1 / 44

Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es

Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es. Synthesis of pyrimidine bases. Transforma tions of pyrimidine bases. Syntheses of puri ne bases. Transforma tions of puri ne bases. Synthesis of nu c leosid es. Synthesis of nu c leosid es.

freira
Télécharger la présentation

Chemical syntheses of nu c leob ases , nu c leosid es , nu c leotid es a oligonu c leotid es

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Chemicalsyntheses of nucleobases, nucleosides, nucleotides a oligonucleotides

  2. Synthesis ofpyrimidine bases

  3. Transformations ofpyrimidine bases

  4. Syntheses of purine bases

  5. Transformations of purine bases

  6. Synthesis of nucleosides

  7. Synthesis of nucleosides

  8. Modifications of Nucleosides and Nucleotides • acyclic nucleoside/nucleotide analogues • cyclonucleosides • fused and bicyclic analogues • homonucleosides • modified oligomucleotides

  9. Biological Activity of Nucleoside Analogues

  10. Synthesis of nucleotides Enzymatic: nucleoside kinase

  11. Synthesis of nucleotides

  12. Synthesis ofoligonucleotides • Phosphodiester method • Phosphotriester method • H-Phosphonate method • Phosphoramidite method

  13. Phosphodiester method History…

  14. Phosphodiester method

  15. Phosphotriester method

  16. Phosphotriester method

  17. Phosphotriester method

  18. O O P R O O Phosphotriester method

  19. H-Phosphonate method

  20. H-Phosphonate method

  21. H-Phosphonate method

  22. H-Phosphonate method

  23. Phosphoramidite method

  24. Phosphoramidite method

  25. Phosphoramidite method

  26. Phosphoramidite method

  27. Protection of bases

  28. Protection of sugar

  29. Protection of sugar

  30. Solid-phase oligodeoxyribonucleotides

  31. Solid-phase oligodeoxyribonucleotides

  32. Solid-phase oligodeoxyribonucleotides • Detritylation • Coupling with phosphoramidite • Capping • Oxidation • Detritylation • ….. • 6. Deprotection and release (aq. NH3, 50°C, 5h) • 7. Purification (HPLC, GEP) Total yield Yn= Yi(n-1) 20-mer 80% -> 1.4% 90% -> 13% 99% -> 82% 99.8% -> 96%

  33. REGULATION OF GENE EXPRESSION ANTISENSE STRATEGY interaction with RNA ANTIGENE STRATEGY interaction with DNA APTAMER STRATEGY interaction with proteins

  34. Translation arest No protein synthesis Hybrid duplex m-RNA*DNA-oligomer

  35. DNA-oligomer RNase H Products of m-RNA cleavage Hybrid duplex m-RNA*DNA-oligomer

  36. REQUIREMENTS FOR MODIFIED OLIGONUCLEOTIDES Resistance against nuclease cleavage High affinity towards target sequences of RNA/DNA Selectivity – discrimination between DNA and RNA Low non-specific binding and high sensitivity to mismatch pairs Activation of RNase H cleavage ability

  37. MOST IMPORTANT MODIFICATIONS OF INTERNUCLEOTIDE LINKAGES ! ! ! !!

  38. Peptide Nucleic Acids

  39. Classical synthesis of genes (duplexes DNA) • Synthesis of oligonucleotide fragments (20-40-mers, cohesive ends) • 5’-Phospohorylation (enzymatic or chemical) • Ligation – T4 DNA ligase

  40. PCR (Polymerase Chain Reaction) • Add primers complementary to flanking sequence • Add all nucleoside triphosphates and thermostable DNA polymerase • Heat 95°C 15s - strand separation • Cool 54°C – hybridization • Heat 72°C (optimal temp.) – DNA synthesis

  41. DNA cloning

More Related