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Creating enzymes not found in nature

Creating enzymes not found in nature. Burckhard Seelig University of Minnesota & Harvard Medical School. How to get new enzymes?. - Isolate enzymes from nature - Enzyme engineering Directed evolution, screen for modified properties Design by computational methods - Catalytic antibodies.

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Creating enzymes not found in nature

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  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. Outline: • Artificial ribozymes • Selection of proteins • De novo protein enzymes

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

  5. In vitro Selection of RNA (1014 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 1014 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. Outline: • Artificial ribozymes • Selection of proteins • De novo protein enzymes Selection in Protein Libraries DNA => RNA => Protein

  10. genotype phenotype Selections for Functional Proteins cell-based droplet-based phage- ribosome- mRNA- screen screen (IVC) display display display complexity~ 1013

  11. P mRNA-Display Protein mRNA • Stable covalent link between protein and gene • Libraries of up to 1013 different proteins in a single tube • Selection of rare, functional molecules Roberts RW & Szostak JW, PNAS 1997(94) 12297.

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

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

  14. Outline: • Artificial ribozymes • Selection of proteins • De novo protein enzymes How to Select for 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 1012 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. Induced Soluble FT Elution kDa: 45 30 20 14 Expression of Ligases in E.coli Ligases fused to maltose binding protein, purification on amylose column.

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

  22. Rate Enhancement & Multiple Turnover Rate enhancements over uncatalyzed background rate > 2 x 106 fold.

  23. Summary • Diels Alderase ribozymes from random RNA library • General scheme for selection of enzymes from • protein libraries > 1012 • Product formation as only selection criterion • Novel RNA-ligases from Zinc-finger library • Rate enhancements 2 x 106 fold + multiple turnover

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

  25. Acknowledgments Diels - Alderase Ribozyme Andres Jäschke and lab members DFG, BMBF Dept. ofBiochemistry, 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

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