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  1. 1. An overview of PNA: structure and applications Chandan Sona NISER National Institue Of Science Education And Research
  2. 2. Orientation of the talk <ul><li>Introduction </li></ul><ul><li>Structures </li></ul><ul><li>Synthesis and Purification </li></ul><ul><li>Its binding and comparison with DNA </li></ul><ul><li>Its therapeutic role with gene expression </li></ul><ul><li>Future role/problems </li></ul><ul><li>Artificial life (small discussion ) </li></ul><ul><li>Origin of life (PNA WORLD) </li></ul><ul><li>Project proposal </li></ul><ul><li>Conclusion </li></ul>
  3. 3. A bit of History <ul><li>PNA was originated from the efforts during 1980’s by Prof. Ole Buchardt together with biochemist Peter Nielsen( inventor of the molecule ). </li></ul><ul><li>Idea : Based on the observation with flow linear dichroism that a-helical poly-gamma - benzyl glutamate (PBG) can form stacked complexes with aromatic chromophores, it was proposed that PBG with alternating nucleobases and acridine moieties instead of phenyls might bind sequence selectively to duplex DNA by combined Hoogsteen base pair formation and binding with the helix backbone in the major groove . </li></ul>
  4. 4. Introduction <ul><li>PNA( Peptide nucleic acid ) is a nucleic acid analog in which sugar phosphate backbone has been replaced by a synthetic peptide backbone formed from N-(2-amino-ethyl)-glycine units . </li></ul><ul><li>PNA are non-ionic , achiral molecule and not susceptible to hydrolytic cleavage </li></ul><ul><li>Despite of all these variations PNA is still capable of sequence specific binding with DNA as well as RNA </li></ul><ul><li>Exhibit extraordinary thermal stability and unique ionic strength properties . </li></ul><ul><li>PNA hybridizes with DNA and RNA in a sequence specific manner by forming Watson-Crick hydrogen bonding scheme . </li></ul><ul><li>Triple helix is also possible by forming Hoogsteen bonding . </li></ul>
  5. 5. Interaction with DNA
  6. 6. Applications <ul><li>Potential drug candidate for gene therapy. </li></ul><ul><li>Molecular tool in molecular biology and biotechnology. </li></ul><ul><li>Lead compound for gene targeted drugs applying antigene and antisense strategy. </li></ul><ul><li>Development of biosensors helping in diagnostic purpose . </li></ul>
  7. 7. Structures of PNA and DNA
  8. 8. Structure of PNA molecule attached to DNA.
  9. 9. Synthesis and Purification <ul><li>Backbone made up of 2-amino-ethyl)-glycine units ,the amino nitrogen connected through a methyl carbonyl linker </li></ul><ul><li>No Stereo selective pathway is required. </li></ul><ul><li>Using the solid phase synthesis protocols these molecules would be constructed </li></ul><ul><li>The most important point in this molecule can be post synthetically modified according to the requirement .eg. bisPNA where cytosine is modified to pseudoisocytosine . </li></ul><ul><li>Can form chimeric structures with DNA called PNA/DNA Chimera. </li></ul><ul><li>Cleaving done by anhydrous hydrogenflouride or trifluromethanesulphonic acid. </li></ul><ul><li>Purification done by HPLC and reverse HPLC. </li></ul>
  10. 10. Differences between the two brothers <ul><li>DNA </li></ul><ul><li>It depurinates on treatment with strong acids </li></ul><ul><li>More water solubility than PNA </li></ul><ul><li>The extinction coefficient is well established </li></ul><ul><li>Base mismatches are less effective for corresponding DNA-DNA hybridization. </li></ul><ul><li>PNA </li></ul><ul><li>Fairly acid stable, amino terminal might collapse under basic condition (very slow). </li></ul><ul><li>Less water soluble than DNA </li></ul><ul><li>The extinction coefficient is not well established </li></ul><ul><li>PNA-DNA hybridization is severely affected by base mismatches and can maintain sequence discrimination up to a single base. </li></ul>
  11. 11. Comparison of PNA-DNA Duplexes <ul><li>PNA-DNA Duplex </li></ul><ul><li>The PNA molecule can form anti parallel duplex with complementary DNA sequence and has Tm of around 70 degree . </li></ul><ul><li>Parallel one Tm value is 56 degrees. </li></ul><ul><li>Low ionic strength provides stability. </li></ul><ul><li>A single mismatch in the hetero duplex of DNA and PNA reduces Tm value from 72.3 deg to 58.1deg for an experimental case. </li></ul><ul><li>DNA-DNA Duplex </li></ul><ul><li>Tm value is 53 degrees </li></ul><ul><li>No parallel binding in general. </li></ul><ul><li>Not stable at slight ionic change </li></ul><ul><li>Note- :Like the DNA-DNA duplex PNA-PNA can also form duplex with its complementary sequence with much more stability. </li></ul>
  12. 12. PNA-DNA Chimera <ul><li>PNA-DNA Chimeras can also form Watson and Crick bonding. </li></ul><ul><li>Tm is in between the DNA-DNA duplex and PNA-DNA duplex. </li></ul><ul><li>Important point : The dependence of Tm is on the length of the molecule. </li></ul><ul><li>Here in Chimeras it depends upon the ratio of PNA and DNA molecule. </li></ul>
  13. 13. To check the geometry <ul><li>Method used is circular dichroism (CD) analyzing a particular PNA sequence. </li></ul><ul><li>But PNA is achiral. </li></ul><ul><li>The chiral orientation of base pairs is the source of CD signal and the measured induced helicity is dependent on nucleobase sequence proximity centre. </li></ul>
  14. 14. Differentiating PNA
