNA > PNA-RNA > PNA-DNA > DNA-DNA

TM of a PNA-DNA-Duplex
Tmpred = 20.79 + 0.83 x TmDNA -26.13 x fpyr + 0.44 x length
(Nielsen et al,1993)

Triple Helix
CH3 N N O H N N N O H N H N O H N O Hoogsteen N N N O CH3 N + N H N H N H O N H N H H H H N N O N

C+

T
A
Watson-Crick

(Nielsen et al,1995)

Molecular complexes formed on binding of PNA to the target DNA

TRIPLEX

TRIPLEX INVASION

DUPLEX INVASION

DOUBLE DUPLEX INVASION (Pellestor et al,2004)

PNA vs. DNA

DNA
Hybridization affinity with DNA Hybridization rate with DNA Salt concentration for hybridization Dependent

PNA
At least 1 oC higher per base 100 - 5000 times Independent

Tm single mismatch
Chemical stability Water solubility Required base length for diagnosis Biological stability Thermal stability

Lowering 10 oC
Unstable in acid and base Soluble 20 - 30 Degradation by nuclease Moderate

Lowering 15 oC
Stable Restricted solubility 13 - 17 Stable 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 Self reporting PNA
Light Up probes Molecular Beacon

PCR Tools
PCR clamping Q PNA

More than 25 probes for clinical and industrial microbiology

4. The study of basic chemistry improvement of basic architecture

ANTIGENE and ANTISENSE ACTION of PNA

NORMAL CELL

DNA

RNA

PROTEIN

ANTIGENE and ANTISENSE STRATEGY

DNA

DNA

RNA NO RNA and NO PROTEIN

NO PROTEIN

ANTIGENE INHIBITION

ANTISENSE INHIBITION

STERIC INTERFERENCE

Chimeric PNA-DNA activates RNase-H

PNA-DNA chimera

RNase H (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

Pre-gel hybridization
A rapid alternative to Southern blotting
(Egholm,1996)

940C
Labelled PNA probe

ssDNA
ds DNA

Excess PNA

ELECTROPHORESIS

PNA/DNA hybrid Excess PNA ssDNA PNA/DNA hybrid ssDNA

PNA-ASSISTED RARE CLEAVAGE SYSTEM

(Veselkov,1996)

Binding of PNA

Methylation

Methylase inactivation and disruption of PNA DNA complex

Cleavage by Restriction enzyme

PNA for SNP GENOTYPING

ARTIFICIAL RESTRICTION ENZYME SYSTEM (Komiyama et al,2004)

SNP site

Enzymatic digestion

Staining dye

Sample DNA

Full match

Mismatch

Enzyme mediated ligation

Linear ds-DNA

Oligonucleotide Biotin

ccDNA

Molecule sorting on Microarrays

(Winssinger et al,2001)

PROTEINS

F
F F

F
F F MOLECULES FROM LIBRARY HYBRIDIZATION

DNA microarray

PCR Clamping
(Orum,1993)
DNA Primer

PNA Binds

PCR
Wild Type DNA

PNA Binds Amplification Blocked

DNA Primer

PNA

PCR
Mutant DNA

Amplification

Analysis of point mutation

Q-PNA
(Fiandaca,2001) Q

DYE

Peptide-PNA conjugates as targeting reagents for plasmids

(Dean,2000)

Targeting peptide/ Ligand PNA Targeting peptide Importin Antennapedia Folate Transferrin Site of action Nuclear import Cell membrane translocation Cancer cell Blood-brain barrier access

Desired gene

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