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
 Search in large libraries
Library size ~ Probability of a hit
Outline:
1. Artificial ribozymes
2. Selection of proteins
3. De novo protein enzymes
RNA
Genetic information
(DNA / PCR)
&
Catalytic properties
(Ribozymes)
=> Selections in RNA libraries possible
In vitro Selection of RNA
(1014 molecules)
Diels-Alder Reaction
O
R2
N
O
N
R2
H
O
+
H
*
*
O
OR1
R1O
- Central reaction in organic synthesis
- Carbon - carbon bond formation / new stereo – centers *
Selection for Diels-Alderase Ribozymes
- New selection scheme
- Library of 2 x 1014 RNAs
- 120 random nucleotides
10 cycles of selection and amplification
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.
Outline:
1.
Artificial ribozymes
2.
Selection of proteins
3.
De novo protein enzymes
Selection in Protein Libraries
DNA
=>
RNA
=>
Protein
Selections for Functional Proteins
genotype
cell-based
screen
droplet-based
screen (IVC)
phenotype
phagedisplay
complexity
ribosomedisplay
mRNAdisplay
~ 1013
mRNA-Display
Protein
mRNA
P
- 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.
Action of Puromycin
messenger RNA
ribosome
P
Puromycin
H3C
CH 3
N
N
nascent
protein
HO H 2C
HN
P
O
N
O
N
N
“Adenine”
moiety
OH
C
CH
NH 2
H2C
“Tyrosine”
moiety
OCH 3
P
truncated protein
mRNA-Display
messenger RNA
DNA
P
Puromycin
ribosome
P
nascent
protein
P
P
P
mRNA-displayed protein
Outline:
1.
Artificial ribozymes
2.
Selection of proteins
3.
De novo protein enzymes
How to Select for Enzymes ?
General Selection Scheme for Enzymes
Selection of RNA-RNA Ligases
- No natural enzymes known
- Artificial ribozymes and deoxyribozymes exist
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.
+
Progress of in vitro Selection
Seelig B & Szostak JW, Nature 2007 (448) 228-31.
In vitro Evolution
=> 100 fold improvement
Elution
FT
Soluble
Induced
Expression of Ligases in E.coli
kDa:
45
30
20
14
Ligases fused to maltose binding protein,
purification on amylose column.
*
5’-HO
5’-P
No splint
10 h
3h
1h
Activity of Free Enzyme
Product
*
Substrate
Ligation of two RNA oligonucleotides by enzyme expressed in E.coli.
Rate Enhancement & Multiple Turnover
Rate enhancements over uncatalyzed background rate
> 2 x 106 fold.
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
Take home message:
We can make new enzymes !
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