Analysis of in vitro evolution reveals the underlying distribution of catalytic activity among random sequences

The emergence of catalytic RNA is believed to have been a key event during the origin of life. Understanding how catalytic activity is distributed across random sequences is fundamental to estimating the probability that catalytic sequences would emerge. Here, we analyze the in vitro evolution of triphosphorylating ribozymes and translate their fitnesses into absolute estimates of catalytic activity for hundreds of ribozyme families. The analysis efficiently identified highly active ribozymes and estimated catalytic activity with good accuracy. The evolutionary dynamics follow Fisher’s Fundamental Theorem of Natural Selection and a corollary, permitting retrospective inference of the distribution of fitness and activity in the random sequence pool for the first time. The frequency distribution of rate constants appears to be log-normal, with a surprisingly steep dropoff at higher activity, consistent with a mechanism for the emergence of activity as the product of many independent contributions.

[1]  Paul J. Atzberger,et al.  Influence of Target Concentration and Background Binding on In Vitro Selection of Affinity Reagents , 2012, PloS one.

[2]  A. Hartmann,et al.  Minimum-free-energy distribution of RNA secondary structures: Entropic and thermodynamic properties of rare events. , 2008, Physical review. E, Statistical, nonlinear, and soft matter physics.

[3]  N. Lehman,et al.  Evolution in vitro of an RNA enzyme with altered metal dependence , 1993, Nature.

[4]  Jeffrey B.-H. Tok,et al.  Massively Parallel Interrogation of Aptamer Sequence, Structure and Function , 2008, PloS one.

[5]  Marie-Luise Winz,et al.  Next-generation sequencing reveals how RNA catalysts evolve from random space , 2013, Nucleic acids research.

[6]  Thomas Bair,et al.  Rapid Identification of Cell-Specific, Internalizing RNA Aptamers with Bioinformatics Analyses of a Cell-Based Aptamer Selection , 2012, PloS one.

[7]  Phuong Dao,et al.  Large scale analysis of the mutational landscape in HT-SELEX improves aptamer discovery , 2015, Nucleic acids research.

[8]  G. F. Joyce,et al.  The effect of cytidine on the structure and function of an RNA ligase ribozyme. , 2001, RNA.

[9]  J. Szostak,et al.  Progress toward synthetic cells. , 2014, Annual review of biochemistry.

[10]  U. F. Müller,et al.  A ribozyme that triphosphorylates RNA 5′-hydroxyl groups , 2014, Nucleic acids research.

[11]  Eric J. Hayden,et al.  Cryptic genetic variation promotes rapid evolutionary adaptation in an RNA enzyme , 2011, Nature.

[12]  Y. Yokobayashi,et al.  High‐Throughput Mutational Analysis of a Twister Ribozyme , 2016, Angewandte Chemie.

[13]  L. Gustafsson.  Lifetime Reproductive Success and Heritability: Empirical Support for Fisher's Fundamental Theorem , 1986, The American Naturalist.

[14]  H. A. Orr,et al.  A General Extreme Value Theory Model for the Adaptation of DNA Sequences Under Strong Selection and Weak Mutation , 2008, Genetics.

[15]  Gregory W. Campbell,et al.  Computational analysis of fitness landscapes and evolutionary networks from in vitro evolution experiments. , 2016, Methods.

[16]  G. F. Joyce,et al.  Randomization of genes by PCR mutagenesis. , 1992, PCR methods and applications.

[17]  James O Lloyd-Smith,et al.  Adaptation in protein fitness landscapes is facilitated by indirect paths , 2016, bioRxiv.

[18]  L. Mirny,et al.  Diffusion in correlated random potentials, with applications to DNA. , 2004, Physical review. E, Statistical, nonlinear, and soft matter physics.

[19]  L. Cook.  The Genetical Theory of Natural Selection — A Complete Variorum Edition , 2000, Heredity.

[20]  Irene A. Chen,et al.  The RNA World as a Model System to Study the Origin of Life , 2015, Current Biology.

[21]  L. Gold,et al.  Theoretical principles of in vitro selection using combinatorial nucleic acid libraries. , 2000, Current protocols in nucleic acid chemistry.

[22]  Lan Huong Lai,et al.  High-Throughput Measurement of Binding Kinetics by mRNA Display and Next-Generation Sequencing. , 2016, Angewandte Chemie.

[23]  W. Ewens.  The Fundamental Theorem of Natural Selection , 1968 .

[24]  T. Bataillon,et al.  Annals of the New York Academy of Sciences Effects of New Mutations on Fitness: Insights from Models and Data , 2022 .

[25]  Gregory W. Campbell,et al.  Comprehensive experimental fitness landscape and evolutionary network for small RNA , 2013, Proceedings of the National Academy of Sciences.

[26]  Eric J. Hayden,et al.  The Effects of Stabilizing and Directional Selection on Phenotypic and Genotypic Variation in a Population of RNA Enzymes , 2014, Journal of Molecular Evolution.

[27]  I. Chen,et al.  Experimental fitness landscapes to understand the molecular evolution of RNA-based life. , 2014, Current opinion in chemical biology.

[28]  J. Krug,et al.  Empirical fitness landscapes and the predictability of evolution , 2014, Nature Reviews Genetics.

[29]  S. Lessard.  Fisher's fundamental theorem of natural selection revisited. , 1997, Theoretical population biology.

[30]  James M. Carothers,et al.  Informational Complexity and Functional Activity of RNA Structures , 2004, Journal of the American Chemical Society.

[31]  D. Bartel,et al.  Isolation of new ribozymes from a large pool of random sequences [see comment]. , 1993, Science.

[32]  N. Lehman,et al.  Non-unity molecular heritability demonstrated by continuous evolution in vitro. , 1999, Chemistry & biology.

[33]  Joshua D. Knowles,et al.  Analysis of a complete DNA–protein affinity landscape , 2010, Journal of The Royal Society Interface.

[34]  Gerald F. Joyce,et al.  Limits of Neutral Drift: Lessons From the In Vitro Evolution of Two Ribozymes , 2014, Journal of Molecular Evolution.

[35]  L. Mirny,et al.  Kinetics of protein-DNA interaction: facilitated target location in sequence-dependent potential. , 2004, Biophysical journal.

[36]  P. V. von Hippel,et al.  Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters. , 1987, Journal of molecular biology.

[37]  A. Ferré-D’Amaré,et al.  Rapid Construction of Empirical RNA Fitness Landscapes , 2010, Science.

[38]  G. F. Joyce,et al.  Mutagenic PCR. , 2006, CSH protocols.

[39]  Zoltán Konthur,et al.  Probing the SELEX Process with Next-Generation Sequencing , 2011, PloS one.

[40]  P. Schuster,et al.  Statistics of RNA melting kinetics , 2004, European Biophysics Journal.

[41]  J W Szostak,et al.  Structurally complex and highly active RNA ligases derived from random RNA sequences. , 1995, Science.

[42]  Y. Yokobayashi,et al.  High-throughput assay and engineering of self-cleaving ribozymes by sequencing , 2015, Nucleic acids research.