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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

InVITRO selection (SELEX)