SMALL INTERFERING RNA
(
si
RNA
)
AND RIBOSOME RNA
(
r
RNA
)
IV
.
1
S
m
a
ll
I
nt
e
rf
e
ring
RNA
(
si
RNA
)
A
.
I
ntro
d
u
c
tion
I
n
2006
,
th
e N
o
be
l
P
riz
e
in physiology or m
ed
i
c
in
e
w
a
s
a
w
a
r
ded
to
A
n
d
r
e
w
Z
.
F
ir
e a
n
d C
r
a
ig
C
.
Me
llo
,
two
A
m
e
ri
ca
n prof
e
ssors from
S
t
a
nfor
d U
niv
e
rsity
a
n
d
th
e U
niv
e
rsity of
Ma
ss
ac
hus
e
tts
,
r
e
sp
ec
tiv
e
ly
.
T
h
e
ir
b
r
ea
kthrough
d
is
c
ov
e
ry from nin
e
y
ea
rs
ea
rli
e
r r
e
v
ea
l
ed
th
a
t
d
ou
b
l
e
-
str
a
n
ded RNA
(
d
s
RNA
)
ca
n suppr
e
ss th
e ac
tivity or
e
xpr
e
ssion
of
a
sp
ec
ifi
c
g
e
n
e
in
a
w
a
y th
a
t
de
p
e
n
d
s on homology
,
a
ph
e
nom
e
non
now r
ec
ogniz
ed a
s
RNA
int
e
rf
e
r
e
n
ce
(
RNA
i
).
T
h
e e
xpr
e
ssion of g
e
n
e
s is
a c
ru
c
i
a
l
a
n
d
fun
da
m
e
nt
a
l pro
ce
ss in
a
ll living org
a
nisms
.
T
ypi
ca
lly
,
g
e
n
e e
xpr
e
ssion o
cc
urs in
c
hromosom
e
s within th
e
nu
c
l
e
us
,
with th
e
n
e
xt ph
a
s
e
involving prot
e
in synth
e
sis in th
e c
ytopl
a
sm
.
Ge
n
e
ti
c
m
a
t
e
ri
a
l
,
i
de
ntifi
ed a
s
de
oxyri
b
onu
c
l
e
i
c ac
i
d
(
DNA
),
w
a
s
d
is
c
ov
e
r
ed
in
1942
,
a
n
d
its
d
ou
b
l
e
-
h
e
lix stru
c
tur
e
w
a
s
e
lu
c
i
da
t
ed
in
1953
.
A
noth
e
r
form of nu
c
l
e
i
c ac
i
d
,
singl
e
-
str
a
n
ded
ri
b
onu
c
l
e
i
c ac
i
d
(
RNA
),
s
e
rv
e
s
a
s
a
m
e
ss
e
ng
e
r th
a
t tr
a
nsl
a
t
e
s
DNA
into its fin
a
l pro
d
u
c
t
,
prot
e
in
,
a
s
outlin
ed
in th
e ce
ntr
a
l
d
ogm
a
of mol
ec
ul
a
r
b
iology
.
T
h
e e
m
e
rg
e
n
ce
of g
e
n
e
th
e
r
a
py
be
g
a
n with
a
pivot
a
l
d
is
c
ov
e
ry
in
2001
,
r
e
v
ea
ling th
a
t
RNA c
oul
d e
ff
ec
tiv
e
ly sil
e
n
ce
g
e
n
e e
xpr
e
ssion
.
U
nlik
e
tr
ad
ition
a
l m
e
tho
d
s th
a
t
a
im
ed
to r
e
pl
ace de
f
ec
tiv
e
g
e
n
e
s
,
g
e
n
e
th
e
r
a
py fo
c
us
e
s on r
e
p
a
iring m
e
ss
e
ng
e
r
RNA
(
m
RNA
)
b
y l
e
v
e
r
a
ging
th
e ce
ll
'
s inh
e
r
e
nt r
e
gul
a
tory m
ec
h
a
nisms to suppr
e
ss h
a
rmful
e
ff
ec
ts
.
T
his inhi
b
ition o
cc
urs
d
uring th
e
tr
a
nsl
a
tion st
a
g
e
,
wh
e
r
e RNA
mol
ec
ul
e
s
d
isrupt prot
e
in synth
e
sis
b
y int
e
rf
e
ring with ri
b
osom
e
r
ec
ognition of m
RNA
s
e
qu
e
n
ce
s
.
T
his
a
ppro
ac
h
,
known
a
s
RNA
int
e
rf
e
r
e
n
ce
(
RNA
i
),
in
c
lu
de
s t
ec
hniqu
e
s su
c
h
a
s
a
ntis
e
ns
e RNA a
n
d
sm
a
ll int
e
rf
e
ring
RNA
(
si
RNA
).
A
lthough r
e
l
a
tiv
e
ly n
e
w
,
RNA
i h
a
s
r
e
volutioniz
ed
mol
ec
ul
a
r
b
iology sin
ce
its
d
is
c
ov
e
ry in n
e
m
a
to
de
s in
1998
,
g
a
ining wi
de
spr
ead ad
option
d
u
e
to its high imp
ac
t
a
n
d be
ing
h
a
il
ed a
s
a b
r
ea
kthrough
.
I
ts import
a
n
ce
in
b
iot
ec
h r
e
s
ea
r
c
h is now
lik
e
n
ed
to th
a
t of th
e
polym
e
r
a
s
e c
h
a
in r
eac
tion
(
PCR
),
a
n
d RNA
i is
e
xp
ec
t
ed
to
bec
om
e a
st
a
n
da
r
d
t
ec
hniqu
e ac
ross lif
e
s
c
i
e
n
ce
s
l
ab
or
a
tori
e
s worl
d
wi
de
.
RNA
int
e
rf
e
r
e
n
ce
(
RNA
i
)
is
a
n
e
volution
a
ry
de
f
e
ns
e
m
ec
h
a
nism
foun
d
in pl
a
nts
a
n
d
low
e
r inv
e
rt
eb
r
a
t
e
s th
a
t h
e
lps prot
ec
t
a
g
a
inst vir
a
l
inf
ec
tions
a
n
d da
m
a
g
e ca
us
ed b
y th
e
intro
d
u
c
tion of for
e
ign g
e
n
e
ti
c
m
a
t
e
ri
a
l
.
