ribozyme course

8/9/2019 Ribozyme Course

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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)



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he he+atitis delta ribozyme (human virus)



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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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,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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23/69


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


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/


45/69


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%% nt

RNA

Identi"iation o" 2I/ER

3ernstein et al0 Nature v '80 ++ 1>1-1>> (%''5)


47/69

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


52/69


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Post-transri+tional

/leava!e o" mRNA

ranslational re+ression

o" the mRNA

ransri+tional

silenin!


54/69

Struture o" the PAK domain

Lin!el et al and an et al0 Nature %>0 ++ >7-6 (%''1)


55/69

Struture o" an


56/69

A++liations:

-enome study (/-ele!ans)

-ene ;no;out


57/69

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

ribozyme course

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