Peptide Nucleic Acid

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Peptide Nucleic Acid

• Stephanie Persson
• April 12, 2007

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Peptide Nucleic Acid (PNA)

• Originally developed as a major-groove binding
  molecule
• Polyamide backbone
• Structural mimic of ribose-phosphate backbone
• Peptide or nucleic acid?
• Functional Duality

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PNA Structure

• 3 Parts
• Alpha-amino acid (glycine)
• Nucleobase attached via an amide linker (acetyl)
• Spacer (ethyl)
• Easy to modify

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Why is PNA a good DNA mimic?

• Favorable geometry of the polyamide backbone
• Constrained flexibility
• Planar amide rigidity
• Adapts to helical structure of DNA (B-form) or
  RNA (A-form) without losing the entire gain in
  binding enthalpy in decreased entropy upon
  formation of the much more rigid duplex
• Cyclohexyl PNA Further rigidified PNA structure
  (linker) to decrease entropy loss during PNA-DNA
  complex formation. However, the binding enthalpy
  was also reduced.

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PNA Complexes

• P-form (PNA-PNA helix)
• Preferred structure
• 28 Å wide
• 18 base pair pitch
• Ethylene linker in gauche conformation
  (stabilizing factor)
• not perfect. room to improve

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Recognition of d.s. DNA
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Triplex Recognition Problem

• Hoogsteen base pairing
• Thymine recognizes A-T base pair
• N3 protonated cytosine recognizes G-C base pair
• Pseudoisocytosines often replace cytosine for pH
  independent base pairing
• No natural nucleobases specifically recognize the
  T-A or C-G base pairs
• H-bonding sites are positioned further out in the
  major groove than the incoming base

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Recognition of the T-A base pair

• E-base
• Recognizes T-A base pair
• Extended linker from base to backbone
• Reaches O4 of thymine
• Avoids steric clashing with 5-methyl group

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Other Nucleobases
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PNA Strand Invasion
X 2-thiouracil D diaminopurine
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Strand Invasion

• Uncharged PNA backbone is not repelled by the
  negatively charged DNA or RNA phosphodiester
  backbone
• Attachment of positively charged amino acids to
  the peptide backbone can improve strand invasion
• Addition of DNA intercalators can also improve
  strand invasion

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PNA Properties

• Resistance to nuclease and protease digestion
• Stability in serum and cell extracts
• High affinity for RNA and DNA targets
• Ability to recognize complementary sequences by
  strand invasion
• Binds with high specificity

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Lots of Applications

• Recognition of cellular nucleic acids by
  synthetic oligonucleotides is a versatile
  strategy for regulating biological processes
• Antisense oligonucleotides target mRNA
• Bind to sense mRNA strand, prevent translation
  into protein
• Antigene oligonucleotides target chromosomal DNA
• Bind to dsDNA, block transcription of a specific
  gene

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Targets for Antigene PNAs

• Disrupt protein binding at transcription factor
  binding sites
• Requires strong binding to prevent progress of
  transcription machinery in mammalian cells

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Activation of Transcription by PNA

• Attach PNA to a peptide or small molecule that
  can bind to a transcription factor
  (transactivation domain)
• PNA complex binds within a promoter region of DNA
• Recruits RNA polymerase and increases
  transcription

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PNAs in Gene Therapy

• Used as delivery vehicles for gene therapy
• High affinity and specificity of binding to DNA
• Covalently link other molecules to the PNA
  polyamide backbone
• Used as adapters
• Link plasmid vectors to peptides, proteins, and
  drugs

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Other Applications of PNA

• Induce mutations
• Nucleic acid biosensors
• Modulations of PCR analyses
• Tools for genome mapping
• Induce gene expression

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Cellular Uptake of PNAs

• Complex Method
• Anneal PNAs to negatively charged DNA
• Complex heteroduplex with cationic lipid
• Lipid binds to DNA and carries it through cell
  membrane
• PNA is release in cell by melting of the complex

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Cellular Uptake of PNAs

• Spontaneous Method
• Attachment of peptide sequences that promote
  translocation across cell membrane
• Peptide may also promote nuclear uptake
• Requires high concentrations of PNA

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Future Work

• Continued development of new PNA designs and new
  strategies for strand invasion
• Improved methods for cellular delivery,
  specifically to the nucleus
• Development of anti-gene PNAs for cell culture,
  animal studies, and clinical development

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Origin of Life Debate

• Current view RNA World
• RNA served purpose of both genetic storage and
  functional enzymes
• PNA World?
• Only amino acids and nucleobases needed
• No ribose or phosphate required
• Chemical transfer of genetic information from PNA
  to PNA, RNA, or DNA (and vice versa) is feasible

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Another Nucleic Acid

• LNA (Locked)
• Modified RNA
• Locked structural conformation
• Increased thermal stability
• Improved recognition properties
• Avid binding

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References

• Dean, D.A. Peptide nucleic acids versatile
  tools for gene therapy strategies. Advanced Drug
  Delivery Reviews. 2000, 44, 81-95.
• Kaihatsu, K. Janowski, B.A. Corey, D.A.
  Recognition of chromosomal DNA by PNAs.
  Chemistry and Biology. 2004, 11, 749-758.
• Nielsen, P.E. Peptide nucleic acid. A molecule
  with two identities. Accounts of Chemical
  Research. 1999, 32, 624-630.

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Exam Questions

• What are the three major parts of PNA?
• Draw the four main ways that PNA can bind to ds
  DNA.
• Describe two ways that PNA can be modified and
  the effects/results of each modification.
• What is the triplex recognition problem?
• Describe one way that PNA could be used to
  regulate a cellular process.