Evolution in vitro and Evolutionary Biotechnology Peter Schuster - - PowerPoint PPT Presentation

Evolution in vitro and Evolutionary Biotechnology

Peter Schuster Institut für Theoretische Chemie und Molekulare Strukturbiologie der Universität Wien RNA Secondary Structures in Dijon Dijon, 24.– 26.06.2002

Generation time 10 000 generations 106 generations 107 generations RNA molecules 10 sec 1 min 27.8 h = 1.16 d 6.94 d 115.7 d 1.90 a 3.17 a 19.01 a Bacteria 20 min 10 h 138.9 d 11.40 a 38.03 a 1 140 a 380 a 11 408 a Higher multicelluar

• rganisms

10 d 20 a 274 a 20 000 a 27 380 a 2 × 107 a 273 800 a 2 × 108 a

Generation times and evolutionary timescales

Evolution of RNA molecules based on Qβ phage

D.R.Mills, R,L,Peterson, S.Spiegelman, An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule. Proc.Natl.Acad.Sci.USA 58 (1967), 217-224 S.Spiegelman, An approach to the experimental analysis of precellular evolution. Quart.Rev.Biophys. 4 (1971), 213-253 C.K.Biebricher, Darwinian selection of self-replicating RNA molecules. Evolutionary Biology 16 (1983), 1-52 C.K.Biebricher, W.C. Gardiner, Molecular evolution of RNA in vitro. Biophysical Chemistry 66 (1997), 179-192 G.Strunk, T. Ederhof, Machines for automated evolution experiments in vitro based on the serial transfer concept. Biophysical Chemistry 66 (1997), 193-202

RNA sample Stock solution: Q RNA-replicase, ATP, CTP, GTP and UTP, buffer

• Time

1 2 3 4 5 6 69 70 The serial transfer technique applied to RNA evolution in vitro

Reproduction of the original figure of the serial transfer experiment with Q RNA β D.R.Mills, R,L,Peterson, S.Spiegelman, . Proc.Natl.Acad.Sci.USA (1967), 217-224 An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule 58

Decrease in mean fitness due to quasispecies formation

The increase in RNA production rate during a serial transfer experiment

G G G G C C C G C C G C C G C C G C C G C C C C G G G G G C G C

Plus Strand Plus Strand Minus Strand Plus Strand Plus Strand Minus Strand

3' 3' 3' 3' 3' 5' 5' 5' 3' 3' 5' 5' 5' +

Complex Dissociation Synthesis Synthesis

Complementary replication as the simplest copying mechanism of RNA

G G G C C C G C C G C C C G C C C G C G G G G C

Plus Strand Plus Strand Minus Strand Plus Strand 3' 3' 3' 3' 5' 3' 5' 5' 5'

Point Mutation Insertion Deletion

GAA AA UCCCG GAAUCC A CGA GAA AA UCCCGUCCCG GAAUCCA

Mutations represent the mechanism of variation in nucleic acids

Ij In I2 I1 Ij Ij Ij Ij Ij

+ + + +

(A) + fj Q1j fj Q2j fj Qjj fj Qnj Q (1-p) p

ij n-d(i,j) d(i,j)

= p .......... Error rate per digit d(i,j) .... Hamming distance between I and I

i j

dx / dt = x - x x

j i i j i i

Σ

; Σ = 1 ; f f x

i i i i

Φ Φ = Σ Qji Qij

Σi

= 1

[A] = a = constant

Chemical kinetics of replication and mutation

space Sequence C

• n

c e n t r a t i

• n

Master sequence Mutant cloud

The molecular quasispecies in sequence space

Decrease in mean fitness due to quasispecies formation

The increase in RNA production rate during a serial transfer experiment

Ronald Fisher‘s conjecture of optimization of mean fitness in populations does not hold in general for replication-mutation systems: In general evolutionary dynamics the mean fitness of populations may also decrease monotonously or even go through a maximum or

• minimum. It does also not hold in general for recombination of many

alleles and general multi-locus systems in population genetics. Optimization of fitness is, nevertheless, fulfilled in most cases, and can be understood as a useful heuristic.

Selection of Q

• RNA through replication in

a capillary

G.Bauer, H.Otten, J.S. McCaskill, Proc.Natl.Acad.Sci.USA 90:4191, 1989

Bacterial Evolution

• S. F. Elena, V. S. Cooper, R. E. Lenski. Punctuated evolution caused by selection of

rare beneficial mutants. Science 272 (1996), 1802-1804

• D. Papadopoulos, D. Schneider, J. Meier-Eiss, W. Arber, R. E. Lenski, M. Blot.

