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

What‘s Evolution? Subway Seminar on Darwinian DNA Machines – Christoph Wienken 22.09.2018

What‘s necessary for Evolution? Darwinian Evolution What‘s necessary for Evolution? Reproducibility Mutation Selection Subway Seminar on Darwinian DNA Machines – Christoph Wienken 22.09.2018

Heterogeneous population Darwinian Evolution Heterogeneous population Mutation & Selection Selection Subway Seminar on Darwinian DNA Machines – Christoph Wienken 22.09.2018

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 22.09.2018

 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 22.09.2018

Under rough Conditions… Amplification of RNA by Qβ Replicase with Ethidium Bromide Subway Seminar on Darwinian DNA Machines – Christoph Wienken 22.09.2018

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 22.09.2018

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 22.09.2018

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 22.09.2018

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 22.09.2018

Intrinsic error of Polymerase enzymes 10-4-10-6 per nucleotide 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 22.09.2018

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 22.09.2018

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 22.09.2018

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 22.09.2018

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 22.09.2018

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 22.09.2018