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Part I: Ribozymes
Part II: SELEX
(RNA in-vitro evolution)
Part III: RNAi
RNA inhibition and silenin!
Pro"# $#Allain %&%&%''
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he RNA *orld hy+othesis
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Part I: Ribozymes
A brie" ,istory
,o* many ribozyme .hy
/atalyti e""iieny0 ondition
12 struture o" ribozyme:And a mehanism o" atalysis
3iolo!ial a++liation
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A brie" ,istory
1982:Self-splicing in Tetrahymena pre-rRNA (group I intron)
4ru!er et al0 and /eh0 /ell 150 56-576 (589%)
1983:RNAse P is a ribo!"e
uerrier-a;ada et al0 and Altman0 /ell0 170 98-976 (5891)
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,o* many ribozyme .hy
- the hammerhead ribozyme (+lant virus)
- the hair+in ribozyme (+lan virus)
- he+atitis delta ribozyme (human virus)
- neuros+ora
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=ne main reation: Nuleolyti leava!e
ransesteri"iation (SN%)
$rom Lilley I3S (%''1)
,ammerhead
,ai+in
,e+atitis delta
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he hammerhead ribozyme (+lant virus)
- disovered in small RNA satellites o" small viruses (589>)
- re+liation by rollin! irle mehanism
Seondary struture
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he hammerhead ribozyme (+lant virus)
- tertiary struture
Sott et al and 4lu!0 Siene 588>
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he hair+in ribozyme (+lant virus)
$rom Lilley I3S (%''1)
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he he+atitis delta ribozyme (human virus)
$rom Lilley I3S (%''1)
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rou+ I ?IIintron ribozyme(rRNA and mt RNA)
2oudna and /eh
Nature0 %''%
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rou+ Iintron ribozyme(rRNA and mt RNA)
olden et al0 and eh
Siene (5889)
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/atalyti e""iieny0 ondition
- ribozyme "ollo*s a @ihaelis-@enten ;inetis
E S ES E P
;5 ;%
;-5
- all ribozyme need ations "or ativity (@!%0@n%)
4mB;-5;%
;5
B 5'-7-5'-6@ ;atB '#7-% min-5
;at& 4mB 5'1-5'> @-5#min-5 ood atalyti e""iienyCC
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12 struture o" ribozyme: mehanism o" atalysis
hair+in ribozyme,e+atitis delta ribozyme
Ru++ert et al0 Nature %''50 Siene %''% $erre dDAmare0 Nature 5889
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$rom Lilley I3S (%''1)
,o* to atalyse the reation
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Struture o" the hair+in ribozyme
hair+in ribozyme
Ru++ert et al0 Nature %''50 Siene %''%
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hair+in ribozyme
#oop A
"ree
bound
#oop $
"ree bound
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hair+in ribozyme
Ru++ert et al0 Siene %''%
ransition state
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Aid-3ase atalysis (tetboo;
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Aid-3ase atalysis
%8 as a
base
A38 as an
aci&
3evilaFua0 3iohemistry %''1
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3iolo!ial a++liation
entative o" !ene thera+y *iththe hair+in and the hammerhead ribozyme
a!ainst viral RNA "or eam+le#
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Re"erene:
Revie*s: Lilley I3S (%''1)
2e Rose /hem ? 3iol (%''%)
$erre dDAmare 3io+olymer (%''1)
Artile: 4ru!er et al0 and /eh0 /ell (589%)
uerrier-a;ada et al0 and Altman0 /ell (5891)
Sott et al Nature (5887) Siene (588>)Ru+ert et al Nature (%''5)0 Siene (%''%)
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SELEX :Systemati Evolution o"Li!ands by EX+onential enrihment
A brie" ,istory
Ellin!ton and Szosta;0 Nature (588')
uer; and old 0 Siene (588')
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In vitroselection 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.