T
his pro
ce
ss fun
c
tions
b
y s
e
l
ec
tiv
e
ly inhi
b
iting g
e
n
e
e
xpr
e
ssion through th
e b
r
ea
k
d
own of m
RNA a
t sp
ec
ifi
c
s
e
qu
e
n
ce
s
.
T
h
e
k
e
y pl
a
y
e
r in
RNA
i
'
s
ac
tion is
a
mol
ec
ul
e
known
a
s sm
a
ll int
e
rf
e
ring
RNA
(
si
RNA
),
whi
c
h m
ed
i
a
t
e
s this t
a
rg
e
t
ed
g
e
n
e
sil
e
n
c
ing
.
C
lini
c
i
a
ns
h
a
v
e be
gun l
e
v
e
r
a
ging
RNA
i in tr
ea
ting
c
on
d
itions lik
e Pa
rkinson
'
s
d
is
ea
s
e
,
h
e
p
a
titis
,
HIV
,
a
n
d
v
a
rious
ca
n
ce
rs
,
b
y
e
ith
e
r
d
ir
ec
tly t
a
rg
e
ting
d
is
ea
s
e
-
ca
using g
e
n
e
s or
a
tt
ac
king g
e
n
e
s th
a
t m
a
k
e ca
n
ce
r
ce
lls
r
e
sist
a
nt to
c
h
e
moth
e
r
a
py
.
Re
s
ea
r
c
h
e
rs fo
c
using on
RNA
mol
ec
ul
e
s h
a
v
e de
monstr
a
t
ed
promising out
c
om
e
s
,
showing th
a
t
RNA
int
e
rf
e
r
e
n
ce
(
RNA
i
)
ca
n
be
e
ff
ec
tiv
e
in m
a
mm
a
li
a
n
ce
lls
.
I
niti
a
l in vitro stu
d
i
e
s h
a
v
e
progr
e
ss
ed
to
in vivo r
e
s
ea
r
c
h to furth
e
r
e
xplor
e RNA
i m
ec
h
a
nisms in th
e
s
e ce
lls
.
RNA
i t
ec
hnology is
ad
v
a
n
c
ing r
a
pi
d
ly
,
p
a
rti
c
ul
a
rly in m
ed
i
c
in
e
,
a
gri
c
ultur
e
,
a
n
d a
qu
ac
ultur
e
.
I
n m
ed
i
c
in
e
,
its
a
ppli
ca
tion is growing
swiftly
,
e
sp
ec
i
a
lly in g
e
n
e
th
e
r
a
py for
c
on
d
itions lik
e
h
e
p
a
titis
A a
n
d B
,
ca
n
ce
r
,
a
n
d
tumors
.
RNA
i is us
ed
for g
e
n
e
sil
e
n
c
ing
,
d
is
ea
s
e
mo
de
l
v
a
li
da
tion
,
d
rug
ac
tivity
a
ss
e
ssm
e
nt
,
a
n
d
th
e
i
de
ntifi
ca
tion of n
e
w
d
rug
ca
n
d
i
da
t
e
s
.
I
t
a
lso shows pot
e
nti
a
l
a
s
a
tr
ea
tm
e
nt for h
e
p
a
titis
B
virus
(
HBV
)
b
y t
a
rg
e
ting vir
a
l
RNA
to r
ed
u
ce
virus pro
d
u
c
tion
a
n
d
improv
e
immun
e
r
e
spons
e
,
l
ead
ing to v
a
rious m
ed
i
ca
l
b
iot
ec
hnology
e
xp
e
rim
e
nts to
de
v
e
lop
RNA
i
a
s
a
tr
ea
tm
e
nt for
HBV
inf
ec
tions
through g
e
n
e
ti
c e
ngin
ee
ring t
ec
hniqu
e
s
.
B
.
S
i
RNA
stru
c
tur
e
si
RNA
mol
ec
ul
e
s
a
r
e
sm
a
ll
,
c
onsisting of only
21
-
25
nu
c
l
e
oti
de
s
with two unp
a
ir
ed
nu
c
l
e
oti
de
s
a
t th
e
3
'
e
n
d
s
.
T
h
e
s
e
mol
ec
ul
e
s
a
r
e
g
e
n
e
r
a
t
ed b
y th
e e
nzym
e D
i
ce
r
,
a
ri
b
onu
c
l
ea
s
e
th
a
t us
e
s
ATP
to
r
ec
ogniz
e a
n
d c
l
ea
v
e d
ou
b
l
e
-
str
a
n
ded RNA
into sm
a
ll
e
r fr
a
gm
e
nts
.
si
RNA ca
n
a
lso
be de
riv
ed
from short h
a
irpin
RNA
,
a
stru
c
tur
e
form
ed
from
a
singl
e RNA
str
a
n
d
th
a
t fol
d
s
bac
k on its
e
lf lik
e a
h
a
irpin
,
whi
c
h
is
a
lso
c
l
ea
v
ed b
y
D
i
ce
r
.
T
h
e
h
e
li
ca
s
e e
nzym
e
unwin
d
s th
e
hy
d
rog
e
n
b
on
d
s of th
e
si
RNA
,
a
llowing th
e a
ntis
e
ns
e
str
a
n
d
to
b
in
d
with
a
prot
e
in to form th
e RNA
-
in
d
u
ced
sil
e
n
c
ing
c
ompl
e
x
(
RISC
).
O
n
ce
ac
tiv
a
t
ed
,
this
c
ompl
e
x
c
l
ea
v
e
s m
RNA
th
a
t
c
ont
a
ins s
e
qu
e
n
ce
s
homologous to th
e
si
RNA
.
H
ow
e
v
e
r
,
th
e
st
ab
ility
a
n
d de
liv
e
ry of si
RNA
in th
e
r
a
p
e
uti
c a
ppli
ca
tions pr
e
s
e
nt
c
h
a
ll
e
ng
e
s
,
a
s si
RNA
is
ea
sily
de
gr
aded
in th
e b
o
d
y
.
