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Allain 2/2/2004
Part I: Ribozymes
1983:RNAse P is a ribozyme
- 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)
Secondary structure
The hammerhead ribozyme (plant virus)
- tertiary structure
k-1+ k2
Km= = 10-5-10-7 M kcat= 0.5-2 min-1
k1
Ruppert et al, Nature 2001, Science 2002 Ferre d’Amare, Nature 1998
How to catalyse the reaction ?
free bound
free
bound
Loop A Loop B
hairpin ribozyme
Transition state
like with
RNAse A
Acid-Base catalysis ?
G8 as a
base
A38 as an
acid
Bevilacqua, Biochemistry 2003
Hepatitis delta ribozyme
The method ?
A few examples.
Biological application ?
SELEX :Systematic Evolution of
Ligands by EXponential enrichment
A brief History
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.
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
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:
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‡
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.
22 nt
RNA
miRNA
in Plants
miRNA
in C.elegans
with homologs
In flies and human
A high number: about 1% of the genes
Translational repression
of the mRNA
Transcriptional
silencing
Structure of the PAZ domain
-Gene knockout
Reference
:
Reviews: Bartel Cell.(2004)
Why ?
Which is the right siRNA sequence ?
How do I get the “siRNA’s” into the cell ?
Practical aspects
RNAi vs Knock-Out
• Ensure that your target sequence is not homologous to any other genes
• secondary structure of the target mRNA does not appear to have a strong effect
on silencing
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
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