Systematic Evolution of Ligands by Exponential Enrichment SELEX

Transcription

1 Systematic Evolution of Ligands by Exponential Enrichment SELEX

2 SELEX Systematic evolution of ligands by exponential amplification * Isolation of new RNs with novel properties * New therapeutics, New diagnostics * Isolation of new ribozymes

3 Strategy for the isolation of aptamers

4 Complexity of SELEX pools Completely random sequences flanked by constant regions required for PCR amplification and transcription. Complexity 4 N, N= length of random sequence N = 25; 10 ng RN N = 40; 25 kg RN N = 100; 50 x kg RN sequence space can be enriched by mutagenic PCR during selection; feasibility molecules

5 ptamers = In vitro selected RNs with high affinity to ligands Nucleotides (TP, guanosine, 7-Methyl-GTP, Theophylline) mino acids (arginine, citrulline, valine, tryptophan) Cofactors (Cyanocobalamine, Flavin, ND) ntibiotics (tobramycin, neomycin, viomycin, streptomycin, chloramphenicol) Dopamin Proteins (thrombin, proeases, cell-surface receptors, autoreactive antibodies, reverse transcriptases, basic fibroplast growth factor, immunoglobulin E, interferon g,...k D 50 pm 50 nm )

6 n RN aptamer, which binds TP Sassanfar & Szostak, (1993) Nature 364:550 Jiang et al., (1996) Nature 383:183

7 Neomycin-binding aptamer HO HO CHR 2 O NH 2 O HOCH 2 O O I HN 2 OH O OH O NH 2 III OH OH CHNH 2 2 IV NH 2 II R neomycin B NH 2 paromomycin I OH Wallis et al. (1995) Chem.& Biol. 2:543

8 Crystal structure of streptomycin binding aptamer has been solved recently

9 SELEX Strategy for the Isolation of an RN Ligase

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11 Synthetic ribozyme which catalyses a Diels-lder reaction Tarasow et al., (1997) Nature 389:54

12 Durch SELEX Ribozyme katalysierte Insertion von Cu 2+ in Mesoporphyrin IX

13 Selection scheme for an UMP Synthetase

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15 StreptoTag, a method for the isolation of RN-binding proteins

16 Tested streptotag variants

17 MicF small 93 nt regulatory RN Induced by termperature shifts, high osmolarity and superoxide stress. Inhibits expression of OmpF outer membrane protein. Binds to ompf mrn SD and blocks ribosome binding. crosslinks to unknown 80kDa protein

18 MicF

19 B Tetracycline binding aptamer P2 U U U G C C U C U C C G P1 G 5 C G U C U G 30 G P3 G G GUG G U U C C C U C C G G G C C G G U pwhe601 pwhe602 B fluorescence / e³ 5e³ 7 OH S S P H 3 C CTTG c (tc) [µm] 6 O OH OH C TG GCT GC TG TG OH N N(CH 3 ) 2 N fluorescence / O 5e³ 2.5e³ N OH CONH c (tc) [µm]

20 pplication of ptamers : In vitro synthesis followed by injection into body, cells, via liposomes,...etc..., modified RN possible, B: Intracellular expression from specialised plasmids, correct localisation necessary, inhibition of the protein targeted, INTRMERS

21 Stability of RN RN is completely unstable in serum This obstacle is overcome by modifications of the RN backbone, which are RNase resistant 2 -fluoro, 2 -amino oligonucleotides, which are accepted by T7 RN polymerase and reverse transcriptase

22 (a) DN aptamer, that binds to neutrophil elastase and acts as an inhibitor a b (b) Inflamation imaging by aptamer NX21909 (right) and IgG (left) Charlton et al. (1997) Chem Biol 4: 809

23 PrP c -specific RN aptamer motifs (a) secondary structure of two cellular prion protein (PrP c )- binding aptamers (b) Recombinant PrP protein. The aptamer recognises the amino terminal domain P Weiss et al., (1997) J. Virol. 71: 8790

24 Selex ptamers vs natural ptamers large number of aptamers have been created for protein and small-molecule targets, which indicates that RN has the structural sophistication that is needed to serve as a sensor element. In several instances, there are both engineered and natural aptamers for the same compound. In each instance, the engineered aptamers are smaller and carry fewer essential nucleotides and structural elements than their natural counterparts. The engineered aptamers are less able to discriminate against closely related analogues, and have poorer binding affinities for their respective targets. These observations imply that there is a higher demand for specificity and affinity with natural aptamers, which is achieved by the formation of more complex motifs.