Almost like this!

1. New ligase-derived RNA polymerase ribozymes M. S. Lawrence and D. P. Bartel RNA (2005), 11:1173-1180 Almost like this! Almost like this!

2. An ’RNA World’ Univ. of Oregon, www.uoregon.edu/...

3. An ’RNA World’ ”Primordial Sea” (Wikimedia Commons)

4. An ’RNA World’ Red Sea ”Primordial Sea” ”[…] the alternative scenario in which oligonucleotides and code protein synthesis emerged together in miraculous concert.” (p.1173)

5. Historical background • 1967: Woese suggests that primordial organisms may have been based on RNA • 1982: Cech et al. finds a self-splicing intron (a ribozyme) • 1986: Gilbert introduces the term ’RNA World Hypothesis’ • 1989-92: Szostak/Doudna develops derivatives of self-splicing introns, but with limited succes • 1992 og forth: Noller et al. demonstrates that rRNA is responsible for the peptidyl transferase activity • 1993: Szostak/Ekland develops RNA-based ligases • 2001: Bartel, Lawrence and others develop • ribozymatic polymerases Pol 1 and Evolved Pol 1

6. Design of the initial library Szostaks/Ekland (1993): • the Class I RNA ligase is almost as • fast as an enzyme (kcat ≈ 1 s-1), • - and it will do several turnovers • - but it requires that the template is base-paired onto it, i.e. it is not generic

7. Design of the initial library Szostaks/Ekland (1993): Lawrence/Bartel (2001): Extending the ligase domain with an auxiliary domain

8. Selection method Goal: Select polymerases that incorporate rNTPs onto their ”template end” Method: 1. Tagged UTP: 4-thio-UTP 2. Retard molecules with 4-thio-UTP on ”mercury gels” (acrylamide gel w/urea+N-acryloyl-aminophenylmercuric acetate) 3. Excise and amplify 4-thio-UTP [Science 2001, 292:1319]

9. Selection method Goal: Select polymerases that incorporate rNTPs onto their ”template end” Method: 1. Tagged UTP: 4-thio-UTP 2. Retard molecules with 4-thio-UTP on ”mercury gels” (acrylamide gel w/urea+N-acryloyl-aminophenylmercuric acetate) 3. Excise and amplify [Science 2001, 292:1319]

10. Selection method Goal: Select polymerases that incorporate rNTPs onto their ”template end” Method: 1. Tagged UTP: 4-thio-UTP 2. Retard molecules with 4-thio-UTP on ”mercury gels” (acrylamide gel w/urea+N-acryloyl-aminophenylmercuric acetate) 3. Excise and amplify • Elongation w/tagged rNTPs (vars: t, • [rNTP], tag, turnovers) • Purification (APM-PAGE 1-2× or • using biotin/streptavidin) • RT-PCR (error prone) • In vitro transcription of cDNA [Science 2001, 292:1319] Cloning and sequencing

11. Original selection (2003) After 8 rounds, a number of distinct types (”families”) begin to emerge: Fig. 2

12. Original selection (2003) After 10 rounds, members of the Pol 1 family dominate: Fig. 2

13. Original selection (2003) Pol 1 optimised to polymerise at up to 14 nt, i.e. beyond one hel. turn! Fig. 2

14. Revising the selection criteria • Lessons learned from that study: • While 4SUTP worked fine, biotin-ATP did not • Mercury gels allow for selection of incorporation of two4SUs • 2-aminopurine in the template helps pairing with 4SU • Moreover: • - competitors (A, C, G) • - lower [4SUTP] • - try various primer-templates

15. New selection (2005) Re-examining an earlier pool for alternative polymerases, now using the new selection criteria: Pol 1 re-emerges, but is then overtaken! Fig. 2

16. New selection (2005) Re-examining an earlier pool for alternative polymerases, now using the new selection criteria: Pol 1 re-emerges, but is then overtaken! Pol 2 is the new Pol 1! 33 new families! Fig. 2

17. Fig. 3 Examining the new polymerases Polymerases are named according to their efficacy Primer only + Ligase core (no aux.) Fig. 4

18. Fig. 3 Examining the new polymerases Polymerases are named according to their efficacy Strong stem structuresappear to be a requirement; computational folding finds that these folds are among the 70% most stable within each sequence space. Conclusion: The ligase core can be extended with an auxiliary domain and become generic!

19. Final notes ”Parasites” are a problem during selection:

20. Final notes • ”Parasites” are a problem during selection • As much as 6 nt extension was achieved with non-optimised RNA polymerases. Authors suggest further experiments: • Optimisation (cf. Evolved Pol 1) • A selection ”battle” between select polymerases • Recombination during selection

21. Final notes ”Parasites” are a problem during selection As much as 6 nt extension was achieved with non-optimised RNA polymerases RNA polymerases are limited by their own degradation: ”The polymerization reaction uses micromolar concentrations of RNA, but [Pol 1] binds its PT substrate with only millimolar affinity” (p. 1178) ”As the ribozyme slowly polymerizes NTPs, it suffers the constant hydrolytic onslaught of the [200 mM Mg2+, pH 8.5] reaction buffer” (p.1178) (Data not shown.)

22. Final notes ”Parasites” are a problem during selection As much as 6 nt extension was achieved with non-optimised RNA polymerases RNA polymerases are limited by their own degradation - Bartel’s current work is focused on improving substrate interactions using micelles. (Wikimedia Commons)

23. Final notes ”Parasites” are a problem during selection As much as 6 nt extension was achieved with non-optimised RNA polymerases RNA polymerases are limited by their own degradation - Bartel’s current work is focused on improving substrate interactions using micelles. RNase A = ”The End” RNase A means ”The End”