Subway Seminar on Darwinian DNA Machines Christoph Wienken

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

What‘s Evolution? Subway Seminar on Darwinian DNA Machines – Christoph Wienken 9/9/2020 2

Darwinian Evolution What‘s necessary for Evolution? § Reproducibility § Mutation § Selection Subway Seminar on Darwinian DNA Machines – Christoph Wienken 9/9/2020 3

Darwinian Evolution Heterogeneous population Selection Subway Seminar on Darwinian DNA Machines – Christoph Wienken Mutation & Selection 9/9/2020 4

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 9/9/2020 5

As fast as you can… First In. Vitro Experiments by Spiegelman et al. „Amplification of RNA by Qβ Replicase“ Reiteration +Qβ Subway Seminar on Darwinian DNA Machines – Christoph Wienken Transfer 9/9/2020 6

Under rough Conditions… Amplification of RNA by Qβ Replicase with Ethidium Bromide Subway Seminar on Darwinian DNA Machines – Christoph Wienken 9/9/2020 7

And then came PCR… In. Vitro 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 9/9/2020 8

And then came PCR… 1. How does PCR help In. Vitro evolution? 2. Additional amplification at the level of ds. DNA using the PCR 3. Mutations can be introduced during PCR Amplification 4. Selection is performed by making only chosen molecules eligible for amplification Subway Seminar on Darwinian DNA Machines – Christoph Wienken 9/9/2020 9

In. Vitro Evolution for Binding Studies The decoupled selection and amplification allows selection due to target ligand (SELEX) the ability of the RNA to bind to a Binding RNA molecules are called „Aptamers“ (lat. Aptus = fit, suitable) Subway Seminar on Darwinian DNA Machines – Christoph Wienken 9/9/2020 10

In. Vitro Evolution and Catalytic Processes RNA molecules can catalyze bond-forming and bond-breaking reactions Bond-forming Subway Seminar on Darwinian DNA Machines – Christoph Wienken Bond-breaking 9/9/2020 11

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 9/9/2020 12

Summing up… In. Vitro Evolution (and SELEX) needs § Heterogen population as a starting point § Methods to Introduce Mutations § Selection processes § Methods to (Self-)Duplicate In. Vitro 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 9/9/2020 13

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 9/9/2020 14

Evolution becomes continous… Most In. Vitro experiments with ribozymes in a stepwise fashion but Continous Evolution possible! More about „Continous In. Vitro Evolution“ on November, 5 th presented by Christof Mast Subway Seminar on Darwinian DNA Machines – Christoph Wienken 9/9/2020 15

In. Vitro 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 NS 3 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 9/9/2020 16

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 9/9/2020 17