Nucleic mimetics (PNA, LNA)
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Transcript of Nucleic mimetics (PNA, LNA)
NUCLEIC ACID MIMETICS (PNA & LNA)
ByAmit Patro
Nucleic Acid MimeticsNucleic Acid Mimetics
It is the phenomenon of imitating the behavior or function of natural occurring nucleic acids (i.e.- DNA and RNA) by some chemical compounds or oligo-nucleotides.
PNA LNA
Peptide Nucleic Acid carries the same bases as DNA (orange), but
has a totally different protein-like backbone (blue) i.e. N-amino-ethyl glycine based polyamide structure.
PNA – A DNA Mimic with Unique Properties
N
N
N
N
NH2
N
N
O
NH2
OO
O
P
O
O O
O
O
P OO
DNA
N
O
NH
O NH
N
N
O
NH2
N
O
O
N
N
N
N
NH2
PNA
Nielsen et al. 1991
PNA featuresPNA features
♦ Neutral backbone
♦ Stronger and faster binding to nucleic acids
♦ Strand invasion into duplex DNA
♦ High sequence-specificity
♦ No peptide ⇒ no degradation by protease
♦ No nucleic acid ⇒ no degradation by nucleases
PNA/DNA chimera
base triadsbase triads
Hoogsteen pairing
Watson-Crick pairing
Hoogsteen pairing
Watson-Crick pairing
PNA openersPNA openersTriplex
InvasionDouble Duplex
Invasion
PseudocomplementarypcPNA
any base composition
Homopyrimidine
PNA
PNA Application:PNA Application:•Regulating biological process: 1- antisense oligonuceotide target mRNA 2- antigene oligonucleotide target chromosomal DNA 3- antigene PNAs disrupt protein binding at transcription factor binding site.• PNA in gene therapy: 1- used as delivery vehicle for gene therapy 2- high affinity and specificity of binding to DNA 3- covalently link other molecules to PNA 4- used as adapter that link plasmid vector to peptides, proteins and drugs.• Other Application: 1- induce mutation 2- nucleic acid biosensor 3- tools for genome mapping 4- modulation of PCR analysis 5- induce gene expression
E.coliB.subtilis S.mutans
AEM 2007
Locked Nucleic AcidsLocked Nucleic AcidsThe furanose ring conformation is restricted in LNA by a
methylene linker that connects the 2'-O position to the 4'-C position. By convenience, all nucleic acids containing one or more LNA modifications are called LNA.
Features of LNAFeatures of LNA•Affinity (Watson-Crick base pairing system )
•LNA:LNA > LNA:RNA > RNA:RNA > RNA:DNA > DNA:DNA•Tm modulation
•LNA/DNA or LNA/RNA duplexes have increased thermal stability compared DNA or RNA. In general, the thermal stability of a LNA/DNA duplex is increased 3°C to 8°C per modified base in the oligonucleotide.•It is possible to fine-tune the placement of LNA bases to reach the desired Tm level without losing specificity.
•Specificity•LNA enhances hybridization performance relative to native DNA and RNA, phosphorothiate or peptide nucleic acid (PNA) probes.•LNA lowers experimental error rates due to better mismatch discrimination.•LNA provides more robust assay conditions.
•Simplicity & Design Flexibility
(A) Crystal structure of an RNA:RNA duplex (left) and an allLNA:LNA duplex (right),
(B) space filling models, derived from crystal structures, of DNA:DNA duplex(left), RNA:RNA duplex(middle) and LNA:LNA duplex (right).
Application
• Hybridization probes• Strand invasion• In-situ hybridization• Triple-helix forming
oligos• Nuclease protection
assays
• Capture probes• Sample preparation
(mRNA)• SNP analysis• Mutation analysis• Allele specific PCR• Antisense Target
Validation
PNA
LNA
References
Nielsen JT, Stein PC, Petersen M; NMR structure of an alpha-L-LNA:RNA hybrid: structural implications for RNase H recognition, Nucleic Acids Res. 2003; 31(20):5858-67Campbell M A. and Wengel J; Locked vs. unlocked nucleic acids (LNA vs. UNA): contrasting structures work towards common therapeutic goals, Chem. Soc. Rev., 2011, 40, 5680–5689.Uhalmann E; Peptide nucleic acid (PNA) & PNA-DNA chimeras; from high binding affinity towards biological function; Biol. Chem., 1998, 379, 1045-1052Nielsen P. E.; Peptide Nucleic Acid, a molecule with two identities; Accounts of chemical Research, 1999, 32, 624-630. Betts L, Josey JA, Veal JM, Jordan SR. A nucleic acid triple helix formed by a peptide nucleic acid-DNA complex. Science, 1995; 270(5243): 1838-41