Prof. F.

Allain 2/2/2004

Part I: Ribozymes

Part II: SELEX

(RNA in-vitro evolution)

Part III: RNAi

RNA inhibition and silencing
The RNA world hypothesis
 Part I: Ribozymes
A brief History

How many ribozyme ? Why ?

Catalytic efficiency, condition

3D structure of ribozyme:
And a mechanism of catalysis
Biological application ?
 A brief History

1982:Self-splicing in Tetrahymena pre-rRNA (group I intron)

Kruger et al, and Cech, Cell 31, 147-157 (1982)

1983:RNAse P is a ribozyme

Guerrier-Takada et al, and Altman, Cell, 35, 849-857 (1983)

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)
One main reaction: Nucleolytic cleavage
Transesterification (SN2)

Hammerhead
Haipin
Hepatitis delta
VS ribozyme From Lilley TIBS
(2003)
 The hammerhead ribozyme (plant virus)

- discovered in small RNA satellites of small viruses (1986)

- replication by rolling circle mechanism

Secondary structure
 The hammerhead ribozyme (plant virus)

- tertiary structure

Scott et al and Klug, Science 1996

The hairpin ribozyme (plant virus)

From Lilley TIBS

(2003)
The hepatitis delta ribozyme (human virus)

From Lilley TIBS

(2003)
Group I &II intron ribozyme (rRNA and mt RNA)

Doudna and Cech

Nature, 2002
Group I intron ribozyme (rRNA and mt RNA)

Golden et al, and cech

Science (1998)
Catalytic efficiency, condition
- ribozyme follows a Michaelis-Menten kinetics
k1 k2
E+S ES E+P
k-1

k-1+ k2
Km= = 10-5-10-7 M kcat= 0.5-2 min-1
k1

kcat/ Km= 103-106 M-1.min-1 Good catalytic efficiency!!

- all ribozyme need cations for activity (Mg2+ ,Mn2+)

 3D structure of ribozyme: mechanism of catalysis
hairpin ribozyme Hepatitis delta ribozyme

Ruppert et al, Nature 2001, Science 2002 Ferre d’Amare, Nature 1998
How to catalyse the reaction ?

From Lilley TIBS

(2003)
Structure of the hairpin ribozyme
hairpin ribozyme

Ruppert et al, Nature 2001, Science 2002

 hairpin ribozyme

Ground state Transition state

Ruppert et al, Nature 2001 Ruppert et al, Science 2002

hairpin ribozyme

free bound
free

bound

Loop A Loop B
hairpin ribozyme
Transition state

Ruppert et al, Science 2002

Acid-Base catalysis ?
(textbook Voet and Voet)

like with
RNAse A
 Acid-Base catalysis ?

G8 as a
base

A38 as an
acid
Bevilacqua, Biochemistry 2003
Hepatitis delta ribozyme

Ferre d’Amare, Nature 1998

Biological application ?

Tentative of gene therapy with

the hairpin and the hammerhead ribozyme
against viral RNA for example.
 Reference:

Reviews: Lilley TIBS (2003)

De Rose Chem & Biol (2002)
Ferre d’Amare 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)
 Part II: SELEX
A brief History

The method ?

A few examples.

Biological application ?
SELEX :Systematic Evolution of
Ligands by EXponential enrichment

A brief History

Ellington and Szostak, Nature (1990)

Tuerk and Gold , Science (1990)

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.
Wilson and Szostak, Ann.Rev.Bioc. (1999)
- Selection against small molecules

- Selection against proteins

- Selection of new ribozymes (RNA world)

The ATP aptamer structure
 Nucleolin RNA Targets
C G C
C C C G C C
C G C A C G
A C
U A C A U G U/G G/A
A U C C
G­C U Nx Ny
A• A C­G N­N
A•U G A­U
G­C N­N
G•A G­C
U­A N­N
G­C G­C
A­U N­N
G­C U­G 5’ 3’ 5’ 3’
5’ 3’ 5’ 3’

 selex B1 B2 Consensus NRE
 NRE 10­50nM 50­100nM
5­20nM (5’ETS) (5’ETS)
Mouse Mouse
 RBD2-RNA-RBD1 “sandwich”
RBD1

RBD2
F56
linker
G16

22
1 3’ 3’
5’ 5’
Allain et al, EMBOJ (2000)
 In vitro
selection of an enzyme
 Reference:

Reviews: Wilson and Szostak Ann.Rev.Bioch.(1999)

Gold et al, Ann.Rev.Bioch.(1995)

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
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‡

Experimental 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
 + DS RNA
against GFP

Fire at al, Nature V391 pp 806-811 (1998)

-C +C

From animal: with AntisensRNA with DS RNA

Fire at al, Nature V391 pp 806-811 (1998)
 RNAi: double-stranded RNA directs the ATP-dependent
cleavage of mRNA at 21 to 23 nucleotide intervals.
Zamore PD, Tuschl T, Sharp PA, Bartel DP.

