The Robustness of Naturally and Artificially Selected Nucleic Acid Secondary Structures
Thermodynamic stability and mutational robustness of secondary structure are critical to the function and evolutionary longevity of RNA molecules. We hypothesize that natural and artificial selection for functional molecules favors the formation of structures that are stable to both thermal and mutational perturbation. There is little direct evidence, however, that functional RNA molecules have been selected for their stability. Here we use thermodynamic secondary structure prediction algorithms to compare the thermal and mutational robustness of over 1000 naturally and artificially evolved molecules. Although we find evidence for the evolution of both types of stability in both sets of molecules, the naturally evolved functional RNA molecules were significantly more stable than those selected in vitro, and artificially evolved catalysts (ribozymes) were more stable than artificially evolved binding species (aptamers). The thermostability of RNA molecules bred in the laboratory is probably not constrained by a lack of suitable variation in the sequence pool but, rather, by intrinsic biases in the selection process.
KeywordsRNA Secondary structure Thermostability Mutational robustness Aptamer Ribozyme In vitro selection
The authors thank Walter Fontana and Rob Knight for technical advice and Kourosh Salehi-Ashtiani and Jack Szostak at Mass General Hospital for providing selected ribozyme sequences. This work was supported in part by the Santa Fe Institute and grants from the NSF (Grant DEB-0303636) to L.A.M., grants from the NSF (Grant EIA-0218447) and the NIH–NIBIB (Grant 8R01EB002043) to A.D.E., and NSF-IGERT fellowships in computational phylogenetics to J.F.L and M.C.
Ancel, LW, Fontana, W 2000Plasticity, evolvability and modularity in RNAJ Exp Zool (Mol Dev Evol)288242283CrossRefGoogle Scholar Bevilacqua, JM, Bevilacqua, PC 1998Thermodynamic analysis of an RNA combinatorial library contained in a short hairpinBiochemistry371587715884CrossRefPubMedGoogle Scholar Bornberg-Bauer, E, Chan, HS 1999Modeling evolutionary landscapes: Mutational stability, topology, and superfunnels in sequence spaceProc Natl Acad Sci USA961068910694CrossRefPubMedGoogle Scholar Bussemaker, HJ, Thirumalai, D, Bhattacharjee, JK 1997Thermodynamic stability of folded proteins against mutationsPhys Rev Lett7935303533CrossRefGoogle Scholar Calcagnile, A, Basic-Zaninovic, T, Plombo, F, Dogliotti, E 1996Misincorporation rate and type on the leading and lagging strands of UV-damaged DNANucleic Acids Res1530053009CrossRefGoogle Scholar Claesson, C, Samuelsson, T, Lustig, F, Boren, T 1990Codon reading properties of an unmodified transfer RNAFEBS Lett273173176CrossRefPubMedGoogle Scholar Cox, EC, Yanofsky, C 1967Altered base ratios in the DNA of an Escherichia coli mutator strainProc Natl Acad Sci USA5818951902PubMedGoogle Scholar Gilbert, W 1986The RNA worldNature.319618Google Scholar 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 Griffiths-Jones, S, Bateman, A, Marshall, M, Khanna, A, Eddy, SR 2003Rfam: An RNA family databaseNucleic Acids Res31439441CrossRefPubMedGoogle Scholar Guo, F, Cech, TR 2002Evolution of Tetrahymena ribozyme mutants with increased structural stabilityNat Struct Biol9855861PubMedGoogle Scholar Harrington, KM, Nazarenko, IA, Dix, DB, Thompson, RC, Uhlenbeck, OC 1993In vitro analysis of translational rate and accuracy with an unmodified tRNABiochemistry3276177622CrossRefPubMedGoogle Scholar Hofacker, IL, Fontana, W, Stadler, PF, Bonhoeffer, LS, Tacker, M, Schuster, P 1994Fast folding and comparison of RNA secondary structuresMonatsh Chem125167199CrossRefGoogle Scholar James, KD, Ellington, AD 1999The fidelity of template-directed oligonucleotide ligation and the inevitability of polymerase functionOrig Life Evol Biosph29375390PubMedGoogle Scholar Lai, EC 2003RNA sensors and riboswitches: Self-regulating messagesCurr Biol13R285R291PubMedGoogle Scholar Lambert, J, Moran, N 1998Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteriaProc Natl Acad Sci USA9544584462PubMedGoogle Scholar Levy, M, Ellington, AD 2001The descent of polymerizationNat