By Davide De Lucrezia THE WORLD OF THE “NEVER BORN RNAs”

by Davide De Lucrezia

THE WORLD OF THE“NEVER BORN RNAs”

4,5bya Earth formation

4 bya End of meteorite

s Bombardment

3,72 bya First fossil

Abiotic synthesis of organic compoundsAbiotic synthesis of biopolymers

First probionts

The origin of life: timeline and main features

Main features of abiotically synthetised biopolymers:

• Thermodynamic driven synthesis

• Combinatorial synthesis

N = m l

where m is number of monomers available and l lenght of the polymers

Combinatorial synthesis of polymers: some calculations...

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

It’s unlikely that Nature explored exhaustively the whole space of sequences

Extant proteins are only an infinitesimal fraction of the theoretically possible ones

radius of the universe

Theoretical space of sequences

10130

(20100)

space of sequences related to extant proteins≈ 1010

A striking discrepancy...

There may be an entire universe of “Never Born Proteins” (NBP), whose properties have never been sampled by Nature

Contingency theory:

extant proteins are the result of the simultaneous interplay of several concomitant causes (Gould, 1994).

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).

How can one explain the striking discrepancy between the theoretical number of sequences and the actual one?

Natural proteinsPossible-protein space

The contingency vs. determinism debate: an old issue….

Il progetto „Never Born Biopolymers“

[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.

“Never Born Proteins”

Studio delle proprietà strutturali di peptidi con sequenza casuale [1]

Spazio delle proteine possibili

Spazio delle proteine naturali

“Never Born RNAs”

Studio delle proprietà strutturali dei corrispondenti RNA a sequenza casuale

Spazio degli RNA possibili

Spazio degli RNA naturali

RNA Sequence space

Which is the fraction of folded RNA?

Folded RNA

Which is the fraction of functional?

Functional RNA

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

S1 mapping

Probing secondary domain by means of single-strand specific RNase S1

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

Susceptibility to S1

Temperature

PlasmidLibrary

E.coli

2. Tranformation

Investigation of the folding properties of random RNA: the experiments

DNA Library

Vector

E.coliE.coli

3. Plating on selective medium

1. Cloning

4. Single colony isolation and plasmid purification

37°C

– S

1-

60°C

– S

1-

37°C

– 1

h

37°C

45°C

50°C

55°C

60°C

RNA Foster (RNA Folding Stability Test)

37°C 45°C 50°C 55°C 60°CNo

rmal

ised

In

ten

sit

y

0

0.5

1

Temperature

RNA

37°C - S1

37°C + S1

45°C + S1

50°C + S1

55°C + S1

60°C + S1

60°C - S1

Ab

s/A

bs°

Abs°

Temperature

60°C

55°C

50°C

45°C

37°C

Matzura O. and Wennborg A. RNAdraw: an integrated program for RNA secondary structure calculation and analysis. Computer Applications in the Biosciences. 1996.

Results: folding and thermal stability

Analisi-Distr

0

2

4

6

8

10

12

37°C 45°C 50°C 55°C 60°C

TemperaturaRNA S1-resistenti RNA S1-sensibili

All RNAs show a stable and compact secondary structure at 37°CThe average Tm is within 45°C – 50°CA “thermostable” RNA was found with a thermal stability over 60°C

Nu

mero

di

clo

ni

Analisi-TvsGC

Tm

RNA 32

RNA 59

RNA 58

RNA 48

RNA 33

RNA 69

RNA 45

RNA 50

RNA 57

RNA 35

RNA 63

RNA 64

% G

C

45

50

55

60

65

37°C-45°C 45°C-50°C 50°C-55°C 55°C-60°C Over 60°C

There is no correlation between GC content and thermal stability

Results: thermal stability and GC content

Concl&Prosp:RNAprp

erties

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

Conclusions

Investigation of the functional properties of random RNA

In order to evaluate the fraction of functional RNAs in RNA sequence space, we plan to:

•Ligation•Phospodiester bond cleavage•Protease activity•Esterase activity

1. Synthetise a DNA library codifing for 60 nt. Long random RNA

2. Screen the RNA library for catalytic activities, such as:

Selection

Investigation of the functional properties of random RNA: the experiments

Recovery & Amplification

Selection

Joyce G.F. Directed evolution of nucleic acids. Annu Rev Biochem. 2004.

5 or more cycles


RNA FosterSelection


MutagenesisSelezione

Recovery & Amplification

Improved efficiency


Investigation of the functional properties of random RNA: the selection criteria

2. Wash unbound RNAs

3. Recover bound RNAs

TSA TSA TSA TSA

1. Biopanning

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

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)

Essential Bibliografy

(continues…)

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

RNA Foster – esperimenti di controllo

•Idrolisi non enzimatica dell’RNA

Ctrlexp-idro

p33 RNA

(178 nt)

37°C

– S

1-

60°C

– S

1-

37°C

– 1

h

37°C

45°C

50°C

55°C

60°C



•Aumento dell’attività enzimatica

0

0,5

1

Inte

nsi

tà n

orm

aliz

zata

37°C 45°C 50°C 55°C 60°C

Temperatura

Ctrlexp-enz

37°C

45°C

50°C

55°C

60°C



•Formazione di dimeri

Random Coiled RNA

(RNAu)

Folded RNA

(RNAf)

Dimero RNA

(RNAd)


Ctrlexp-dim1



•Formazione di dimeri


2

2

][][

][][

][2][][

udd

uff

dufu

RNAkdt

RNAd

RNAkdt

RNAd

RNAukRNAkdt

RNAd

0,0

0,1

0,20,3

0,4

0,5

0,6

0,70,8

0,9

1,0

0 100 200 300 400 500 600

time (S)

Mo

lar

Fra

ctio

n

[RNAu] [RNAf] [RNAd]

Draper DE. Strategies for RNA folding. Trends Biochem Sci. 1996

RNA World

Documents

By Davide De Lucrezia THE WORLD OF THE “NEVER BORN RNAs”