Another Realization of Aqueous Computing with Peptide Nucleic Acid
Peptide Nucleic Acid
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Transcript of 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