1. Chemical syntheses of nucleobases, nucleosides, nucleotides a oligonucleotides

2. Synthesis of pyrimidine bases

3. Transformations of pyrimidine 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 of oligonucleotides
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. 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
…..
6. Deprotection and release (aq. NH3, 50°C, 5h)
7. Purification (HPLC, GEP)
Total yield Yn= Yi(n-1) mer 80% -> 1.4%
90% -> 13%
99% -> 82%
99.8% -> 96%

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

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

35. Products of m-RNA cleavage
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

44. DNA cloning