1. InVITRO selection (SELEX)
Subway Seminar on Darwinian DNA Machines
Christoph Wienken
InVITRO selection (SELEX)
Systematic Evolution of Ligands by EXponential Enrichment

2. What‘s Evolution?
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

3. What‘s necessary for Evolution?
Darwinian Evolution
What‘s necessary for Evolution?
Reproducibility
Mutation
Selection
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

4. Heterogeneous population
Darwinian Evolution
Heterogeneous population
Mutation
& Selection
Selection
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

5. Torwards Evolution of Molecules…
Can we transfer this biological principle
to the level of molecules?
YES, WE CAN!
(Self-)Duplication
Mutations
Selection
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

6.  First InVitro Experiments by Spiegelman et al.
As fast as you can…
First InVitro Experiments by Spiegelman et al.
„Amplification of RNA by Qβ Replicase“
Reiteration 
+Qβ
Transfer
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

7. Under rough Conditions…
Amplification of RNA by Qβ Replicase with Ethidium Bromide
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

8. Qβ replicase taken from bacteriophage Qβ
And then came PCR…
InVitro Evolution works on the level of molecules with many different possible selective pressures
But various problems:
Qβ replicase taken from bacteriophage Qβ
 Selectively replicates only viral RNA but no other RNA
Selection, mutation, and amplification are not independent
(Selection is coupled to the speed of amplification)
 PCR decouples the amplification from the selection process
and allows many more experiments
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

9. How does PCR help InVitro evolution?
And then came PCR…
How does PCR help InVitro evolution?
Additional amplification at the level of dsDNA using the PCR
Mutations can be introduced during PCR Amplification
Selection is performed by making only chosen molecules eligible for amplification
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

10. InVitro Evolution for Binding Studies
The decoupled selection and amplification allows selection due to the ability of the RNA to bind to a target ligand (SELEX)
Binding RNA molecules are called „Aptamers“ (lat. Aptus = fit, suitable)
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

11. InVitro Evolution and Catalytic Processes
RNA molecules can catalyze bond-forming and bond-breaking reactions
Bond-forming
Bond-breaking
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

12. Intrinsic error of Polymerase enzymes 10-4-10-6 per nucleotide
How bad is bad enough…
Intrinsic error of Polymerase enzymes per nucleotide
 too good to maintain a heterogen population of RNA sequences
Ideal error rate: about 1 mutation per copy
Techniques for introducing random mutations:
„Error-prone PCR“ (error rate: 0.007/nucleotide)
„hypermutagenic PCR“ (error rate: 0.1/nucleotide)
„thermostable DNA polymerase“(error rate: /nucleotide)
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

13. InVitro Evolution (and SELEX) needs
Summing up…
InVitro Evolution (and SELEX) needs
Heterogen population as a starting point
Methods to Introduce Mutations
Selection processes
Methods to (Self-)Duplicate
InVitro Evolution can
Optimize molecular processes under given selective pressures
Find suitable Aptamers for binding reactions
Catalyze bond-forming and bond-breaking reactions
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

14. Can we evolve a RNA-Ribozyme that catalyzes the binding of NTPs?
Torwards an RNA World…
Can we evolve a RNA-Ribozyme that catalyzes the binding of NTPs?
 A RNA replicase made out of RNA
Experiments by Bartel & Szostak:
Starting with 1015 different RNAs
Selection with substrate specific
oligo affinity column
Amplification with PCR using substrate
oligo as a primer
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

15. Evolution becomes continous…
Most InVitro experiments with ribozymes in a stepwise fashion
but Continous Evolution possible!
More about „Continous InVitro Evolution“ on November, 5th
presented by Christof Mast
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

16. InVitro Evolution – Today and Tomorrow
RNA Aptamers as therapeuticals due to their high specific binding
Inhibit von Willebrand Factor (responsible for adhesion of blood platelets)
Binding to Hepatitis C Virus NS3
Developing a self-sustained system that evolves without the aid of protein polymerases
Problems:
Microfluidics for automated Serial dilutions
No ligase ribozyme with a catalytic rate as fast as Class I Ligase
Subway Seminar on Darwinian DNA Machines – Christoph Wienken

17. Thanks for your attention!
References
[1] C . R. Darwin, On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life, John Murray, London, 1859.
[2] D . R. Mills, R. L. Peterson, S. Spiegelman, Proc. Natl. Acad. Sci. USA 1967, 58, 217 – 224.
[3] I. Haruna, S. Spiegelman, Science 1965, 150, 884 – 886.
[4] R. Levisohn, S. Spiegelman, Proc. Natl. Acad. Sci. USA 1969, 63, 805 – 811.
[5] R. Saffhill, H. Schneider-Bernloehr, L. E. Orgel, S. Spiegelman, J. Mol. Biol. 1970, 51, 531 – 539.
[6] G.F. Joyce, Angew. Chem. Int. Ed. 2007, 46, 6420 – 6436
[7]
Thanks for your attention!
Subway Seminar on Darwinian DNA Machines – Christoph Wienken