T
o
add
r
e
ss this
,
si
RNA
st
ab
ility
ca
n
be e
nh
a
n
ced
through
c
h
e
mi
ca
l mo
d
ifi
ca
tions to its ri
b
os
e
group
a
n
d ba
s
e
s
,
or
b
y
de
liv
e
ring si
RNA d
ir
ec
tly or vi
a
pl
a
smi
d
s or virus
e
s
e
n
c
o
d
ing th
e
t
a
rg
e
t
s
e
qu
e
n
ce
.
I
n si
RNA
mol
ec
ul
e
s
,
hy
d
rog
e
n
b
on
d
ing
be
tw
ee
n
ba
s
e
s
ca
n o
cc
ur
through non
-
Wa
tson
-
C
ri
c
k or non
-
ca
noni
ca
l
ba
s
e
p
a
iring
,
whi
c
h pl
a
ys
a c
ru
c
i
a
l rol
e
in sh
a
ping th
e
stru
c
tur
a
l
a
rr
a
ng
e
m
e
nt of si
RNA
.
U
nlik
e
DNA
mol
ec
ul
e
s
,
wh
e
r
e ba
s
e
p
a
iring typi
ca
lly follows th
e
st
a
n
da
r
d
Wa
tson
-
C
ri
c
k mo
de
l
,
si
RNA e
xhi
b
its uniqu
e
stru
c
tur
a
l
c
h
a
r
ac
t
e
risti
c
s
.
W
h
e
n si
RNA
is mo
d
ifi
ed
,
its s
ec
on
da
ry stru
c
tur
e
is form
ed
,
typi
ca
lly
a
s
a d
ou
b
l
e
h
e
lix or
d
ou
b
l
e
str
a
n
d
,
whi
c
h is
e
ss
e
nti
a
l for int
e
r
ac
tions with
oth
e
r mol
ec
ul
e
s
.
T
h
e
s
e
mo
d
ifi
ca
tions
a
r
e
sp
ec
ifi
ca
lly
de
sign
ed
to
e
nh
a
n
ce
th
e
fun
c
tion
a
l prop
e
rti
e
s of si
RNA
.
T
h
e
ri
b
os
e
group
ca
n
be
mo
d
ifi
ed b
y lo
c
king it into
a
st
ab
l
e c
onform
a
tion
,
forming
a L
o
c
k
ed
N
u
c
l
e
i
c Ac
i
d
(
LNA
)
through
a
m
e
thyl
e
n
e b
ri
d
g
e
th
a
t
c
onn
ec
ts th
e
2
'-
4
oxyg
e
n with th
e
4
'-
ca
r
b
on on th
e
ri
b
os
e
ring
,
k
ee
ping it in
a C
3
'-
e
n
d
o
RNA c
onform
a
tion
.
Add
ition
a
lly
,
ba
s
e
p
a
ir mo
d
ifi
ca
tions
ca
n
e
nh
a
n
ce
si
RNA
st
ab
ility
b
y r
e
pl
ac
ing non
-
ca
noni
ca
l
CU ba
s
e
p
a
irs with
ca
noni
ca
l
CG ba
s
e
p
a
irs
,
l
ead
ing to strong
e
r hy
d
rog
e
n
b
on
d
ing
.
T
his
e
x
c
h
a
ng
e
is
ba
s
ed
on th
e
f
ac
t th
a
t
CG ba
s
e
p
a
irs
,
with thr
ee
hy
d
rog
e
n
b
on
d
s
,
a
r
e
mor
e
st
ab
l
e
th
a
n
CU
p
a
irs
,
whi
c
h only h
a
v
e
two
.
Re
s
ea
r
c
h
h
a
s shown th
a
t su
c
h mo
d
ifi
ca
tions improv
e
si
RNA d
upl
e
x st
ab
ility
a
n
d
e
nh
a
n
ce
th
e a
ffinity
be
tw
ee
n
LNA
-
mo
d
ifi
ed
si
RNA a
n
d
th
e A
rgon
a
ut
prot
e
in
.
F
urth
e
rmor
e
,
RNA
r
e
si
d
u
e
s p
a
ir
ed
with
GC
str
a
n
d
s
de
monstr
a
t
e
gr
ea
t
e
r
c
onv
e
rg
e
n
ce
th
a
n thos
e
p
a
ir
ed
with
CG
str
a
n
d
s
.
T
his stu
d
y inv
e
stig
a
t
e
s th
e d
o
c
king of
A
rgon
a
ut prot
e
in with
b
oth
si
RNA a
n
d
its mo
d
ifi
ed
forms
,
a
iming to
e
lu
c
i
da
t
e
th
e
int
e
r
ac
tion
a
n
d
positioning
be
tw
ee
n th
e
m
.
C
.
Ba
si
c Mec
h
a
nisms of
RNA
i
T
h
e RNA
int
e
rf
e
r
e
n
ce
(
RNA
i
)
m
ec
h
a
nism involv
e
s s
e
v
e
r
a
l k
e
y
st
e
ps
,
st
a
rting with th
e e
ntry of
a d
ou
b
l
e
-
str
a
n
ded RNA
(
d
s
RNA
)
into th
e ce
ll
'
s
c
ytopl
a
sm
,
e
ith
e
r n
a
tur
a
lly or synth
e
ti
ca
lly
.
T
his
d
s
RNA
is qui
c
kly i
de
ntifi
ed b
y
a
n
e
nzym
e ca
ll
ed D
i
ce
r
,
whi
c
h
sli
ce
s th
e d
s
RNA
into short s
e
gm
e
nts of
ab
out
21
ba
s
e
p
a
irs
,
eac
h
e
n
d
ing with two nu
c
l
e
oti
de
s
a
t th
e
3
'-
e
n
d
.
A
long with
v
a
rious
c
o
-
f
ac
tors
,
D
i
ce
r r
e
m
a
ins highly
ac
tiv
e
in
c
utting th
e
d
s
RNA
into m
a
ny sm
a
ll fr
a
gm
e
nts known
a
s sm
a
ll int
e
rf
e
ring
RNA
(
si
RNA
),
whi
c
h r
e
t
a
in th
e
ir
d
ou
b
l
e
-
str
a
n
ded
stru
c
tur
e
.