Genomic evolution during a 10,000-generation experiment with bacteria. Proc.Natl.Acad.Sci.USA 96 (1999), 3807-3812

Epochal evolution of bacteria in serial transfer experiments under constant conditions

• S. F. Elena, V. S. Cooper, R. E. Lenski. Punctuated evolution caused by selection of rare beneficial mutants.

Science 272 (1996), 1802-1804

2000 2000 4000 4000 6000 6000 8000 8000 10000 10000 Time (Generations) Time (Generations) 5 10 15 20 25 Distance to ancestor Distance within sample 2 4 6 8 10 12

Variation of genotypes in a bacterial serial transfer experiment

• D. Papadopoulos, D. Schneider, J. Meier-Eiss, W. Arber, R. E. Lenski, M. Blot. Genomic evolution during a

10,000-generation experiment with bacteria. Proc.Natl.Acad.Sci.USA 96 (1999), 3807-3812

Evolutionary design of RNA molecules

D.B.Bartel, J.W.Szostak, In vitro selection of RNA molecules that bind specific ligands. Nature 346 (1990), 818-822 C.Tuerk, L.Gold, SELEX - Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249 (1990), 505-510 D.P.Bartel, J.W.Szostak, Isolation of new ribozymes from a large pool of random

• sequences. Science 261 (1993), 1411-1418

R.D.Jenison, S.C.Gill, A.Pardi, B.Poliski, High-resolution molecular discrimination by

• RNA. Science 263 (1994), 1425-1429

yes

Selection Cycle

no

Genetic Diversity

Desired Properties ? ? ? Selection Amplification Diversification

Selection cycle used in applied molecular evolution to design molecules with predefined properties

Retention of binders Elution of binders C h r

• m

a t

• g

r a p h i c c

• l

u m n

The SELEX technique for the evolutionary design of aptamers

A A A A A U U U U U U C C C C C C C C G G G G G G G G A U C G

= adenylate = uridylate = cytidylate = guanylate

Combinatorial diversity of sequences: N = 4{ 4 = 1.801 10 possible different sequences

27 16

• 5’-
• 3’

Combinatorial diversity of heteropolymers illustrated by means of an RNA aptamer that binds to the antibiotic tobramycin

A A A A A C C C C C C C C G G G G G G G G U U U U U U

5’- 3’-

A A A A A U U U U U U C C C C C C C C G G G G G G G G

5’-

• 3’

Formation of secondary structure of the tobramycin binding RNA aptamer

• L. Jiang, A. K. Suri, R. Fiala, D. J. Patel, Chemistry & Biology 4:35-50 (1997)

The three-dimensional structure of the tobramycin aptamer complex

• L. Jiang, A. K. Suri, R. Fiala, D. J. Patel,

Chemistry & Biology 4:35-50 (1997)

A ribozyme switch

E.A.Schultes, D.B.Bartel, One sequence, two ribozymes: Implication for the emergence of new ribozyme folds. Science 289 (2000), 448-452

Two ribozymes of chain lengths n = 88 nucleotides: An artificial ligase (A) and a natural cleavage ribozyme of hepatitis-

• virus (B)

The sequence at the intersection: An RNA molecules which is 88 nucleotides long and can form both structures

Reference for the definition of the intersection and the proof of the intersection theorem

Two neutral walks through sequence space with conservation of structure and catalytic activity

Sequence of mutants from the intersection to both reference ribozymes

Reference for postulation and in silico verification of neutral networks

No new principle will declare itself from below a heap of facts.

Sir Peter Medawar, 1985

Coworkers

Walter Fontana, Santa Fe Institute, NM Christian Reidys, Christian Forst, Los Alamos National Laboratory, NM Peter Stadler, Universität Leipzig, GE Ivo L.Hofacker, Christoph Flamm, Universität Wien, AT Bärbel Stadler, Andreas Wernitznig, Universität Wien, AT Michael Kospach, Ulrike Langhammer, Ulrike Mückstein, Stefanie Widder Jan Cupal, Kurt Grünberger, Andreas Svrček-Seiler, Stefan Wuchty Ulrike Göbel, Institut für Molekulare Biotechnologie, Jena, GE Walter Grüner, Stefan Kopp, Jaqueline Weber