Struct Biol8580582PubMedGoogle Scholar Lowe, TM, Eddy, SR 1997tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequenceNucleic Acids Res25955964PubMedGoogle Scholar Lynch, M 1996Mutation accumulation in transfer RNAs: molecular evidence for Muller’s ratchet in mitochondrial genomes Mol Biol Evol13209202PubMedGoogle Scholar Lynch, M 1997Mutation accumulation in nuclear, organelle, and prokaryotic transfer RNA genesMol Biol Evol14914925PubMedGoogle Scholar Mathews, DH, Sabina, J, Zucker, M, Turner, M 1999Expanded sequence dependence of thermodynamic parameters provides robust prediction of RNA secondary structureJ Mol Biol288911940PubMedGoogle Scholar McCaskill, JS 1990The equilibrium partition function and base pair binding probabilities for RNA secondary structureBiopolymers2911051119PubMedGoogle Scholar Moody, EM, Bevilacqua, PC 2003Thermodynamic coupling of the loop and stem in unusually stable DNA hairpins closed by CG base pairsJ Am Chem Soc12520322033PubMedGoogle Scholar Nakano, M, Moody, EM, Liang, J, Bevilacqua, PC 2002Selection for thermodynamically stable DNA tetraloops using temperature gradient gel electrophoresis reveals four motifs: d(cGNNAg), d(cGNABg), d(cCNNGg), and d(gCNNGc)Biochemistry411428114292PubMedGoogle Scholar Pestova, TV, Hellen, CU 2001Preparation and activity of synthetic unmodified mammalian tRNAi(Met) in initiation of translation in vitroRNA714961505PubMedGoogle Scholar Proctor, DJ, Schaak, JE, Bevilacqua, JM, Falzone, CJ, Bevilacqua, PC 2002Isolation and characterization of a family of stable RNA tetraloops with the motif YNMG that participate in tertiary interactionsBiochemistry411206212075PubMedGoogle Scholar Repsilber, D, Wiese, S, Rachen, M, Schroder, AW, Riesner, D, Steger, G 1999Formation of metastable RNA structures by sequential folding during transcription: time-resolved structural analysis of potato spindle tuber viroid (−)-stranded RNA by temperature-gradient gel electrophoresisRNA5574584PubMedGoogle Scholar Rocha, E, Danchin, A 2002Base composition bias might result from competition for metabolic resourcesTrends Genet18291294PubMedGoogle Scholar Salehi-Ashtiani, K, Szostak, JW 2001In vitro evolution suggests multiple origins for the hammerhead ribozymeNature4148284PubMedGoogle Scholar Sampson, JR, Uhlenbeck, OC 1988Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitroProc Natl Acad Sci USA8510331037PubMedGoogle Scholar Schmitt, T, Lehman, N 1999Non-unity molecular heritability demonstrated by continuous evolution in vitroChem Biol6857869PubMedGoogle Scholar Schultes, EA, Bartel, DP 2000One sequence, two ribozymes: Implications for the emergence of new ribozyme foldsScience289448452PubMedGoogle Scholar Shu, Z, Bevilacqua, PC 1999Isolation and characterization of thermodynamically stable and unstable RNA hairpins from a triloop combinatorial libraryBiochemistry381536915379PubMedGoogle Scholar Smith, JB, Gottlieb, PA, Dinter-Gottlieb, G 1992A sequence element necessary for self-cleavage of the antigenomic hepatitis delta RNA in 20 M formamideBiochemistry3196299635PubMedGoogle Scholar Szymanski, M, Barciszewska, MZ, Barciszewski, J, Erdmann, VA 20005S ribosomal RNA database Y2KNucleic Acids Res28166167PubMedGoogle Scholar Takai, K, Takaku, H, Yokoyama, S 1996Codon-reading specificity of an unmodified form of Escherichia coli tRNA1Ser in cell-free protein synthesisNucleic Acids Res2428942899PubMedGoogle Scholar van Nimwegen, E, Crutchfield, JP, Huynen, MA 1999Neutral evoltuion of mutational robustnessProc Natl Acad Sci USA9697169720PubMedGoogle Scholar Vendruscolo, M, Maritan, A, Banavar, JR 1997Stability threshold as a selection principle for protein designPhys Rev Lett7839673970Google Scholar Wagner, GP, Booth, G, Bagheri-Chaichian, H 1997A population genetic theory of canalizationEvolution51329347Google Scholar Walter, A, Turner, D, Kim, J, Lyttle, M, Müller, P, Mathews, D, Zuker, M 1994Coaxial stacking of helices enhances binding of oligoribonucleotidesProc Natl Acad Sci USA9192189222PubMedGoogle Scholar Wuchty, S, Fontana, W, Hofacker, IL, Schuster, P 1999Complete suboptimal folding of RNA and the stability of secondary structuresBiopolymers49145165PubMedGoogle Scholar