T
h
e
s
e
si
RNA
s
a
r
e
th
e
n r
ec
ogniz
ed b
y th
e RNA
-
I
n
d
u
ced S
il
e
n
c
ing
C
ompl
e
x
(
RISC
),
c
ont
a
ining th
e A
rgon
a
ut
e
nzym
e
,
wh
e
r
e
th
e
si
RNA
is
c
l
ea
v
ed
into
a
singl
e
str
a
n
d
th
a
t
ac
tiv
a
t
e
s th
e RISC
c
ompl
e
x
.
O
n
ce a
n
ac
tiv
e RISC
is form
ed
,
it imm
ed
i
a
t
e
ly s
ee
ks
out m
e
ss
e
ng
e
r
RNA
(
m
RNA
)
th
a
t h
a
s r
ece
ntly
e
xit
ed
th
e ce
ll
nu
c
l
e
us following tr
a
ns
c
ription from
DNA
.
T
h
e
singl
e
-
str
a
n
ded
si
RNA
within
RISC acc
ur
a
t
e
ly i
de
ntifi
e
s th
e
t
a
rg
e
t m
RNA a
n
d
b
in
d
s to it through
c
ompl
e
m
e
nt
a
ry
ba
s
e
p
a
iring
.
Eac
h
RISC
c
ont
a
ins
e
n
d
onu
c
l
ea
s
e e
nzym
e ac
tivity
,
sp
ec
ifi
ca
lly within th
e
A
rgon
a
ut su
b
unit
,
whi
c
h is r
e
sponsi
b
l
e
for
c
l
ea
ving th
e
t
a
rg
e
t
m
RNA
into fr
a
gm
e
nts
,
th
e
r
eb
y
d
isrupting th
e
tr
a
nsf
e
r of g
e
n
e
ti
c
inform
a
tion from
DNA
to prot
e
in
.
T
h
e
s
e
m
RNA
fr
a
gm
e
nts
a
r
e
th
e
n n
a
tur
a
lly
de
gr
aded b
y
e
n
d
og
e
nous m
ec
h
a
nisms
.
D
.
RNA I
nt
e
rf
e
r
e
n
ce Tec
hnology
RNA
int
e
rf
e
r
e
n
ce
is
a
pro
ce
ss th
a
t pr
e
v
e
nts g
e
n
e e
xpr
e
ssion
b
y
l
e
v
e
r
a
ging th
e c
ompl
e
m
e
nt
a
ry n
a
tur
e
of
RNA
to sm
a
ll
RNA
fr
a
gm
e
nts
,
typi
ca
lly
21
-
23
nu
c
l
e
oti
de
s long
.
T
h
e
s
e
sm
a
ll
RNA
fr
a
gm
e
nts
a
r
e
g
e
n
e
r
a
t
ed
wh
e
n th
e D
i
ce
r
e
nzym
e a
n
d A
rgon
a
ut
2
prot
e
in
c
l
ea
v
e
long
d
ou
b
l
e
-
str
a
n
ded RNA
pr
e
s
e
nt in
ce
lls
.
T
h
e
r
e
sulting
RNA
th
e
n
b
in
d
s to
homologous s
ec
tions of m
RNA
,
trigg
e
ring th
e RNA
s
e H e
nzym
e
to
de
gr
ade
th
e
m
RNA
or o
b
stru
c
t its tr
a
nsl
a
tion
.
T
his m
ec
h
a
nism s
e
rv
e
s
a
s
a de
f
e
ns
e
syst
e
m in
e
uk
a
ryoti
c
org
a
nisms
a
g
a
inst for
e
ign
RNA
mol
ec
ul
e
s
,
su
c
h
a
s virus
e
s
a
n
d
tr
a
nspos
ab
l
e e
l
e
m
e
nts
.
I
n pl
a
nts
,
RNA
int
e
rf
e
r
e
n
ce
prot
ec
ts
a
g
a
inst vir
a
l inf
ec
tions
,
whil
e
in oth
e
r org
a
nisms
,
it limits th
e
spr
ead
of tr
a
nspos
ab
l
e e
l
e
m
e
nts
.
A
ft
e
r th
e d
ou
b
l
e
-
str
a
n
ded RNA
is
c
ut
,
it s
e
p
a
r
a
t
e
s into singl
e
str
a
n
d
s th
a
t
c
om
b
in
e
with
th
e A
rgon
a
ut
2
prot
e
in to form th
e RNA
-
in
d
u
ced
sil
e
n
c
ing
c
ompl
e
x
(
RISC
).
T
his
c
ompl
e
x gui
de
s th
e RNA
to
b
in
d
with th
e c
ompl
e
m
e
nt
a
ry
m
RNA
s
e
qu
e
n
ce
,
in
ac
tiv
a
ting th
e
g
e
n
e
.
RNA
int
e
rf
e
r
e
n
ce ca
n
in
ac
tiv
a
t
e
g
e
n
e
s through thr
ee
prim
a
ry m
ec
h
a
nisms
:
de
gr
ada
tion of
th
e RNA
-
g
e
n
e c
ompl
e
x
,
inhi
b
ition of tr
a
nsl
a
tion
b
y pr
e
v
e
nting
ri
b
osom
a
l
acce
ss to th
e
g
e
n
e
s
e
qu
e
n
ce
,
or
c
hrom
a
tin mo
d
ifi
ca
tion th
a
t
l
ead
s to g
e
n
e
in
ac
tiv
a
tion
.
T
o h
e
lp un
de
rst
a
n
d RNA
int
e
rf
e
r
e
n
ce
(
RNA
i
)
a
n
d
its rol
e
in g
e
n
e
in
ac
tiv
a
tion
,
F
igur
e
s
7
a
n
d
8
illustr
a
t
e
th
e
pro
ce
ss
b
y whi
c
h m
RNA
is
hy
b
ri
d
iz
ed b
y short
RNA
(
s
RNA
).
T
his pro
ce
ss is f
ac
ilit
a
t
ed b
y th
e RNA
-
in
d
u
ced
sil
e
n
c
ing
c
ompl
e
x
(
RISC
),
whi
c
h is
a
prot
e
in
c
ompl
e
x in
c
lu
d
ing
th
e
s
RNA
-
AGO e
nzym
e
,
forming
a c
om
b
in
a
tion th
a
t pr
e
v
e
nts th
e
tr
a
nsl
a
tion of m
RNA
into prot
e
ins
.
