Part I: Ribozymes

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Prof. F.Allain 2/2/2004
Part I Ribozymes
Part II SELEX (RNA in-vitro evolution)
Part III RNAi RNA inhibition and silencing
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The RNA world hypothesis
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Part I Ribozymes
A brief History
How many ribozyme ? Why ?
Catalytic efficiency, condition
3D structure of ribozyme And a mechanism of
catalysis
Biological application ?
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A brief History
1982Self-splicing in Tetrahymena pre-rRNA
(group I intron) Kruger et al, and Cech, Cell
31, 147-157 (1982)
1983RNAse P is a ribozyme Guerrier-Takada et
al, and Altman, Cell, 35, 849-857 (1983)
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How many ribozyme ? Why ?
- the hammerhead ribozyme (plant virus)
- the hairpin ribozyme (plan virus)
- hepatitis delta ribozyme (human virus)
- neurospora VS ribozyme (mitochondrial RNA)
- group I and group II intron ribozyme (rRNA and
mt RNA)
- RNAse P (tRNA maturation)
- Ribosome (translation)
- Spliceosome ?? (splicing)
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One main reaction Nucleolytic cleavage Transester
ification (SN2)
Hammerhead Haipin Hepatitis delta VS ribozyme
From Lilley TIBS (2003)
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The hammerhead ribozyme (plant virus)
- discovered in small RNA satellites of small
viruses (1986)
- replication by rolling circle mechanism
Secondary structure
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The hammerhead ribozyme (plant virus)
- tertiary structure
Scott et al and Klug, Science 1996
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The hairpin ribozyme (plant virus)
From Lilley TIBS (2003)
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The hepatitis delta ribozyme (human virus)
From Lilley TIBS (2003)
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Group I II intron ribozyme (rRNA and mt RNA)
Doudna and Cech Nature, 2002
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Group I intron ribozyme (rRNA and mt RNA)
Golden et al, and cech Science (1998)
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Catalytic efficiency, condition
- ribozyme follows a Michaelis-Menten kinetics
k1
k2
E S
ES
E P
k-1
k-1 k2
Km
kcat 0.5-2 min-1
10-5-10-7 M
k1
kcat/ Km 103-106 M-1.min-1
Good catalytic efficiency!!
- all ribozyme need cations for activity (Mg2
,Mn2)
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3D structure of ribozyme mechanism of catalysis
hairpin ribozyme
Hepatitis delta ribozyme
Ferre dAmare, Nature 1998
Ruppert et al, Nature 2001, Science 2002
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How to catalyse the reaction ?
From Lilley TIBS (2003)
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Structure of the hairpin ribozyme
hairpin ribozyme
Ruppert et al, Nature 2001, Science 2002
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hairpin ribozyme
Transition state
Ground state
Ruppert et al, Nature 2001
Ruppert et al, Science 2002
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hairpin ribozyme
free
bound
free
bound
Loop A
Loop B
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hairpin ribozyme
Transition state
Ruppert et al, Science 2002
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Acid-Base catalysis ? (textbook Voet and Voet)
like with RNAse A
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Acid-Base catalysis ?
G8 as a base
A38 as an acid
Bevilacqua, Biochemistry 2003
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Hepatitis delta ribozyme
Ferre dAmare, Nature 1998
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Biological application ?
Tentative of gene therapy with the hairpin and
the hammerhead ribozyme against viral RNA for
example.
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Reference
Reviews Lilley TIBS (2003) De Rose Chem
Biol (2002) Ferre dAmare Biopolymer
(2003) Article Kruger et al, and Cech, Cell
(1982) Guerrier-Takada et al, and Altman, Cell
(1983) Scott et al Nature (1995) Science
(1996) Rupert et al Nature (2001), Science
(2002)
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Part II SELEX
A brief History
The method ?
A few examples.
Biological application ?
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SELEX Systematic Evolution of Ligands by
EXponential enrichment
A brief History Ellington and Szostak, Nature
(1990) Tuerk and Gold , Science (1990)
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In vitro selection of RNA molecules that bind
specific ligands Andrew D. Ellington  Jack
W. Szostak Subpopulations of RNA molecules that
bind specifically to a variety of organic dyes
have been isolated from a population of random
sequence RNA molecules. Roughly one in 1010
random sequence RNA molecules folds in such a way
as to create a specific binding site for small
ligands. Systematic evolution of ligands by
exponential enrichment RNA ligands to
bacteriophage T4 DNA polymerase.Tuerk C, Gold
L.High-affinity nucleic acid ligands for a
protein were isolated by a procedure that depends
on alternate cycles of ligand selection from
pools of variant sequences and amplification of
the bound species. Multiple rounds exponentially
enrich the population for the highest affinity
species that can be clonally isolated and
characterized. In particular one eight-base
region of an RNA that interacts with the T4 DNA
polymerase was chosen and randomized. Two
different sequences were selected by this
procedure from the calculated pool of 65,536
species. One is the wild-type sequence found in
the bacteriophage mRNA one is varied from wild
type at four positions. The binding constants of
these two RNA's to T4 DNA polymerase are
equivalent. These protocols with minimal
modification can yield high-affinity ligands for
any protein that binds nucleic acids as part of
its function high-affinity ligands could
conceivably be developed for any target
molecule.
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Wilson and Szostak, Ann.Rev.Bioc. (1999)
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- Selection against small molecules
- Selection against proteins
- Selection of new ribozymes (RNA world)
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The ATP aptamer structure
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Nucleolin RNA Targets