S!ste"atic e'olution of ligan&s b! eponential enric"ent: RNA ligan&s tobacteriopage *+ ,NA pol!"erase
*uer. /0 %ol& #
ig-affinit! nucleic aci& ligan&s for a protein ere isolate& b! a proce&ure tat &epen&s on alternate
c!cles of ligan& selection fro" pools of 'ariant seuences an& a"plification of te boun& species
@ulti+le rounds e+onentially enrih the +o+ulation "or the hi!hest a""inity s+eies that an be lonally
isolated and haraterized# In +artiular one ei!ht-base re!ion o" an RNA that interats *ith the 2NA
+olymerase *as hosen and randomized# *o di""erent seFuenes *ere seleted by this +roedure "rom the
alulated +ool o" >7071> s+eies# =ne is the *ild-ty+e seFuene "ound in the baterio+ha!e mRNAG one is
varied "rom *ild ty+e at "our +ositions# he bindin! onstants o" these t*o RNAHs to 2NA +olymerase are
eFuivalent# *ese protocols it "ini"al "o&ification can !iel& ig-affinit! ligan&s for an! protein tat
bin&s nucleic aci&s as part of its function4 ig-affinit! ligan&s coul& concei'abl! be &e'elope& for an!
target "olecule
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.ilson and Szosta;0 Ann#Rev#3io# (5888)
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- Seletion a!ainst small moleules
- Seletion a!ainst +roteins
- Seletion o" ne* ribozymes (RNA *orld)
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he AP a+tamer struture
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Nucleolin RNA Targets
B110-50nM(5ETS)Mouse
B250-100nM(5ETS)Mouse
G-C
A-UG-CG-CU-G5 3
UC
C
C
A
G
AC
5 3
C-GG-CU-AA-U
C
U
C
CC
A
G
G
U
Consensus NRE
5 3
N-NN-NN-NN-N
Nx
U/G
C
C C
G/A
G
Ny
selexNRE5-20nM
5 3
C
AAUGAG-CG-C
G
A
U
C
C
A
G
A
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RBD2-RNA-RBD1sandwich
5
31
G16
22
F56
5
3
RBD2
RBD1
linker
Allain et al0 E@3= (%''')
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In vitro
seletion o" an enzyme
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Re"erene:
Re'ies: 5ilson an& Sosta. AnnRe'$ioc(1999)
%ol& et al0 AnnRe'$ioc(1996)
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Part III: Introdution to RNAi
A brie" ,istory
SiRNA and miRNA
RNAi @ehanism
A "e* very reent strutures
3iolo!ial a++liation
A +ratial eam+le o" siRNA
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Potent and specifc genetic intererence by double-stranded RNA in Caenorhabditis elegans
ANDREW FIRE*, SIQUN XU*, MARY K. MONTGOMERY*, STEVEN A. KOSTAS*,
SAMUEL E. DRIVER & CRAIG C. MELLO
Experimental introduction o RNA into cells can be used in certain biologicalsystems to interere with the unction o an endogenous gene,. uch e!ectsha"e been proposed to result rom a simple antisense mechanism thatdepends on hybridi#ation between the in$ected RNA and endogenousmessenger RNA transcripts. RNA intererence has been used in thenematode Caenorhabditis elegansto manipulate gene expression,. %ere wein"estigate the re&uirements or structure and deli"ery o the intereringRNA. 'o our surprise, we ound that double-stranded RNA was substantially
more e!ecti"e at producing intererence than was either strand indi"idually.Ater in$ection into adult animals, purifed single strands had at most amodest e!ect, whereas double-stranded mixtures caused potent and specifcintererence. 'he e!ects o this intererence were e"ident in both thein$ected animals and their progeny.(nly a ew molecules o in$ected double-stranded RNA were re&uired per a!ected cell, arguing against stochiometricintererence with endogenous mRNA and suggesting that there could be a
catalytic or amplifcation component in the intererence process.
