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Cene Lechnology and Molecular blology, 881110
2012-10-31
Lmma Lundberg, hu
k1P, Sclence for Llfe LaboraLory, Sweden
emma.lundberg[sclllfelab.se
CuLllne
1. 8nA lnLerference
2. 8nAl as a bloLechnologlcal Lool
3. 1herapeuuc appllcauons
1
8nA lnLerference
1he cenLral dogma
DNA
Replication
Transcription Translation
mRNA Protein
8nA ls noL slmply a messenger for converung unA lnLo
proLelns
8nA have lmporLanL regulaLory funcuons for conLrolllng gene
expresslon

8nA anusense / 8nA lnLerference
8nA anusense - Anusense 8nA blnds Lo Lhe
complemenLary m8nA and blocks Lranslauon.

8nA lnLerference - Small non-codlng 8nAs
ldenufy speclc m8nAs and Lrlgger Lhelr
degradauon (enzymaucally)

8nA anusense
Slngle-sLranded 8nA (ss8nA)

Anusense 8nA ls synLheslzed
uslng Lhe sense sLrand as
LemplaLe - Lherefore lL has a
sequence complemenLarlLy Lo
m8nA.
Anusense 8nA ls someumes
produced ln normal cells of
many dlerenL organlsms.
Companlon slLe for
8loLechnology. by Clark
CopyrlghL 2009 by Academlc ress.
All rlghLs reserved.
8nA anusense
A) 1he complemenLary sequence of anusense 8nA blnds Lo speclc
reglons of m8nA. 1hls can block Lhe rlbosome blndlng slLe or
spllce [uncuons.
8) Anusense unA LargeLs m8nA for degradauon. 1he heLeroduplex
Lrlggers 8nase P Lo degrade Lhe m8nA.
A) 8)
Lxamples of naLural anusense conLrol
ConLrol of ColL1 lasmld repllcauon: 8nAl and
8nAll m8nA are anusense/sense parLners LhaL
prevenL unA polymerase from lnluaung plasmld
repllcauon. 1he amounL of anusense 8nAl
conLrols how oen repllcauon ls lnluaLed.

ConLrol of x-Chromosome lnacuvauon: Anusense
8nA blnds Lo Lhe !"#$ gene Lo prevenL lLs
expresslon and Lhus prevenL x-chromosome
lnacuvauon.
Mammallan x-chromosome lnacuvauon
Figure 7-90 Molecular Biology of the Cell ( Garland Science 2008) Figure 7-89 Molecular Biology of the Cell ( Garland Science 2008)
8nA lnLerference (8nAl)
uouble-sLranded 8nA (ds8nA) segmenLs
aLhway for endogenous gene regulauon where shorL
8nA segmenLs Lrlgger an enzyme complex Lo degrade a
LargeL m8nA.
1wo Lypes of small 8nA molecules are cenLral Lo 8nAl
mlcro8nA (ml8nA)
small lnLerferlng 8nA (sl8nA)

8nA lnLerference - An evoluuonary
conserved paLhway
1he 8nAl paLhway ls found ln many eukaryoLes such as slngle-
celled fungl, planLs, worms, les and mammals.
1he dlerenL organlsms have varlauons of Lhe same response.
8nAl has an lmporLanL funcuon as a cell defence mechanlsm.
Many Lransposable elemenLs and vlruses produce ds8nA ln
Lhelr llfe cycles and 8nAl helps Lo defend agalnsL Lhem and
oLher forelgn 8nA molecules.
8nAl ls also lmporLanL ln dlrecung developmenL as well as
gene expresslon ln general.
ulerenL sources of ds8nA
ds8nA Lrlggers 8nA lnLerference.
ds8nA can come from dlerenL sources, for example
8nA vlruses
mlcro8nA ls encoded
by Lhe genome
overexpresslon
of Lransgenes.
8nAl paLhway
Can be dlvlded lnLo Lwo phases
lnluauon phase: lormauon of ds8nA, cumng lnLo
smaller pleces of 21-30 nL by ulcer.
LecLor phase: Assoclauon wlLh 8lSC/8l1S enzyme
complex. uesLrucuon of complemenLary m8nA or
Lranslauonal sllenclng.
8nAl paLhway
LecLor
phase:
lnluauon
phase:
sl8nA
ml8nA
recursor
ml8nA
vlral
repllcauon
ds8nA
8l1S
8lSC
Lnzyme
complex
m8nA
degradauon
1ranslauon
block
1ranscrlpuonal sllenclng
(unA/hlsLone meLhylauon)
key players ln 8nAl process
,%!"# (mlcro 8nA)
A large class of evoluuonarlly conserved, non-codlng 8nA, orlglnaung from
longer LranscrlpLs characLerlzed by lmperfecL halrpln sLrucLures. ml8nAs are
21-23 nL 8nAs processed from pre-ml8nA precursors by ulcer.

