Creating enzymes not found in nature

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

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Outline

• Artificial ribozymes
• Selection of proteins
• De novo protein enzymes

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RNA
Genetic information Catalytic
properties (DNA / PCR)
(Ribozymes) gt Selections in RNA
libraries possible
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In vitro Selection of RNA
(1014 molecules)
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Diels-Alder Reaction


- Central reaction in organic synthesis -
Carbon - carbon bond formation / new stereo
centers
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Selection for Diels-Alderase Ribozymes
- New selection scheme - Library of 2 x 1014
RNAs - 120 random nucleotides 10 cycles of
selection and amplification
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Diels-Alderase Ribozymes

• 20,000 fold rate acceleration
• Enantioselectivity gt 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.
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Selection in Protein Libraries

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

DNA gt RNA gt Protein
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Selections for Functional Proteins
cell-based droplet-based phage- ribosome- mR
NA- screen screen (IVC) display display dis
play
complexity 1013
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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.
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Action of Puromycin
messenger RNA
ribosome
Puromycin
Adenine moiety Tyrosine moiety
nascent protein
P
truncated protein
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mRNA-Display
messenger RNA
DNA
Puromycin
ribosome
nascent protein
P
mRNA-displayed protein
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How to Select for Enzymes ?

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

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General Selection Scheme for Enzymes
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Selection of RNA-RNA Ligases

• No natural enzymes known
• Artificial ribozymes and deoxyribozymes exist

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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.
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Progress of in vitro Selection

Seelig B Szostak JW, Nature 2007 (448) 228-31.
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In vitro Evolution
gt 100 fold improvement
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Expression of Ligases in E.coli
Ligases fused to maltose binding protein,
purification on amylose column.
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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.
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Rate Enhancement Multiple Turnover
Rate enhancements over uncatalyzed background
rate gt 2 x 106 fold.
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Summary

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

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Take home message
We can make new enzymes !
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Acknowledgments
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