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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.
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Chemicalsyntheses of nucleobases, nucleosides, nucleotides a oligonucleotides
Modifications of Nucleosides and Nucleotides • acyclic nucleoside/nucleotide analogues • cyclonucleosides • fused and bicyclic analogues • homonucleosides • modified oligomucleotides
Synthesis of nucleotides Enzymatic: nucleoside kinase
Synthesis ofoligonucleotides • Phosphodiester method • Phosphotriester method • H-Phosphonate method • Phosphoramidite method
Phosphodiester method History…
O O P R O O Phosphotriester method
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%
REGULATION OF GENE EXPRESSION ANTISENSE STRATEGY interaction with RNA ANTIGENE STRATEGY interaction with DNA APTAMER STRATEGY interaction with proteins
Translation arest No protein synthesis Hybrid duplex m-RNA*DNA-oligomer
DNA-oligomer RNase H Products of m-RNA cleavage Hybrid duplex m-RNA*DNA-oligomer
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
MOST IMPORTANT MODIFICATIONS OF INTERNUCLEOTIDE LINKAGES ! ! ! !!
Classical synthesis of genes (duplexes DNA) • Synthesis of oligonucleotide fragments (20-40-mers, cohesive ends) • 5’-Phospohorylation (enzymatic or chemical) • Ligation – T4 DNA ligase
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