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  1. THE WORLD OF THE “NEVER BORN RNAs” by Davide De Lucrezia

  2. The origin of life: timeline and main features First probionts Abiotic synthesis of biopolymers Abiotic synthesis of organic compounds • Main features of abiotically synthetised biopolymers: • Thermodynamic driven synthesis • Combinatorial synthesis N = m l where m is number of monomers available and llenght of the polymers 4,5bya Earth formation 4 bya End of meteorites Bombardment 3,72 bya First fossil

  3. Combinatorial synthesis of polymers: some calculations... It’s unlikely that Nature explored exhaustively the whole space of sequences Extant proteins are only an infinitesimal fraction of the theoretically possible ones Given the 20 natural amino acids and assuming an average length of 50 residues, there are 2050 ≈ 1065 theoretically different sequences Given the 4 natural nucleotides and assuming an average length of 150 residues, there are 4150 ≈ 1090 theoretically different sequences

  4. A striking discrepancy... space of sequences related to extant proteins≈ 1010 radius of the universe Theoretical space of sequences 130 10 100 (20 )

  5. There may be an entire universe of “Never Born Proteins” (NBP), whose properties have never been sampled by Nature The contingency vs. determinism debate: an old issue…. How can one explain the striking discrepancy between the theoretical number of sequences and the actual one? Possible-protein space Natural proteins Determinist theory: The life constituents are the result of an evolutive fine work; what we see is the better possible solution for the biological needs (de Duve, 1995). Contingency theory: extant proteins are the result of the simultaneous interplay of several concomitant causes(Gould, 1994).

  6. Il progetto „Never Born Biopolymers“ Spazio degli RNA possibili “Never Born RNAs” Studio delle proprietà strutturali dei corrispondenti RNA a sequenza casuale Spazio degli RNA naturali Spazio delle proteine possibili “Never Born Proteins” Studio delle proprietà strutturali di peptidi con sequenza casuale [1] Spazio delle proteine naturali [1] Vrijbloed JW.; Chiarabelli C.; De Lucrezia D.; Thomas RM.; Luisi PL.On the Frequency of folded polypeptides in a random sequence production.COST D27. 2002.

  7. Which is the fraction of folded RNA? Folded RNA Which is the fraction of functional? Functional RNA RNA Sequence space Some basic features of biopolymers.... Biopolymers exert their biological function due to their tri-dimensional structures

  8. Investigation of the folding properties of random RNA: the methodology Susceptibility to S1 Temperature RNA Folding Stability Test (RNA Foster) Probing secondary domain and their stability by means of single-strand specific RNase S1 at different temperatures Susceptibility to S1 S1 mapping Probing secondary domain by means of single-strand specific RNase S1

  9. PlasmidLibrary Investigation of the folding properties of random RNA: the experiments Vector DNA Library 1. Cloning 2. Tranformation E.coli E.coli E.coli 3. Plating on selective medium 4. Single colony isolation and plasmid purification

  10. RNA Foster (RNA Folding Stability Test) 37°C – S1- 37°C – 1h 37°C 45°C 50°C 55°C 60°C 60°C – S1- RNA 55°C 45°C 37°C - S1 37°C + S1 45°C + S1 50°C + S1 55°C + S1 60°C + S1 60°C - S1 37°C 60°C 50°C 1 Normalised Intensity 0.5 Abs° Abs/Abs° Temperature 0 37°C 45°C 50°C 55°C 60°C Temperature Matzura O. and Wennborg A. RNAdraw: an integrated program for RNA secondary structure calculation and analysis. Computer Applications in the Biosciences. 1996.

  11. Analisi-Distr 12 10 8 6 4 2 0 37°C 45°C 50°C 55°C 60°C Temperatura RNA S1-resistenti RNA S1-sensibili • All RNAs show a stable and compact secondary structure at 37°C • The average Tm is within 45°C – 50°C • A “thermostable” RNA was found with a thermal stability over 60°C Results: folding and thermal stability Numero di cloni

  12. Analisi-TvsGC Results: thermal stability and GC content 65 RNA 59 60 RNA 48 RNA 50 RNA 33 RNA 58 % GC 55 RNA 57 RNA 69 RNA 64 RNA 63 50 RNA 35 RNA 32 RNA 45 45 37°C-45°C 45°C-50°C 50°C-55°C 55°C-60°C Over 60°C Tm • There is no correlation between GC content and thermal stability