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RBD2-RNA-RBD1 sandwich
RBD1
RBD2
F56
linker
G16
22
3
3
1
5
5
Allain et al, EMBOJ (2000)
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In vitro selection of an enzyme
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Reference
Reviews Wilson and Szostak Ann.Rev.Bioch.(1999)
Gold et al, Ann.Rev.Bioch.(1995)
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Part III Introduction to RNAi
A brief History
RNAi Mechanism
SiRNA and miRNA
A few very recent structures
Biological application
A practical example of siRNA
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Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis
elegans ANDREW FIRE, SIQUN XU,
MARY K. MONTGOMERY, STEVEN A. KOSTAS,
SAMUEL E. DRIVER CRAIG C. MELLO Experimenta
l introduction of RNA into cells can be used in
certain biological systems to interfere with the
function of an endogenous gene,. Such effects
have been proposed to result from a simple
antisense mechanism that depends on hybridization
between the injected RNA and endogenous messenger
RNA transcripts. RNA interference has been used
in the nematode Caenorhabditis elegans to
manipulate gene expression,. Here we investigate
the requirements for structure and delivery of
the interfering RNA. To our surprise, we found
that double-stranded RNA was substantially more
effective at producing interference than was
either strand individually. After injection into
adult animals, purified single strands had at
most a modest effect, whereas double-stranded
mixtures caused potent and specific interference.
The effects of this interference were evident in
both the injected animals and their progeny. Only
a few molecules of injected double-stranded RNA
were required per affected cell, arguing against
stochiometric interference with endogenous mRNA
and suggesting that there could be a catalytic or
amplification component in the interference
process. Nature V391 pp 806-811 (1998)
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DS RNA against GFP
Fire at al, Nature V391 pp 806-811 (1998)
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In situ mRNA hybridization of Mex3 RNA in Embryo
-C
C
with DS RNA
From animal with AntisensRNA
Fire at al, Nature V391 pp 806-811 (1998)
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dsRNAi is cut in 21-23 nt fragments
Zamore et al, Cell, v101 pp25-33 (2000)
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The mRNA is cut in 21-23 nt fragments by the siRNA
Zamore et al, Cell, v101 pp25-33 (2000)
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A first model for the mechanism RNAi
Zamore et al, Cell, v101 pp25-33 (2000)
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Identification of DICER
22 nt RNA
Bernstein et al, Nature v 409, pp 363-366 (2001)
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The RISC complex
Elbashir et al, GD, v18 pp188-200 (2001)
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SiRNA and miRNA
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First miRNA in C.elegans
miRNA in Plants
miRNA in C.elegans with homologs In flies and
human
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A high number about 1 of the genes
Human 200-255 miRNA C.elegans 103-120
miRNA Drosophila 96-124 miRNA
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Functions ??
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miRNA
siRNA
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Post-transcriptional Cleavage of mRNA
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Structure of the PAZ domain
Lingel et al and Yan et al, Nature 426, pp
465-474 (2003)
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Structure of an Viral siRNA suppressor
Vargason et al, Cell 115, pp 799-811 (2003)
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• Applications
• Genome study (C-elegans)
• Gene knockout

