By Davide De Lucrezia THE WORLD OF THE “NEVER BORN RNAs”
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Transcript of 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:
Recovery & Amplification
Selection
Joyce G.F. Directed evolution of nucleic acids. Annu Rev Biochem. 2004.
5 or more cycles
Investigation of the functional properties of random RNA: the experiments
MutagenesisSelezione
Recovery & Amplification
Improved efficiency
Investigation of the functional properties of random RNA: the experiments
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
RNA Foster – esperimenti di controllo
•Idrolisi non enzimatica dell’RNA
•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
RNA Foster – esperimenti di controllo
•Idrolisi non enzimatica dell’RNA
•Formazione di dimeri
Random Coiled RNA
(RNAu)
Folded RNA
(RNAf)
Dimero RNA
(RNAd)
•Aumento dell’attività enzimatica
Ctrlexp-dim1
RNA Foster – esperimenti di controllo
•Idrolisi non enzimatica dell’RNA
•Formazione di dimeri
•Aumento dell’attività enzimatica
2
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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