  15. 15. PNA-DNA-PNA triplexes <ul><li>Tm value greater than 70 degrees. </li></ul><ul><li>Increase in Tm with, around 10 degrees per base pair </li></ul><ul><li>DNA-PNA-DNA triplexes can also form with C rich PNA and GC rich DNA duplex. </li></ul><ul><li>Here in this triplex a protonated cytosine is involved into base triplet formation and hence it shows a pH dependence. (best stable at pH 5 - 5.5). </li></ul><ul><li>So to overcome this pH dependence we can use bisPNA which has pseudoisocytosine in place of cytosine so no charge is there. </li></ul><ul><li>It was shown using linear dichroism that this triplex is right handed triple helix. </li></ul>
  16. 16. Triple bond formation
  17. 17. Binding DNA <ul><li>Homo pyrimidine PNA binds with Homo purine DNA by strand invasion mechanism. </li></ul><ul><li>The homopyrimidine PNA recognizes its complementary site in the dsDNA and displaces the pyrimidine strand of dsDNA, forming a stable internal PNA–DNA–PNA triplex involving the homopurine strand and a looped-out, single-stranded structure containing pyrimidine bases. </li></ul><ul><li>Strand invasion favoured in A rich and at low ionic strength (<50 mM NaCl). </li></ul>
  18. 18. PNA-DNA-PNA triplex <ul><li>The purine DNA strand forms a Watson Crick base pair with anti parellel orientation . </li></ul><ul><li>And Hoogsteen bonds with the second PNA strand of identical sequence running parallel to each other . </li></ul><ul><li>Diameter is around 26 Angstrom. </li></ul><ul><li>And pitch is around 16 (triplet) bases. </li></ul>
  19. 19. Strand invasion structures
  20. 20. Antigene and antisense application <ul><li>Antigene </li></ul><ul><li>Oligonucleotides designed to recognize a particular gene , so that could interfere with the transcription of that particular gene. </li></ul><ul><li>Antisense </li></ul><ul><li>Nucleic acid analogs can be designed to recognize and hybridize to complementary sequence in mRNA and thereby inhibiting translation. </li></ul>
  21. 21. Differentiating antisense and antigene
  22. 22. Antigene strategy <ul><li>Stable triplex or strand invasion or strand displacement complex with DNA. </li></ul><ul><li>These block stable function of RNA polymerase. </li></ul><ul><li>Best is to target promoter region . </li></ul><ul><li>And downstream target leads to truncated mRNA synthesis. </li></ul><ul><li>Difficulty is strand displacement complex is rather slow at physiological salt concentrations. </li></ul><ul><li>Binding of PNA to super coiled plasmid DNA is faster than linear DNA . </li></ul><ul><li>PNA binding to DNA is enhanced when dsDNA is transcribed and if situated on non template strand . </li></ul>
  23. 23. Gene regulation
  24. 24. Antisense strategy <ul><li>The PNA antisense strategy is based on RNA processing transport into the cytoplasm or translation. </li></ul><ul><li>Experiments done in vitro suggest its potential use as antesense agent </li></ul><ul><li>Examples-:: </li></ul><ul><li>experiments involving rabbit reticulocyte lysates that both duplex- (mixed sequence) and triplex-forming (pyrimidine-rich) PNAs are capable of inhibiting translation at targets overlapping the AUG start codon. </li></ul>
  25. 25. Antisense strategy <ul><li>Duplex forming PNA can inhibit translation at initiation codon targets but triplex forming does it at initiation as well as coding region of m-RNA. </li></ul><ul><li>Mologni et al. showed effects of three different types of antisense. </li></ul><ul><li>first one was complementary to the first AUG (initiation) site </li></ul><ul><li>The second could bind to a sequence in the coding region </li></ul><ul><li>The third PNA was targeted against the 5’-untranslated region (UTR) of the mRNA , the point of assembly of the translation machinery. </li></ul>
  26. 26. Antisense strategy <ul><li>These three PNA’s could efficiently inhibit the translation. </li></ul><ul><li>Targeting 5’ UTR shows efficient inhibition </li></ul><ul><li>Important point -: </li></ul><ul><li>The transcription and translation was arrested at sub-micro molar range of PNA concentration . </li></ul>
  27. 27. Blocking translation
  28. 28. Inhibiting replication <ul><li>By strand invasion </li></ul><ul><li>Taylor et .al showed efficient binding of the PNA molecules to extrachromosomal mitDNA which remains single stranded during replication </li></ul><ul><li>A novel (and also potential) approach toward the treatment of patients suffering from ailments related to the heteroplasmy of mitochondrial DNA. </li></ul><ul><li>Where wild-type and mutated DNA are both present in the same cell. So the mutated one is targeted and replication is inhibited. </li></ul>
  29. 29. Artificial life <ul><li>What makes us think about this artificial life? </li></ul><ul><li>RNA is ahead, having the ability of autocatalytic activity and PNA yet to be discovered. </li></ul><ul><li>Self replicating short nucleotide sequence have been discovered . </li></ul><ul><li>Now the replicating system is only one part of life but the essence of life is a network of chemical reactions functioning in a state that is relatively stable. </li></ul>
  30. 30. Artificial life continues <ul><li>Steen Rasmussen of Los Alamos National Laboratory suggested a primitive protocell design based on PNA. </li></ul><ul><li>The protocell container self-assembles out of surfactant molecules—lipid chains with hydrophilic, or water loving, heads. The PNA’s backbone is modified to be lipophilic, or oil-loving, so that the PNA embeds itself in the protocell’s surface. </li></ul><ul><li>Short pieces of PNA pair up with the protocell’s PNA to form a second strand with the complementary sequence. </li></ul><ul><li>A light sensitive molecule powers the production of more surfactant molecules, which increases the protocell’s size. When it grows large enough, the protocell becomes unstable and naturally fissions. </li></ul>
  31. 31. Diagrammatic representation of artificial life .
