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Journal of Molecular Evolution

, Volume 61, Issue 2, pp 192–206 | Cite as

Diverse Evolutionary Trajectories Characterize a Community of RNA-Cleaving Deoxyribozymes: A Case Study into the Population Dynamics of In Vitro Selection

  • Kenny Schlosser
  • Yingfu LiEmail author
Article

Abstract

Two parallel in vitro selections (denoted Selection A and Selection B) were conducted under different selection-pressure regimes, yielding a diverse community of RNA-cleaving deoxyribozymes. In Selection A, the reaction time was reduced four times (from 5 h to 5 s) over the course of 24 generations, while in Selection B the reaction time was maintained at 5 h for 30 rounds of selective amplification. Sequence alignment was conducted on more than 800 clones assembled from 18 generations that span both selections. Many prominent catalytic sequence classes, including some that extend across both selections, were identified and used to construct fitness landscapes depicting their rise and fall over time. The landscapes from both selections exhibit similar global trends despite differences in population dynamics. Some deoxyribozymes were predominant in the early rounds of selection but gave way to other species that dominated in the middle rounds. Ultimately, these middle classes disappeared from the landscape in favor of new and presumably more fit deoxyribozyme sequence classes. The shape of these landscapes alludes to the presence of many latent deoxyribozymes in the initial library, which can only be accessed by changes in the selection pressure and/or by adaptive mutations. Basic computer simulations provide theoretical corroboration of the experimentally observed pattern of staggered sequence-class transitions across the fitness landscapes. These simulations model the influence of one or more contributing factors, including catalytic rate, folding efficiency, PCR amplification efficiency, and random mutagenesis. This is the first study which thoroughly documents the topography of a deoxyribozyme fitness landscape over many generations of in vitro selection.

Keywords

Deoxyribozyme Population dynamics RNA cleavage In vitro selection Sequence diversity Reaction time 

References

  1. Bartel, DP, Szostak, JW 1993Isolation of new ribozymes from a large pool of random sequencesScience26114111418PubMedGoogle Scholar
  2. Beaudry, AA, Joyce, GF 1992Directed evolution of an RNA enzymeScience257635641PubMedGoogle Scholar
  3. Been, MD, Perrotta, AT, Rosenstein, SP 1992Secondary structure of the self-cleaving RNA of hepatitis delta virus: applications to catalytic RNA designBiochemistry311184311852CrossRefPubMedGoogle Scholar
  4. Breaker, RR, Joyce, GF 1994A DNA enzyme that cleaves RNAChem Biol1223229CrossRefPubMedGoogle Scholar
  5. Carrigan, MA, Ricardo, A, Ang, DN, Benner, S A 2004Quantitative analysis of a RNA-cleaving DNA catalyst obtained via in vitro selectionBiochemistry431144611459CrossRefPubMedGoogle Scholar
  6. Charlton, J, Smith, D 1999Estimation of SELEX pool size by measurement of DNA renaturation ratesRNA513261332CrossRefPubMedGoogle Scholar
  7. Ellington, AD, Szostak, JW 1990In vitro selection of RNA molecules that bind specific ligandsNature346818822CrossRefPubMedGoogle Scholar
  8. Gottlieb, PA, Prasad, Y, Smith, JB, Williams, AP, Dinter-Gottlieb, G 1994Evidence that alternate foldings of the hepatitis delta RNA confer varying rates of self-cleavageBiochemistry3328022808CrossRefPubMedGoogle Scholar
  9. Irvine, D, Tuerk, C, Gold, L 1991SELEXION. Systematic evolution of ligands by exponential enrichment with integrated optimization by non-linear analysisJ Mol Biol222739761CrossRefPubMedGoogle Scholar
  10. Lehman, N 2003A case for the extreme antiquity of recombinationJ Mol Evol56770777CrossRefPubMedGoogle Scholar
  11. Lehman, N 2004Assessing the likelihood of recurrence during RNA evolution in vitroArtif Life10122CrossRefPubMedGoogle Scholar
  12. Lehman, N, Joyce, GF 1993aEvolution in vitro of an RNA enzyme with altered metal dependenceNature361182185CrossRefGoogle Scholar
  13. Lehman, N, Joyce, GF 1993bEvolution in vitro: analysis of a lineage of ribozymesCurr Biol3723734CrossRefGoogle Scholar
  14. Lehman, N, Donne, MD, West, M, Dewey, TG 2000The genotypic landscape during in vitro evolution of a catalytic RNA: implications for phenotypic bufferingJ Mol Evol50481490PubMedGoogle Scholar
  15. Meyerhans, A, Vartanian, JP, Wain-Hobson, S 1990DNA recombination during PCRNucleic Acids Res1816871691PubMedGoogle Scholar
  16. Odelberg, SJ, Weiss, RB, Hata, A, White, R 1995Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase INucleic Acids Res2320492057PubMedGoogle Scholar
  17. Robertson, DL, Joyce, GF 1990Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNANature344467468Google Scholar
  18. Santoro, SW, Joyce, GF 1997A general purpose RNA-cleaving DNA enzymeProc Natl Acad Sci USA9442624266CrossRefPubMedGoogle Scholar
  19. Schlosser, K, Li, Y 2004Tracing sequence diversity change of RNA-cleaving deoxyribozymes under increasing selection pressure during in vitro selectionBiochemistry4396959707CrossRefPubMedGoogle Scholar
  20. Schmitt, T, Lehman, N 1999Non-unity molecular heritability demonstrated by continuous evolution in vitroChem Biol6857869CrossRefPubMedGoogle Scholar
  21. Sun, F, Galas, D, Waterman, MS 1996A mathematical analysis of in vitro molecular selection–amplificationJ Mol Biol258650660CrossRefPubMedGoogle Scholar
  22. Tuerk, C, Gold, L 1990Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymeraseScience249505510PubMedGoogle Scholar
  23. Uhlenbeck, OC 1995Keeping RNA happyRNA146PubMedGoogle Scholar
  24. Vant-Hull, B, Payano-Baez, A, Davis, RH, Gold, L 1998The mathematics of SELEX against complex targetsJ Mol Biol278579597CrossRefPubMedGoogle Scholar
  25. Wright, S 1932The roles of mutation, inbreeding, cross-breeding, and selection in evolutionProc 6th Int Congr Genet1356366Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  1. 1.Department of Biochemistry and Biomedical Sciences and Department of ChemistryMcMaster UniversityHamiltonCanada

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