Drosophila in vitro systemRNAi is ATP dependentDuring the RNAi

reaction, both strands of the dsRNA are processed to RNA segments
21-23 nucleotides in length.The mRNA is cleaved only within the
region of identity with the dsRNA. Cleavage occurs at sites 21-23
nucleotides apart, the same interval observed for the dsRNA itself,
suggesting that the 21-23 nucleotide fragments from the dsRNA are
guiding mRNA cleavage

Cell, v101 pp25-33 (2000)

dsRNAi is cut in 21-23 nt fragments

Zamore et al, Cell, v101 pp25-33 (2000)

The mRNA is cut in 21-23 nt fragments by the siRNA

Zamore et al, Cell, v101 pp25-33 (2000)

A first model for the mechanism RNAi

Zamore et al, Cell, v101 pp25-33 (2000)

Role for a bidentate ribonuclease in the initiation step of RNA
interference.
Bernstein E, Caudy AA, Hammond SM, Hannon GJ..

Here we identify an enzyme, Dicer, which can produce putative guide RNAs.
The enzyme has a distinctive structure, which includes a helicase domain and
dual RNase III motifs. Dicer also contains a region of homology to the
RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.

Nature v 409, pp 363-366 (2001)

 Identification of DICER

22 nt
RNA

Bernstein et al, Nature v 409, pp 363-366 (2001)

 The RISC
complex

Elbashir et al, G&D, v18 pp188-200 (2001)

 SiRNA and miRNA

MicroRNAs. Genomics, Biogenesis, Mechanism, and Function.

Bartel DP.
 First miRNA
in C.elegans

miRNA
in Plants
miRNA
in C.elegans
with homologs
In flies and human
A high number: about 1% of the genes

Human: 200-255 miRNA

C.elegans: 103-120 miRNA

Drosophila: 96-124 miRNA

Functions ??
miRNA siRNA
 Post-transcriptional
Cleavage of mRNA

Translational repression
of the mRNA

Transcriptional
silencing
 Structure of the PAZ domain

Lingel et al and Yan et al, Nature 426, pp 465-474 (2003)

Structure of an Viral siRNA suppressor

Vargason et al, Cell 115, pp 799-811 (2003)

Applications:
-Genome study (C-elegans)

-Gene knockout
 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, G&D, v18 pp188-200 (2001)

RNAi as a tool for knock down
in mammalian cells

Why ?
Which is the right siRNA sequence ?
How do I get the “siRNA’s” into the cell ?
Practical aspects
 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 siRNA’s is not always easy
negative controls
 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 > 4 T's or A's

• secondary structure of the target mRNA does not appear to have a strong effect
on silencing

• Designing several siRNA’s helps to find a highly efficient one

 Example for siRNA’s

Lamin A/C
targeted region (cDNA): 5' AACTGGACTTCCAGAAGAACATC
sense siRNA: 5' CUGGACUUCCAGAAGAACAdTdT
antisense siRNA: 5' UGUUCUUCUGGAAGUCCAGdTdT

GL2 Luciferase
targeted region (cDNA): 5' AACGTACGCGGAATACTTCGATT
sense siRNA: 5' CGUACGCGGAAUACUUCGAdTdT
antisense siRNA: 5' UCGAAGUAUUCCGCGUACGdTdT

Elbashir,
 Delivering Double Stranded RNA

Hannon,
 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
EXAMPLE:
combinatorial
control of splicing
in the c-src N1
exon

Black
 EXAMPLE:
combinatorial control of splicing in the c-src N1 exon
minigene

whole proteom
1775 or 1808 isolation
hPTB siRNA 96 h
in 3’UTR check protein
expression
2. Transfection 48 h via western
(Lipofectamin 2000)

1. Transfection
(Lipofectamin 2000)
cytoplasmatic
RNA isolation
96 h
HeLa-cell-line

1775 or 1808
hPTB siRNA
in 3’UTR check alternative
spliced exon
Wagner via RT-PCR
Plasmid Approach

Ambion Inc
Lentivirus-Based Approach:
shRNA-expressing vector

Rubinson,
Functional silencing of genes in mice
by Lentivirus-infection
 Summary
Pros Cons
•Fast •Non-inducible
•Effective •Most effective in embr. System
•Works in many systems •Time dependent

•Stable •Time consuming to generate

•Inducible •Cloning can be problematic
•Tissue specific

•Stable •Time consuming to generate

•Inducible •Promotor can silence each other
•Tissue specific

•Most commen technique in plants •Resistance

•Also in non cycling cells possible •Not well established
•Therapeutically useful •Difficult to work with