-%!"# (shorL lnLerferlng 8nA)
sl8nAs are 21-23 bp ds8nA wlLh dlnucleoude 3' overhangs LhaL are
processed from longer ds8nA by ulcer ln Lhe 8nA lnLerference paLhway.
.%+()
A 8nase lll famlly enzyme LhaL processes ds8nA Lo sl8nA and assembly of
Lhe gulde sLrand lnLo 8lSC. ulcer cleaves ds8nA aL 21-23 bp lnLervals Lo
generaLe sl8nAs wlLh 2-nL 3' overhangs and phosphorylaLed 3' ends.

#)/0&12'(
A proLeln found ln all 8lSC/8l1S and ml8nA-conLalnlng rlbonucleoproLeln
complexes and have been shown Lo be Lhe caLalyuc slLe of m8nA cleavage
ln 8lSC. ArgonauLes are ~100 kua proLelns characLerlzed by conserved
domalns called AZ and lWl.
mlcro8nA (ml8nA)
lmporLanL ln anlmals and planLs.
Lvoluuonary conserved non-codlng 8nAs
MaLure ml8nA are ss-8nA molecules of 21-23
nucleoudes. CrlglnaLe from prl-m8nA lmperfecL
halrpln sLrucLures.
Puman expresses more Lhan 400 ml8nAs LhaL
regulaLe more Lhan 1/3 of all human proLeln-codlng
genes
ShorL lnLerferlng 8nA (sl8nA)
Cen of exogenous orlgln

uouble-sLranded 8nA molecules of 21-23 nL
(mosL oen 21 nL)
ulnucleoude 3' overhang
Lach sLrand has a 3' phosphaLe group and a 3' hydroxyl group
very eecuve - As few as 30 coples of sl8nA can desLroy Lhe
enure cellular conLenL of a LargeL m8nA.
lnl1lA1lCn PASL:
ml8nA and sl8nA processlng
llg 08:19 CLLL
LllLC1C8 PASL:
1hrough Lhe 8lSC complex.
31)415
,1'+6
3()*(+' ,1'+6
ml8nA/sl8nA ln anlmals/planLs
31)415
,1'+6
3()*(+' ,1'+6
MosL anlmal ml8nAs MosL planL ml8nAs, and sl8nAs
sl8nA ls a very eecuve process
8ecycllng of 8lSC/sl8nA complex
As few as 30 molecules of sl8nA
may desLroy Lhe enure cellular
conLenL of LargeL m8nA.
Ampllcauon by Lhe enzyme
8nA-dependenL 8nA polymerase
(8d8) ln planLs.
sl8nA ls a very eecuve process
8ecycllng of 8lSC/sl8nA complex
As few as 30 molecules of sl8nA
may desLroy Lhe enure cellular
conLenL of LargeL m8nA.
Ampllcauon by Lhe enzyme
8nA-dependenL 8nA polymerase
(8d8) ln planLs.
llCu8L 3.11
Selecuvely shuL o Lhe
synLhesls of LargeL 8nAs