I
niti
a
l
RNA
i stu
d
i
e
s r
e
v
ea
l
ed
th
a
t
inj
ec
ting
d
ou
b
l
e
-
str
a
n
ded RNA
into th
e
n
e
m
a
to
de
*
Cae
norh
abd
itis
e
l
e
g
a
ns
*
e
ff
ec
tiv
e
ly in
ac
tiv
a
t
ed
g
e
n
e
s with homologous s
e
qu
e
n
ce
s to
th
e
inj
ec
t
ed RNA
,
sp
a
rking furth
e
r r
e
s
ea
r
c
h into
RNA
i for t
a
rg
e
t
ed
g
e
n
e
in
ac
tiv
a
tion
.
RNA
i is pr
e
s
e
nt
ac
ross v
a
rious org
a
nisms
,
in
c
lu
d
ing
fungi
,
pl
a
nts
,
worms
,
mi
ce
,
a
n
d
pot
e
nti
a
lly hum
a
ns
.
T
h
e
r
e a
r
e
two
prim
a
ry typ
e
s of
RNA
involv
ed
in
RNA
i
:
mi
c
ro
RNA
(
mi
RNA
)
a
n
d
short
int
e
rf
e
ring
RNA
(
si
RNA
).
mi
RNA
,
a
short
RNA
of
ab
out
21
-
23
nu
c
l
e
oti
de
s
,
origin
a
t
e
s from within
ce
lls
a
n
d ca
n in
ac
tiv
a
t
e
g
e
n
e
s
d
u
e
to its
c
ompl
e
m
e
nt
a
ry nu
c
l
e
oti
de
s
e
qu
e
n
ce
s
.
T
h
e
s
e
mi
RNA
s
a
r
e
pro
d
u
ced
through tr
a
ns
c
ription of th
e ce
ll
'
s int
e
rn
a
l
DNA
,
whi
c
h th
e
n
fol
d
s to form
a
h
a
irpin stru
c
tur
e
.
T
h
e ac
tion m
ec
h
a
nism of mi
RNA d
iff
e
rs from th
a
t of si
RNA
,
a
s
mi
RNA d
o
e
s not
d
ir
ec
tly inhi
b
it th
e
tr
a
nsl
a
tion pro
ce
ss
b
ut inst
ead
b
in
d
s to th
e e
n
d
of th
e
m
RNA
in th
e
untr
a
nsl
a
t
ed
r
e
gion
(
UTR
),
s
e
rving
m
e
r
e
ly
a
s
a
sign
a
l to imp
ede
m
RNA
tr
a
nsl
a
tion
.
I
n
c
ontr
a
st
,
si
RNA
,
whi
c
h
c
onsists of short
d
ou
b
l
e
-
str
a
n
ded RNA a
pproxim
a
t
e
ly
20
-
30
nu
c
l
e
oti
de
s long
,
is g
e
n
e
r
a
t
ed
wh
e
n th
e D
i
ce
r
e
nzym
e c
l
ea
v
e
s long
e
xog
e
nous
d
ou
b
l
e
-
str
a
n
ded RNA
,
su
c
h
a
s th
a
t from virus
e
s
,
e
nt
e
ring
th
e ce
ll
.
T
his
e
nzym
e
,
sp
ec
ifi
c
to
d
ou
b
l
e
-
str
a
n
ded RNA
,
pro
d
u
ce
s
si
RNA
with
eac
h str
a
n
d
f
ea
turing
a
5
'-
phosph
a
t
e a
n
d a
3
'
hy
d
roxyl
group
,
l
ea
ving two unp
a
ir
ed
nu
c
l
e
oti
de
s
(
ov
e
rh
a
ngs
)
a
t th
e
3
'
e
n
d
.
A
mong th
e
s
e
str
a
n
d
s
,
th
e
on
e
r
e
sponsi
b
l
e
for sil
e
n
c
ing is r
e
f
e
rr
ed
to
a
s
th
e
gui
de
,
whil
e
th
e
oth
e
r
,
whi
c
h is
de
gr
aded
,
is known
a
s th
e
m
e
ss
e
ng
e
r
.
I
n r
ece
nt y
ea
rs
,
RNA
int
e
rf
e
r
e
n
ce
(
RNA
i
)
h
a
s
e
m
e
rg
ed a
s
a
promising m
e
tho
d
for inhi
b
iting g
e
n
e e
xpr
e
ssion
ac
ross v
a
rious
org
a
nisms
,
in
c
lu
d
ing pl
a
n
a
ri
a
,
T
ryp
a
nosom
a
,
ins
ec
ts
,
m
a
mm
a
ls
,
pl
a
nts
,
a
n
d a
nim
a
ls
.
T
h
e a
vi
a
n l
e
ukosis virus
(
ALV
)
r
e
m
a
ins
a
glo
ba
l
c
on
ce
rn
for poultry
d
u
e
to th
e
l
ac
k of
a
n
e
ff
ec
tiv
e
v
acc
in
e a
n
d
th
e e
m
e
rg
e
n
ce
of virus mut
a
tions th
a
t
c
ontri
b
ut
e
to r
e
sist
a
n
ce
.
C
ons
e
qu
e
ntly
,
RNA
i
t
ec
hnology is
be
ing
ad
v
a
n
ced a
s
a
pot
e
nti
a
l
a
ntivir
a
l tr
ea
tm
e
nt for
ALV
inf
ec
tion
.
Add
ition
a
lly
,
th
e
us
e
of
e
xpr
e
ssion v
ec
tors to g
e
n
e
r
a
t
e RNA
c
ompl
e
m
e
nt
a
ry to th
e ALV
virus h
a
s shown signifi
ca
nt
e
ff
ec
tiv
e
n
e
ss in
r
ed
u
c
ing p
a
thog
e
ni
c
prot
e
in l
e
v
e
ls in
b
ir
d ce
ll
c
ultur
e
s
.