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2S RNA
a!ainst $P
$ire at al0 Nature
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In situ mRNA hybridization of Mex3 RNA in Embryo
-/ /
$rom animal: *ith AntisensRNA *ith 2S RNA
$ire at al0 Nature
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RNAi: &ouble-stran&e& RNA &irects te A*P-&epen&ent
clea'age of "RNA at 21 to 23 nucleoti&e inter'als
7a"ore P,0 *uscl *0 Sarp PA0 $artel ,P
Double-stranded RNA (dsRNA) directs the seuence-s!ecific de"radation
of mRNA throu"h a !rocess #no$n as RNA interference (RNAi)% &sin" a
recently de'elo!ed Drosophila in i!ro s"s!e# $e examined the
molecular mechanism underlyin" RNAi% e find that RNAi is A$%
dependen!yet uncou!led from mRNA translation% D&rin' !he RNAireac!ion( )o!h s!rands o* !he dsRNA are processed !o RNA se'#en!s
21-23 n&cleo!ides in len'!h+*rocessin" of the dsRNA to the small RNA
fra"ments does not reuire the tar"eted mRNA% $he #RNA is cleaed
onl" wi!hin !he re'ion o* iden!i!" wi!h !he dsRNA+ ,leaa'e occ&rs a!
si!es 21-23 n&cleo!ides apar!( !he sa#e in!eral o)sered *or !hedsRNA i!sel*( s&''es!in' !ha! !he 21-23 n&cleo!ide *ra'#en!s *ro# !he
dsRNA are '&idin' #RNA cleaa'e
,ell( 11 pp25-33 .2/
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dsRNAi is ut in %5-%1 nt "ra!ments
0a#ore e! al( ,ell( 11 pp25-33 .2/
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he mRNA is ut in %5-%1 nt "ra!ments by the siRNA
0a#ore e! al( ,ell( 11 pp25-33 .2/
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A "irst model "or the mehanism RNAi
0a#ore e! al( ,ell( 11 pp25-33 .2/
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%% nt
RNA
Identi"iation o" 2I/ER
3ernstein et al0 Nature v '80 ++ 1>1-1>> (%''5)
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Elbashir et al0 ?20 v59 ++599-%'' (%''5)
he RIS/
om+le
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SiRNA and miRNA
icroRNAs %eno"ics0 $iogenesis0 ecanis"0 an& ;unction
$artel ,P
MicroRNAs (miRNAs) are endo"enous a!!roximately ,, nt
RNAs that can !lay im!ortant re"ulatory roles in animals and!lants by tar"etin" mRNAs for clea'a"e or translational
re!ression%Althou"h they esca!ed notice until relati'ely
recently miRNAs com!rise one of the more abundant classes
of "ene re"ulatory molecules in multicellular or"anisms andli#ely influence the out!ut of many !rotein-codin" "enes%
/ell '001 !! ,20-,4 (,556) (re'ie$)
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$irst miRNA
in C.elegans
miRNA
in C.elegans
*ith homolo!sIn "lies and human
miRNA
in Plants
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A hi!h number: about 5J o" the !enes
,uman: %''-%77 miRNA
/#ele!ans: 5'1-5%' miRNA
2roso+hila: 8>-5% miRNA
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$untions
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Post-transri+tional
/leava!e o" mRNA
ranslational re+ression
o" the mRNA
ransri+tional
silenin!
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Struture o" the PAK domain
Lin!el et al and an et al0 Nature %>0 ++ >7-6 (%''1)
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Struture o" an
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A++liations:
-enome study (/-ele!ans)
-ene ;no;out
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Re"erene
:Re'ies: $artel /ell(2
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RNAi as a tool for .noc. &onin "a""alian cells
Why ?
Which is the right siRNA sequence ?
How do I get the siRNAs into the cell ?