vla 8l1S complex Largeung
LargeL gene
(vla 8lSC complex Largeung
repeLeuve unA)
LllLC1C8 PASL:
heLerochromaun formauon
8nAl as a cell defense mechanlsm
lmporLanL as proLecuon agalnsL Lransposable
elemenLs, vlruses and oLher forelgn 8nA molecules.
ln some organlsms (e.g. planLs) can Lhe 8nAl acuvlLy
Lransfer from cell Lo cell.
8nAl dlrecLed heLerochromaun formauon ls an
lmporLanL cell defense mechanlsm LhaL llmlLs Lhe
accumulauon of Lransposable elemenLs ln Lhe genome
by malnLalnlng Lhem ln a Lranscrlpuonally sllenL form.
8nAl ls also lmporLanL ln dlrecung developmenL as well
as gene expresslon ln general.
1he 8nAl machlnery malnLalns Lhe heLerochromaun
formed around cenLromeres.
PlsLory of 8nAl
1ransgenlc planLs
AlLer ower color ln peLunlas (1990s) -> 1CS
lungus %&'()#*)(+ -> Cuelllng
lanLs showlng Lolerance Lo vlruses -> vlCS

1he nobel rlze ln Medlclne 2006
ur. llre and ur. Mello
dlscovered LhaL 8nA
small 8nA sequences
can lnLerfere wlLh gene
funcuon ln -. &/&0+1#

23)( $4&"( 5"#6)7&(8 )3
9%: "1$&(3&(&16& ; 0&1&
#"/&16"10 <8 5)'</&;
#$(+15&5 9%:2
www.nobelprlze.org
Summary 8nA lnLerference
www.nobelprlze.org
2
8nAl as a bloLechnologlcal Lool
8nAl as a bloLechnologlcal Lool
8nAl can be used as a Lool Lo sLudy gene expresslon/
gene funcuon
8y lnhlblung Lranslauon 8nAl provldes Lhe ablllLy Lo
remove/reduce a parucular proLeln from an
organlsm wlLhouL Lhe need for geneuc modlcauons.
Anusense ollgonucleoudes
ln Lhe laboraLory anusense 8nA can be made by Lwo
dlerenL meLhods
1. Chemlcally synLheslzed ollgonucleoudes
2. Clonlng of Lhe gene of lnLeresL ln Lhe opposlLe dlrecuon
(Lranscrlpuon glves anusense)
8nAl as a Lool
ln Lhe laboraLory 8nAl can be made by Lhree dlerenL
meLhods
1. Chemlcally synLheslzed as 21-23 nL ds-8nA wlLh a 3'
dlnucleoude overhang.
2. 1argeL m8nA can aer ampllcauon wlLh C8 be LreaLed
wlLh purled ulcer Lo produce sl8nA.
3. Lxpresslon of shorL halrpln 8nA (sh8nA) LhaL mlmlc Lhe
sLrucLure of ml8nA
8nAl as a Lool
2.
3.
roblems assoclaLed wlLh 8nA
lnLerference/anusense ollgonucleoudes
ueslgn of anusense ollgonucleoude
non-speclc lnLeracuon wlLh nonLargeL
molecules (o-LargeL eecL)
uellvery Lo LargeL cells

uellvery of 8nA anusense/
lnLerference Lo cells/anlmals
Cell membranes are lmpermeable Lo ollgonucleoudes.
Pave Lo geL Lhe ollgonucleoude Lo Lhe correcL cellular
comparLmenL.
All proLelns can noL be knocked down slnce Lhey are
essenual Lo Lhe cell.
8nAl for sLudylng -.&/&0+1# developmenL
1hree ways for 8nAl Lakeup
1. Absorb ds8nA expressed ln bacLerla Lhey eaL.
2. 1akeup from surroundlng
3. ln[ecuon of ds8nA Lo egg
A) Llposomes
8) 8aslc pepudes
C) SLrepLolysln C
uellvery of 8nA anusense/
lnLerference Lo cells
u) Mlcroln[ecuon
L) Scrape loadlng
uellvery of 8nA anusense/
lnLerference Lo cells
luncuonal screenlng wlLh 8nAl llbrarles
8nAl can llnk a parucular gene Lo a speclc cellular
process.
Cenome wlde 8nAl llbrarles have been creaLed for
several organlsms
86 of Lhe genome of C.elegans
91 of all genes ln urosophlla
AlmosL enure Puman genome
Cbserves phenoLyplc eecLs. roLelns LhaL glve rlse Lo
slmllar phenoLyplc eecL are llkely Lo have a slmllar
funcuon or be lnvolved ln Lhe same paLhways.

