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

1. Artificial ribozymes

2. Selection of proteins

3. De novo protein enzymes

Outline:

RNA

Genetic information & Catalytic properties

(DNA / PCR) (Ribozymes)

=> Selections in RNA libraries possible

In vitro Selection of RNA

(1014 molecules)

Diels-Alder Reaction

- Central reaction in organic synthesis

- Carbon - carbon bond formation / new stereo – centers *

HH

N

O

ON

O

O

+

OR1

R2

R2

R1O

**

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.

Selection in Protein Libraries

DNA => RNA => Protein

Outline:

1. Artificial ribozymes

2. Selection of proteins

3. De novo protein enzymes

Selections for Functional Proteins

cell-based droplet-based phage- ribosome- mRNA-

screen screen (IVC) display display display

complexity ~ 1013

genotype phenotype

mRNA-Display

P

- Stable covalent link between protein and gene

- Libraries of up to 1013 different proteins in a single tube

- Selection of rare, functional molecules

mRNA

Protein

Roberts RW & Szostak JW, PNAS 1997(94) 12297.

messenger RNA

PuromycinP

P

ribosome

truncated protein

Action of Puromycin

nascentprotein

N

NN

NO

N

CH3H3C

OHHN

CO

CH NH2

H2C

OCH3

HO H2C

“Adenine” moiety

“Tyrosine” moiety

P

Pmessenger RNA DNA

Puromycin

PP

P

ribosome

mRNA-displayed protein

mRNA-Display

nascentprotein

P

P

How to Select for Enzymes ?

Outline:

1. Artificial ribozymes

2. Selection of proteins

3. De novo protein 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

Expression of Ligases in E.coli

Ind

uc

ed

So

lub

le

FT

Elu

tio

n

kDa:

45

30

20

14

Ligases fused to maltose binding protein,

purification on amylose column.

Activity of Free Enzyme

Ligation of two RNA oligonucleotides by enzyme expressed in E.coli.

1 h

3 h

10

h

No

sp

lin

t

5’-

P

5’-

HO

Product

Substrate*

*

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 !

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