S
imil
a
rly
,
th
e
H
5
N
1
su
b
typ
e
of th
e a
vi
a
n flu virus pos
e
s
a
s
e
v
e
r
e
thr
ea
t not only to
poultry f
a
rms
b
ut
a
lso to hum
a
n h
ea
lth
,
with no
e
ff
ec
tiv
e
v
acc
in
e
or
a
ntivir
a
l tr
ea
tm
e
nt
c
urr
e
ntly
a
v
a
il
ab
l
e
.
P
r
e
v
e
nting hum
a
n inf
ec
tion
r
e
li
e
s on
c
ontrolling th
e
virus within poultry popul
a
tions
,
b
ut tr
ad
ition
a
l
m
ea
sur
e
s su
c
h
a
s string
e
nt surv
e
ill
a
n
ce a
n
d b
ir
d c
ulling h
a
v
e
prov
e
n
insuffi
c
i
e
nt
.
T
h
e
r
e
for
e
,
e
xploring nov
e
l tr
ea
tm
e
nts lik
e a
ntivir
a
l
c
h
e
moth
e
r
a
py
a
n
d RNA
int
e
rf
e
r
e
n
ce
pr
e
s
e
nts
a
promising
a
lt
e
rn
a
tiv
e
to
add
r
e
ss this
c
h
a
ll
e
ng
e
.
B
ovin
e
vir
a
l
d
i
a
rrh
ea
virus
(
BVDV
)
is
a
signifi
ca
nt p
a
thog
e
n
r
e
sponsi
b
l
e
for
c
onsi
de
r
ab
l
e
loss
e
s in th
e
glo
ba
l
ca
ttl
e
f
a
rming s
ec
tor
.
C
urr
e
nt tr
ea
tm
e
nt m
e
tho
d
s for this
d
is
ea
s
e
h
a
v
e
not yi
e
l
ded
s
a
tisf
ac
tory out
c
om
e
s
.
Rece
nt r
e
s
ea
r
c
h h
a
s
de
monstr
a
t
ed
th
a
t using
a
pl
a
smi
d ca
p
ab
l
e
of g
e
n
e
r
a
ting short
RNA
s
e
qu
e
n
ce
s
,
whi
c
h
ca
n
b
in
d
to
sp
ec
ifi
c
r
e
gions of th
e
virus g
e
nom
e
,
inhi
b
its th
e
virus
.
Add
ition
a
lly
,
e
mploying pl
a
smi
d
s th
a
t pro
d
u
ce
two
d
istin
c
t short
RNA
s h
a
s
bee
n
shown to
e
nh
a
n
ce
th
e e
ff
ec
tiv
e
n
e
ss of this inhi
b
ition
.
E
.
SINGLE STRAND
-
SMALL INTERFERING RNA TECHNOLOGY
I
n
c
ontr
a
st to si
RNA
,
whi
c
h is
a
short
d
ou
b
l
e
-
str
a
n
ded RNA
,
ss
-
si
RNA
is
a
mo
d
ifi
ed
short singl
e
-
str
a
n
ded RNA de
sign
ed
to
e
nh
a
n
ce
its
sp
ec
ifi
c
ity for
b
in
d
ing to t
a
rg
e
t m
RNA
.
T
his t
ec
hnology h
a
s
bee
n
shown to
be
signifi
ca
ntly mor
e
pot
e
nt
a
n
d
s
e
l
ec
tiv
e
—
b
y
a
f
ac
tor of
100
a
n
d
30
,
r
e
sp
ec
tiv
e
ly
—
c
omp
a
r
ed
to unmo
d
ifi
ed
mi
RNA
or si
RNA
.
T
h
e
m
ec
h
a
nism of
ac
tion for ss
-
si
RNA
mirrors th
a
t of
RNA
int
e
rf
e
r
e
n
ce
(
RNA
i
),
f
ac
ilit
a
t
ed b
y th
e AGO e
nzym
e
.
W
h
e
n using
a
ntis
e
ns
e
oligonu
c
l
e
oti
de
s
(
ASO
),
th
e
form
a
tion of
a
n
ASO
-
m
RNA d
upl
e
x
prompts
RNA
s
e ac
tivity to
de
gr
ade
th
e
m
RNA
or inhi
b
it tr
a
nsl
a
tion
through
ASO
int
e
rf
e
r
e
n
ce
.
F
or mi
RNA ac
tion
,
th
e
pr
ec
ursor mi
RNA
(
a
pproxim
a
t
e
ly on
e
kilo
ba
s
e
p
a
ir
)
is pro
ce
ss
ed b
y th
e RNA
s
e D
ros
a
e
n
d
onu
c
l
ea
s
e III e
nzym
e
into pr
e
-
mi
RNA
(
ab
out
60
-
70
ba
s
e
s
),
whi
c
h is
th
e
n tr
a
nsport
ed
into th
e c
ytopl
a
sm
.
T
h
e
r
e
,
th
e RNA
s
e III e
n
d
onu
c
l
ea
s
e
e
nzym
e D
i
ce
r
c
l
ea
v
e
s th
e
pr
e
-
mi
RNA
,
l
ea
ving phosph
a
t
e
groups
a
t th
e
e
n
d
s
a
n
d
pro
d
u
c
ing m
a
tur
e
mi
RNA
.
A
ssist
ed b
y
AGO
,
th
e
m
a
tur
e
mi
RNA
p
a
irs with t
a
rg
e
t m
RNA
to
b
lo
c
k tr
a
nsl
a
tion
,
in
d
u
ce c
l
ea
v
a
g
e
,
or
de
st
ab
iliz
e
th
e
m
RNA
.
T
h
e
m
ec
h
a
nism of
ac
tion for singl
e
-
str
a
n
ded
si
RNA
(
ss
-
si
RNA
),
whi
c
h r
e
s
e
m
b
l
e
s mi
RNA
,
is illustr
a
t
ed
in
F
igur
e
5
.
S
in
ce
th
e
fun
c
tion
a
l form of mi
RNA
or si
RNA
th
a
t
e
ff
ec
tiv
e
ly int
e
r
ac
ts
with t
a
rg
e
t m
RNA
is singl
e
-
str
a
n
ded
,
th
e
stu
d
y
b
y
Y
u
a
n
d c
oll
ea
gu
e
s
(
2012
)
utiliz
ed
singl
e
-
str
a
n
ded
si
RNA
,
now known
a
s ss
-
si
RNA
.