luncuonal screenlng wlLh 8nAl llbrarles
Lxample: Cenome wlde sl8nA
screenlng (MlLoCheck)
hup://people.pwf.cam.ac.uk/[w366/
research/mlLocheck/lndex.hLm

Cenome-wlde funcuonal
screenlng for genes lnvolved
ln mlLosls

1herapeuuc appllcauons
3
lmporLanL aspecLs for Lherapeuuc
8nAl appllcauons
SpeclclLy
no o-LargeL eecLs
uellvery
Crgan/cell-selecuve expresslon/dellvery
SafeLy
Should noL ellclL unwanLed lmmune responses
uellvery of exogenous ds8nA or sh8nA
lssues Lo overcome
harmacoklneucs: ShorL
hallfe lf lngesLed or
ln[ecLed

C-LargeL eecLs:
Pybrldlsauon lsn'L a
perfecL process

1rlggerlng of lmmune
sysLem
osslble soluuons
lorm complexes wlLh oLher
molecules or chemlcally modlfy
dellvered molecule Lo avold
degradauon
Chemlcally modlfy Lhe dellvered
molecule for lncreased speclclLy
Avold Lrlgger-happy sequences
LncapsulaLe or con[ugaLe
dellvered molecule
SLraLegles for sl8nA dellvery
lrom Aagaard & 8ossl, 8nAl Lherapeuucs: rlnclples,
prospecLs and challenges, Adv. urug uellv. 8ev., 2007
llrsL approved anusense drug
lormlvlrsen
lnLraocular ln[ecuon for LreaLmenL of
cyLomegalovlrus (CMv) reunlus
Approved by luA ln 1998
SynLheuc 21 nL (3'-CCC 111 CC1 C11 C11 C11 CCC-3')
8locks Lranslauon of vlral m8nA by blndlng Lo a
codlng segmenL of Lhe CMv gene.
upcomlng anusense drug
Mlpomersen
Weekly ln[ecuon Lo LreaL famlllal hypercholesLrolemla
luA advlsory board recenLly (CcL 2012) voLed ln favor
Lo recommend approval.
SynLheuc 20 nL (Cenzyme)
AccumulaLes ln Lhe llver where lL LargeLs
ApollpoproLeln 8 and reduces Lhe formauon of Low-
denslLy-LlpoproLeln (bad cholesLerol").
1herapeuuc anusense
8eLa-Lhalassemla ls a blood dlsorder where red blood
cells cannoL carry enough oxygen because of defecuve
hemoglobln.
1he dlsease someumes sLem from aberranL spllclng of
Lhe beLa-globulln pre-m8nA
Anusense ollgonucleoudes have been deslgned LhaL
LargeL Lhe muLanL spllce slLe beLween exons Lwo and
Lhree ln Lhe beLa-globulln gene.
1he anusense ollgonucleoude correcLs Lhe spllclng
pauern and resLores Lhe correcL proLeln ln red blood
cells Laken from pauenLs wlLh beLa-Lhalessemla.
8eLa-Lhalassemla
1herapeuuc anusense
Lxample: Plv LreaLmenL uslng 8nA
Puman
lmmunodeclency vlrus
1argeLs Cu4
+
-1 cells and
Lakes over Lhe cellular
machlnery
Leads Lo low levels of
Cu4
+
-1 cells whlch leads Lo
loss of cell-medlaLed
lmmunlLy (AluS)
8apldly muLaung
A slngle lndlvldual usually
carrles several varlanLs of
Lhe vlrus
8nAl as LreaLmenL
LxLracL sLem cells from Lhe
blood
lnfecL exLracLed cells wlLh
a lenuvlrus carrylng an
Plv-Largeung sh8nA ln lLs
genome
knock ouL Lhe lmmune
sysLem of Lhe pauenL
uL Lhe lnfecLed sLem cells
back ln
Should resulL ln Plv-
reslsLanL 1-cells
llrsL lenuvlrus-sh8nA sLem cell LreaLed
man - leb 19, 2008
8ossl &$ +/., Ceneuc Lheraples agalnsL Plv, naL. 8loLechnol. 2007