Subway Seminar on Darwinian DNA Machines by Christoph Wienken

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

4. Darwinian Evolution Mutation & Selection Heterogeneous population 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. 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. 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. 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. How bad is bad enough… • Intrinsic error of Polymerase enzymes 10-4-10-6 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: 0.001-0.007/nucleotide) Subway Seminar on Darwinian DNA Machines – Christoph Wienken

13. 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. 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. 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