M
o
d
ifi
ca
tions to ss
-
si
RNA a
r
e de
sign
ed
to
e
nh
a
n
ce
r
e
sist
a
n
ce
to
nu
c
l
ea
s
e e
nzym
e
s
,
prolong its pr
e
s
e
n
ce
in tissu
e
s
(
h
a
lf
-
lif
e
),
a
n
d b
oost
its
a
ffinity
a
n
d
pot
e
nti
a
l for
b
in
d
ing to
ce
lls in vivo
.
F
igur
e
6
de
pi
c
ts th
e
stru
c
tur
e
of mo
d
ifi
ed
ss
-
si
RNA
,
in
c
orpor
a
ting
add
itions su
c
h
a
s
2
'-
F
luoro
,
2
'-
O
-
Me
thyl
,
2
'-
O
-
Me
thoxy
e
thyl
,
thymi
d
in
e
,
a
n
d
phosph
a
t
e
or
vinylphosphon
a
t
e
.
T
h
e
in
c
lusion of vinylphosphon
a
t
e
h
e
lps pr
e
v
e
nt
nu
c
l
ea
s
e
-
m
ed
i
a
t
ed c
l
ea
v
a
g
e
in
ce
lls
a
n
d
improv
e
s th
e
nu
c
l
e
oti
de
’
s
st
ab
ility in vivo
.
F
urth
e
rmor
e
,
th
e
pr
e
s
e
n
ce
of phosph
a
t
e
groups is
be
li
e
v
ed
to
e
nh
a
n
ce
th
e b
in
d
ing
a
ffinity of ss
-
si
RNA
to s
e
rum prot
e
ins
,
th
e
r
eb
y in
c
r
ea
sing its
b
iologi
ca
l
a
v
a
il
ab
ility
.
C
h
e
mi
ca
l mo
d
ifi
ca
tions
m
a
k
e
ss
-
si
RNA
mor
e
st
ab
l
e
within
ce
lls
c
omp
a
r
ed
to unmo
d
ifi
ed
v
e
rsions
,
whi
c
h
de
gr
ade
mor
e
r
a
pi
d
ly
a
ft
e
r
ce
ll
e
ntry
,
a
s shown in
F
igur
e
7
.
T
his im
a
g
e a
lso in
d
i
ca
t
e
s th
a
t mo
d
ifi
ed
ss
-
si
RNA a
n
d d
ou
b
l
e
-
str
a
n
ded RNA
(
d
upl
e
x
RNA
)
int
e
r
ac
t with th
e AGO e
nzym
e
to form th
e
RISC c
ompl
e
x
,
whi
c
h th
e
n t
a
rg
e
ts
a
n
d
in
ac
tiv
a
t
e
s sp
ec
ifi
c
m
RNA
,
l
ead
ing to r
ed
u
ced
or
ab
s
e
nt prot
e
in
e
xpr
e
ssion
,
a
s
de
pi
c
t
ed
in
F
igur
e
F
.
RNA
i
a
s
a
th
e
r
a
p
e
uti
c
tool in mo
de
rn m
ed
i
c
in
e
Re
s
ea
r
c
h
e
rs
a
r
e
not only
e
xploring
RNA
int
e
rf
e
r
e
n
ce
(
RNA
i
)
in
low
e
r pl
a
nts
a
n
d a
nim
a
ls
b
ut
a
r
e a
lso striving to impl
e
m
e
nt this
fun
da
m
e
nt
a
l knowl
ed
g
e
in
c
lini
ca
l s
e
ttings
,
moving from l
ab
or
a
tory
r
e
s
ea
r
c
h to pr
ac
ti
ca
l
a
ppli
ca
tions
.
T
h
e
y
a
im to gr
a
sp th
e
mol
ec
ul
a
r
m
ec
h
a
nisms of
RNA
i whil
e
simult
a
n
e
ously
de
v
e
loping th
e
r
a
p
e
uti
c
int
e
rv
e
ntions
.
C
lini
c
i
a
ns h
a
v
e
initi
a
t
ed
th
e
us
e
of
RNA
i for tr
ea
ting
de
g
e
n
e
r
a
tiv
e d
is
ea
s
e
s su
c
h
a
s
a
g
e
-
r
e
l
a
t
ed
m
ac
ul
a
r
de
g
e
n
e
r
a
tion
(
AMD
)
a
n
d Pa
rkinson
'
s
,
a
s w
e
ll
a
s inf
ec
tious
d
is
ea
s
e
s lik
e
h
e
p
a
titis
a
n
d HIV
.
T
ri
a
ls h
a
v
e a
lso
be
gun for m
a
lign
a
nt
d
is
ea
s
e
s
,
fo
c
using on t
a
rg
e
ting
ca
n
ce
r
-
ca
using g
e
n
e
s or thos
e c
ontri
b
uting to
c
h
e
moth
e
r
a
py
r
e
sist
a
n
ce
.
F
or inst
a
n
ce
,
b
y l
a
t
e
2004
,
th
e FDA a
pprov
ed a c
lini
ca
l tri
a
l
for
RNA
i in
AMD
p
a
ti
e
nts
,
a
l
ead
ing
ca
us
e
of irr
e
v
e
rsi
b
l
e b
lin
d
n
e
ss
.
I
n
th
e
"
w
e
t
"
form of
AMD
,
whi
c
h involv
e
s
ab
norm
a
l
b
loo
d
v
e
ss
e
l growth
be
hin
d
th
e
r
e
tin
a
,
RNA
i is us
ed
to inhi
b
it th
e
ov
e
rpro
d
u
c
tion of
VEGF
(
v
a
s
c
ul
a
r
e
n
d
oth
e
li
a
l growth f
ac
tor
),
pot
e
nti
a
lly pr
e
v
e
nting
c
ompl
e
t
e
b
lin
d
n
e
ss
.
T
his tri
a
l r
e
pr
e
s
e
nts th
e
first hum
a
n
a
ppli
ca
tion of
RNA
i for
AMD
,
a
n
d
futur
e
tri
a
ls
a
r
e e
xp
ec
t
ed
to
add
r
e
ss oth
e
r
c
h
a
ll
e
nging
d
is
ea
s
e
s with this
a
ppro
ac
h
.