  32. 32. Problems in artificial life <ul><li>Valid for small PNA molecules. </li></ul><ul><li>a basic problem about the stability of PNA double stranded which inhibits its separation into two daughter cells. </li></ul>
  34. 34. Origin of life (PNA world/Pre RNA world) <ul><li>RNA world </li></ul><ul><li>It seems very clear that RNA is probably more primordial and central to life than DNA and proteins. This one molecule can carry both the genotype (the genetic sequence information) of an organism and the phenotype (catalytic functions). </li></ul><ul><li>PNA world or Pre RNA world. </li></ul><ul><li>It is very unclear how primitive prebiotic conditions could have produced RNA molecules, in particular the sugar ribose in the RNA backbone. </li></ul><ul><li>Further, even if RNA molecules were produced, RNA’s very poor chemical stability hardly would have allowed the molecules to survive unprotected long enough to play a central role in the initial chemical evolution of life. </li></ul>
  35. 35. Supporting Pre RNA world <ul><li>Thus, a molecule like PNA appears very attractive as a candidate for a pre-RNA world: it is extremely stable and chemically simple and it carries sequence information. </li></ul><ul><li>Millers experiment more than 50 yrs ago suggested that amino acids could be formed under primitive conditions, identified precursors of PNA. </li></ul><ul><li>Researchers have also shown that sequence information in a PNA oligomer can be transferred by “chemical copying” to another PNA oligomer or to an RNA molecule. </li></ul><ul><li>These evidence builds a strong case of Pre RNA world based on PNA. </li></ul>
  36. 36. Still a Problem <ul><li>Most the work has been done in vitro. </li></ul><ul><li>Effect on intact cells and delivery into to the cell. Cellular uptake very slow </li></ul><ul><li>So a lot of work is being going on by modifying this molecule so that it could be taken up. </li></ul>
  37. 37. My project proposal <ul><li>I would like to work on the uptake of the molecule. </li></ul><ul><li>For uptake there is an idea </li></ul><ul><li>Chitosan, it is molecule which could encapsulate DNA could adhere itself to the mucous layers and Also can help in transformation when they are in nano particle size, this is possible as DNA-PNA chimeras are possible and potential candidate for antisense and antigene strategy. </li></ul><ul><li>So I would like to study the interaction of chitosan with PNA and if all things comes positive then I think it could be very easy for the body to uptake it. </li></ul>
  38. 38. Conclusion <ul><li>Peptide nucleic acids provide a novel class of compounds with wide biological potential . Their very specific interactions with DNA and RNA and their chemical and biological stability make them promising both as therapeutic lead compounds and agents for diagnostic applications . The application of PNA as a genetic therapeutic agent has to await the development of efficient and safe methods for its uptake and cell penetration. By contrast, its application as a recognition molecule has already led to promising developments in many areas of chemistry, biology, and biotechnology. Recent efforts to provide further applications of this exciting nucleic acid analog include modifications of backbone and the development of novel base analogs . </li></ul>
  39. 39. Acknowledgement <ul><li>Mrs. Rona Banerjee for giving me this wonderful topic , and articles to read. </li></ul><ul><li>Mr. Himanshu Raj for giving me colored pictures. </li></ul><ul><li>ALL THE AUDIENCE FOR LISTENING </li></ul>
  40. 40. References <ul><li>Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future ARGHYA RAY AND BENGT NORDE´N1 </li></ul><ul><li>Inhibition of RNA polymerase III elongation by a T10 peptide nucleic acid Giorgio Dieci*‡, Roberto Corradini†, Stefano Sforza†, Rosangela Marchelli† and Simone Ottonello </li></ul><ul><li>Scientific american december 2008 edition. </li></ul>