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

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By davide de lucrezia

THE WORLD OF THE

“NEVER BORN RNAs”

by Davide De Lucrezia


By davide de lucrezia

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


By davide de lucrezia

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


By davide de lucrezia

A striking discrepancy...

space of sequences related to extant proteins≈ 1010

radius of the

universe

Theoretical space of sequences

130

10

100

(20

)


By davide de lucrezia

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).


Il progetto never born biopolymers

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.


By davide de lucrezia

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


Investigation of the folding properties of random rna the methodology

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


By davide de lucrezia

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


Rna foster rna folding stability test

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.


Analisi distr

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


Analisi tvsgc

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


Concl prosp rnaprperties

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


By davide de lucrezia

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


By davide de lucrezia

Selection

Investigation of the functional properties of random RNA: the experiments


By davide de lucrezia

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.


By davide de lucrezia

Selection

RNA Foster

Investigation of the functional properties of random RNA: the experiments


By davide de lucrezia

Investigation of the functional properties of random RNA: the experiments

Recovery & Amplification

Improved efficiency

Selezione

Mutagenesis


By davide de lucrezia

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


By davide de lucrezia

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…)


Essential bibliografy

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


Ctrlexp idro

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


Ctrlexp enz

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


Ctrlexp dim1

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


Ctrlexp dim2

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


By davide de lucrezia

RNA World


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