Practical asects
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RNAi 's =noc.->ut
RNAi: relatively easy to +er"orm
not so time onsumin!
no real trans!eni ells or animals
-RNAi: its Must a ;no; do*n
"indin! siRNADs is not al*ays easyne!ative ontrols
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,esigning siRNA he tar!et seFuene should be 7'-5'' b+ do*nstream o" start odon or in the 1D
OR
Searh "or a %1nt lon! seFuene *ith a AA(N58) or NA(N%5) moti"
Ensure that your tar!et seFuene is not homolo!ous to any other !enes
Avoid more than three !uanosines or three ytosines in a ro*
avoid strethes o" Hs or AHs
seondary struture o" the tar!et mRNA does not a++ear to have a stron! e""eton silenin!
2esi!nin! several siRNADs hel+s to "ind a hi!hly e""iient one
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#a"in A?/
tar!eted re!ion (2NA): 7H AA/A///AAAAA/A/
sense siRNA: 7H /OA/OO//AAAAA/Add
antisense siRNA: 7H OOO/OO/OAAO//Add
a"ple for siRNA@s
%#2 #uciferase
tar!eted re!ion (2NA): 7H AA/A//AAA//A
sense siRNA: 7H /OA//AAOA/OO/Add
antisense siRNA: 7H O/AAOAOO///OA/dd
4l)ashir(Nature% ,550 May ,6+600(1231)766-2%
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,eli'ering ,ouble Stran&e& RNA
annon(Nat Re' 8enet% ,550 9eb+,(,)7005-
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&sRNA Approac
It is +ossible to !et dsRNA ommerially0 either as t*o sin!le strandedRNAs or already annealed
/ommerially available RNAs are +rodued by solid +hase synthesis
Another +ossibility is to !et dsRNA by 6 in vitro transri+tion:
2NA oli!os are the tem+latesAnnealin! o" antisense and sense +rodut *ill !ive dsRNAA"ter +uri"iation they are useable
Normally the 6 +roedure is hea+er and even "aster (inl# oli!oorderin!)
$or one trans"etion reation around '#% m@ siRNA is neessary
rans"etions are arried out by ationi li+ids
2ue to seondary struture dsRNA is rather stable0 om+ared to ssRNA
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AP#:
co"binatorial
control of splicingin te c-src N1
eon
Black Annu Re' :iochem% ,553+4,7,0-331
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HeLa-cell-line
1775 or 1808
hPTB siRNA
in 3UTR
;
minigene
1. Transfection
(Lipofectamin 2000)
2. Transfection 48 h
(Lipofectamin 2000)
whole proteom
isolation
96 h
cytoplasmatic
RN isolation
96 h
check ro!ein
e"ression
#ia $es!ern
1775 or 1808
hPTB siRNA
in 3UTR
check al!erna!i#e
slice% e"on
#ia RT-P&R
AP#:
co"binatorial control of splicing in te c-src N1 eon
>a'ner Mol /ell% ,55,
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Plas"i& Approac
A#)ion nc% Austin exas &.A
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#enti'irus-$ase& Approac:
sRNA-epressing 'ector
R&)inson(Nat 8enet% ,553 Mar+33(3)7650-1
.elf-in
acti'
atin
"
lon"
termin
alre!e
ats
=I>
!ac#i
n"s
i"nal
control!
urin
etra
c#
*rom
otor
/yt
ome"
alo'iru
s
!rom
ooto
r
)oo
dchu
c#he!
atitis
>irusres!
onse
ele
ment
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;unctional silencing of genes in "ice
b! #enti'irus-infection
8eneration of lenti'irus
infected zy"otes
.ilencin" of !?37
issue $as har'estedfrom 2-$#-old mices
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Su""ar!Pros /ons
$astE""etive
.or;s in many systems
Non-induible@ost e""etive in embr# System
ime de+endent
Stable
Induible
issue s+ei"i
ime onsumin! to !enerate
/lonin! an be +roblemati
Stable
Induible
issue s+ei"i
ime onsumin! to !enerate
Promotor an silene eah other
@ost ommen tehniFue in +lants
Also in non ylin! ells +ossible
hera+eutially use"ul
Resistane
Not *ell established
2i""iult to *or; *ith