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Reference
Reviews Bartel Cell.(2004) Hannon Nature
(2002)
Article Fire at al, Nature V391 pp 806-811
(1998)
Bernstein et al, Nature v 409, pp 363-366 (2001)
Elbashir et al, GD, v18 pp188-200 (2001)
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RNAi as a tool for knock down in mammalian cells
Why ? Which is the right siRNA sequence ? How do
I get the siRNAs into the cell ? Practical
aspects
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RNAi vs Knock-Out

• RNAi relatively easy to perform
• not so time consuming
• no real transgenic cells or animals
• RNAi its just a knock down
• finding siRNAs is not always easy
• negative controls

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Designing siRNA

• The target sequence should be 50-100 bp
  downstream of start codon or in the 3 UTR
• Search for a 23nt long sequence with a AA(N19)TT
  or NA(N21) motif
• Ensure that your target sequence is not
  homologous to any other genes
• Avoid more than three guanosines or three
  cytosines in a row
• avoid stretches of gt 4 T's or A's
• secondary structure of the target mRNA does not
  appear to have a strong effect on silencing
• Designing several siRNAs helps to find a highly
  efficient one

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Example for siRNAs

• Lamin A/Ctargeted region (cDNA) 5'
  AACTGGACTTCCAGAAGAACATCsense siRNA 5'
  CUGGACUUCCAGAAGAACAdTdTantisense siRNA 5'
  UGUUCUUCUGGAAGUCCAGdTdT

GL2 Luciferasetargeted region (cDNA) 5'
AACGTACGCGGAATACTTCGATTsense siRNA 5'
CGUACGCGGAAUACUUCGAdTdTantisense siRNA 5'
UCGAAGUAUUCCGCGUACGdTdT
Elbashir, Nature. 2001 May 24411(6836)494-8.
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Delivering Double Stranded RNA
Hannon, Nat Rev Genet. 2001 Feb2(2)110-9
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dsRNA Approach

• It is possible to get dsRNA commercially, either
  as two single stranded RNAs or already annealed
• Commercially available RNAs are produced by solid
  phase synthesis
• Another possibility is to get dsRNA by T7 in
  vitro transcription
• DNA oligos are the templates Annealing of
  antisense and sense product will give
  dsRNA After purification they are useable
• Normally the T7 procedure is cheaper and even
  faster (incl. oligo ordering)
• For one transfection reaction around 0.2 mM siRNA
  is necessary
• Transfections are carried out by cationic lipids
• Due to secondary structure dsRNA is rather
  stable, compared to ssRNA

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EXAMPLEcombinatorial control of splicing in the
c-src N1 exon
Black, Annu Rev Biochem. 200372291-336
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EXAMPLEcombinatorial control of splicing in the
c-src N1 exon
minigene

whole proteom isolation96 h
1775 or 1808 hPTB siRNAin 3UTR
check protein expression via western
2. Transfection 48 h (Lipofectamin 2000)
1. Transfection (Lipofectamin 2000)
cytoplasmatic RNA isolation96 h
HeLa-cell-line
1775 or 1808 hPTB siRNA in 3UTR
check alternative spliced exon via RT-PCR
Wagner, Mol Cell. 2002 Oct10(4)943-9
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Plasmid Approach
Ambion Inc. Austin, Texas, USA
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Lentivirus-Based ApproachshRNA-expressing vector
Self-inactivating long terminal repeats
Cytomegalovirus promootor
Woodchuck hepatitis Virus response element
Promotor
HIV packing signal
control purine track
Rubinson, Nat Genet. 2003 Mar33(3)401-6
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Functional silencing of genes in miceby
Lentivirus-infection
Generation of lentivirus infected zygotes
Silencing of p53 Tissue was harvestedfrom
8-wk-old mices
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Summary
Pros Cons
Fast Effective Works in many systems Non-inducible Most effective in embr. System Time dependent
Stable Inducible Tissue specific Time consuming to generate Cloning can be problematic
Stable Inducible Tissue specific Time consuming to generate Promotor can silence each other
Most commen technique in plants Also in non cycling cells possible Therapeutically useful Resistance Not well established Difficult to work with