  13. Concl&Prosp:RNAprperties Conclusions • The preliminary investigation of the structural properties of Random RNA shows that: • RNAs have an intrinsic properties to fold into stable secondary structures • RNAs have a surprising thermal stability with an average Tm within 45°C – 50°C • The thermal stability is not directly correlated with the GC content • Thermo stable structures seem to be common in RNA sequences space

  14. Investigation of the functional properties of random RNA In order to evaluate the fraction of functional RNAs in RNA sequence space, we plan to: 1. Synthetise a DNA library codifing for 60 nt. Long random RNA 2. Screen the RNA library for catalytic activities, such as: • Ligation • Phospodiester bond cleavage • Protease activity • Esterase activity

  15. Selection Investigation of the functional properties of random RNA: the experiments

  16. Recovery & Amplification Selection Investigation of the functional properties of random RNA: the experiments 5 or more cycles Joyce G.F.Directed evolution of nucleic acids. Annu Rev Biochem. 2004.

  17. Selection RNA Foster Investigation of the functional properties of random RNA: the experiments

  18. Investigation of the functional properties of random RNA: the experiments Recovery & Amplification Improved efficiency Selezione Mutagenesis

  19. 3. Recover bound RNAs 1. Biopanning TSA TSA TSA TSA 2. Wash unbound RNAs TSA: Transition state analogue The TSA is a organic compound that ressembles the transition state of a chemical reaction. Potential catalysts bind the TSA but are unaable to cleave it. Consequently, they are retained onto the matrix surface Investigation of the functional properties of random RNA: the selection criteria

  20. Essential Bibliografy The discovery of catalytic RNAs and their physiological roles introduce a new level of control in gene expression • Introns transposition and gene inactivation (Lambowitz et al. 1993) • Splicing alteration and proteins defects (Vader et al. 2002; Decatur et al. 2002) • Plant pathology (Smith et al. 1992; Wilson, 1993) The discovery that RNA is capable of both information storage and catalysis, suggested its implication for the origin of life • The chicken and the egg dilemma (The RNA world. Edited by Gesteland and Atkins. 1993; Schwartz, 1995; Joyce, 2002, Lazcano and Miller, 2003) • Eigen’s Hypercycle (Eigen and Schuster, 1978, Cronhjort, 1995; Szathmary, 2002) (continues…)

  21. Essential Bibliografy For an historical approach to Ribozyme • Kruger, Cech et al. Self-splicing RNA. Cell, 1982 • Gurrier, Altaman et al. The RNA moiety of ribonuclease P. Cell, 1983 Mehanisms and Structures details • Cech, TR. Self-splicing of group I introns. Ann Rev. Biochem., 1990 • Scott et al.Ribozymes:structure and mechanism in RNA catalysis.TrendsBioch,1996 Theoretical implications • Roman et al. Group I reverse self-splicing in vivo. PNAS, 1998. • Matsuura et al. Encoding introns. Genes Dev., 1997 Biotechnological implications • Marshall et al.Inhibition of gene expression with ribozymes.Cell.Mol.Neur,1994 • Kijima et al. Therapeutic applications of ribozymes. Pharmacol.Ther., 1995 • Sullenger et al. Rybozime trans-splicing. Nature, 1994 Reviews • Tanner NK. Rybozymes. FEMS Micr. Reviews, 1999

  22. Ctrlexp-idro 37°C – S1- 37°C – 1h 37°C 45°C 50°C 55°C 60°C 60°C – S1- p33 RNA (178 nt) RNA Foster – esperimenti di controllo • Idrolisi non enzimatica dell’RNA

  23. Ctrlexp-enz 1 Intensità normalizzata 0,5 0 37°C 45°C 50°C 55°C 60°C Temperatura RNA Foster – esperimenti di controllo 37°C 45°C 50°C 55°C 60°C • Idrolisi non enzimatica dell’RNA • Aumento dell’attività enzimatica

  24. Ctrlexp-dim1 Random Coiled RNA (RNAu) Dimero RNA (RNAd) Folded RNA (RNAf) RNA Foster – esperimenti di controllo • Idrolisi non enzimatica dell’RNA • Aumento dell’attività enzimatica • Formazione di dimeri

  25. Ctrlexp-dim2 RNA Foster – esperimenti di controllo • Idrolisi non enzimatica dell’RNA • Aumento dell’attività enzimatica • Formazione di dimeri • Draper DE.Strategies for RNA folding. Trends Biochem Sci. 1996

  26. RNA World