1. Creating enzymes not found in nature Burckhard Seelig University of Minnesota & Harvard Medical School

2. How to get new enzymes? - Isolate enzymes from nature - Enzyme engineering Directed evolution, screen for modified properties Design by computational methods - Catalytic antibodies  Search in large libraries Library size ~ Probability of a hit

3. 1.Artificial ribozymes 2.Selection of proteins 3.De novo protein enzymes Outline:

4. RNA Genetic information & Catalytic properties (DNA / PCR) (Ribozymes) => Selections in RNA libraries possible

5. In vitro Selection of RNA (10 14 molecules)

6. Diels-Alder Reaction - Central reaction in organic synthesis - Carbon - carbon bond formation / new stereo – centers * * *

7. Selection for Diels-Alderase Ribozymes - New selection scheme - Library of 2 x 10 14 RNAs - 120 random nucleotides 10 cycles of selection and amplification

8. Diels-Alderase Ribozymes -20,000 fold rate acceleration -Enantioselectivity > 95% ee -Minimal structural motif of 49 nucleotides Seelig B et. al. Angew. Chem. Int. Ed. 2000 (39) 4576-4579. Stucture: Serganov A et. al. Nat. Struct. Mol. Biol. 2005, 12,218-24.

9. Selection in Protein Libraries DNA => RNA => Protein Outline: 1.Artificial ribozymes 2.Selection of proteins 3.De novo protein enzymes

10. Selections for Functional Proteins cell-based droplet-based phage-ribosome-mRNA- screenscreen (IVC) displaydisplaydisplay complexity ~ 10 13 genotype phenotype

11. mRNA-Display P - Stable covalent link between protein and gene - Libraries of up to 10 13 different proteins in a single tube - Selection of rare, functional molecules mRNA Protein Roberts RW & Szostak JW, PNAS 1997(94) 12297.

12. messenger RNA Puromycin P P ribosome truncated protein Action of Puromycin nascent protein “Adenine” moiety “Tyrosine” moiety P

13. P messenger RNADNA Puromycin P P P ribosome mRNA-displayed protein mRNA-Display nascent protein P P

14. How to Select for Enzymes ? Outline: 1.Artificial ribozymes 2.Selection of proteins 3.De novo protein enzymes

15. General Selection Scheme for Enzymes

16. Selection of RNA-RNA Ligases - No natural enzymes known - Artificial ribozymes and deoxyribozymes exist

17. Protein Library - Zinc-finger scaffold = common structural motif - Not taking part in catalysis in natural proteins - Library complexity: 3.9 x 10 12 Library design & synthesis: Cho GS & Szostak JW, Chem. Biol. 2006 (13) 139.

18. Progress of in vitro Selection + Seelig B & Szostak JW, Nature 2007 (448) 228-31.

19. In vitro Evolution => 100 fold improvement

20. Expression of Ligases in E.coli Induced Soluble FT Elution kDa: 45 30 20 14 Ligases fused to maltose binding protein, purification on amylose column.

21. Activity of Free Enzyme Ligation of two RNA oligonucleotides by enzyme expressed in E.coli. 1 h 3 h 10 h No splint 5’-P 5’-HO Product Substrate * *

22. Rate Enhancement & Multiple Turnover Rate enhancements over uncatalyzed background rate > 2 x 10 6 fold.

23. Summary - Diels Alderase ribozymes from random RNA library - General scheme for selection of enzymes from protein libraries > 10 12 - Product formation as only selection criterion - Novel RNA-ligases from Zinc-finger library - Rate enhancements 2 x 10 6 fold + multiple turnover

24. Take home message: We can make new enzymes !

25. Diels - Alderase Ribozyme Andres Jäschke and lab members DFG, BMBF Dept. of Biochemistry, Free University of Berlin, Germany RNA - Ligase Jack W. Szostak and lab members, Glen Cho, Anthony D. Keefe, Glenn F. Short III, HHMI, NASA, DFG Dept. of Molecular Biology, Harvard Medical School Acknowledgments