G
.
Ba
rri
e
rs to
T
h
e
r
a
p
e
uti
c U
s
e
T
h
e
pot
e
nti
a
l for
a
pplying
RNA
mol
ec
ul
e
s in m
ed
i
c
in
e
is
a
nti
c
ip
a
t
ed
to
bec
om
e a
r
ea
lity soon
.
N
on
e
th
e
l
e
ss
,
e
xp
e
rts
ac
knowl
ed
g
e
th
a
t
c
h
a
ll
e
ng
e
s must
be add
r
e
ss
ed
to
ac
hi
e
v
e
optim
a
l
r
e
sults with th
e
s
e RNA
mol
ec
ul
e
s
.
O
n
e
signifi
ca
nt
c
h
a
ll
e
ng
e
is th
e
n
eed
to intro
d
u
ce a
ntis
e
ns
e RNA a
n
d
si
RNA
into living
ce
lls r
a
th
e
r th
a
n
ce
ll
-
fr
ee e
nvironm
e
nts
.
A
ntis
e
ns
e RNA
must
a
voi
d de
gr
ada
tion
b
y
nu
c
l
ea
s
e e
nzym
e
s
,
whi
c
h
a
r
e
pr
e
v
a
l
e
nt in
b
oth th
e b
loo
d
str
ea
m
a
n
d
within
ce
lls
.
T
o
e
nh
a
n
ce
r
e
sist
a
n
ce
to th
e
s
e e
nzym
e
s
,
str
a
t
e
gi
e
s su
c
h
a
s r
e
pl
ac
ing oxyg
e
n in th
e ba
s
e b
ri
d
g
e
with sulfur to
c
r
ea
t
e a
mor
e
st
ab
l
e
phosphorothio
a
t
e b
ri
d
g
e
h
a
v
e bee
n
de
v
e
lop
ed
.
Add
ition
a
lly
,
e
ff
ec
tiv
e de
liv
e
ry of th
e
th
e
r
a
py into
ce
lls r
e
m
a
ins
a c
l
a
ssi
c
issu
e
in
g
e
n
e
ti
c
m
ed
i
c
in
e
.
Re
s
ea
r
c
h
e
rs h
a
v
e
work
ed
on
e
ngin
ee
ring
de
liv
e
ry
syst
e
ms for
ce
ll
c
ultur
e
,
with si
RNA de
liv
e
ry th
e
or
e
ti
ca
lly
ac
hi
e
v
ab
l
e
in
two w
a
ys
:
d
ir
ec
t intro
d
u
c
tion of synth
e
ti
c
si
RNA
or th
e
us
e
of pl
a
smi
d
s
or virus
e
s to pro
d
u
ce
th
e de
sir
ed
si
RNA
.
T
h
e
l
a
tt
e
r m
e
tho
d
is g
e
n
e
r
a
lly
pr
e
f
e
rr
ed d
u
e
to its long
e
r
-
l
a
sting
e
ff
ec
ts
.
F
r
ee
nu
c
l
e
i
c ac
i
d
s poss
e
ss
a
strong n
e
g
a
tiv
e c
h
a
rg
e d
u
e
to th
e
phosph
a
t
e
groups
a
long th
e
ir
bac
k
b
on
e
,
whi
c
h m
a
k
e
s th
e
m highly
solu
b
l
e
in w
a
t
e
r
b
ut not in th
e
lipi
d
-
ri
c
h
ce
ll m
e
m
b
r
a
n
e
.
T
o
e
nh
a
n
ce
th
e
ir
ce
llul
a
r
de
liv
e
ry
,
nu
c
l
e
i
c ac
i
d
s
ca
n
be c
om
b
in
ed
with
ca
rri
e
rs
de
sign
ed
to f
ac
ilit
a
t
e
th
e
ir tr
a
nsport into
ce
lls or
e
n
ca
psul
a
t
ed
in lipi
d
stru
c
tur
e
s su
c
h
a
s liposom
e
s
,
whi
c
h h
a
v
e bee
n
e
ff
ec
tiv
e
ly us
ed
for
tr
a
nsporting
a
mphot
e
ri
c
in
a
n
d ce
rt
a
in
ca
n
ce
r
d
rugs
.
Add
ition
a
lly
,
a
promising
a
ppro
ac
h involv
e
s using p
e
pti
de
lig
a
n
d
s from s
e
rpin
e
nzym
e
c
ompl
e
x r
ece
ptors to form
c
ompl
e
x
e
s with th
e
g
e
n
e
ti
c
m
a
t
e
ri
a
l
,
e
n
ab
ling t
a
rg
e
t
ed de
liv
e
ry to v
a
rious
ce
lls
.
De
spit
e
ongoing
c
h
a
ll
e
ng
e
s
in fin
d
ing
a
suit
ab
l
e de
liv
e
ry syst
e
m
,
e
xp
e
rts r
e
m
a
in hop
e
ful
.
S
om
e
sugg
e
st th
a
t
a
ntis
e
ns
e RNA a
n
d
si
RNA
might
be de
liv
e
r
ed
without vir
a
l
v
ec
tors or sp
ec
i
a
liz
ed
syst
e
ms
,
a
s si
RNA c
oul
d
pot
e
nti
a
lly
be
intro
d
u
ced d
ir
ec
tly into tissu
e
s
.
H
ow
e
v
e
r
,
this m
e
tho
d
f
ace
s issu
e
s
beca
us
e
si
RNA
is qui
c
kly
de
gr
aded b
y th
e b
o
d
y
,
n
ece
ssit
a
ting
e
fforts
to
e
nh
a
n
ce
its st
ab
ility
,
su
c
h
a
s through
c
h
e
mi
ca
l mo
d
ifi
ca
tions
.
W
ithin th
e ce
ll
,
a
ntis
e
ns
e a
n
d
si
RNA
n
eed
to
be
tr
a
nsport
ed
acc
ur
a
t
e
ly
.
I
niti
a
lly
,
RNA
is
ca
ptur
ed
within
e
n
d
osom
e
s
a
n
d
su
b
s
e
qu
e
ntly
e
n
c
ount
e
rs lysosom
e
s for intr
ace
llul
a
r
b
r
ea
k
d
own
.
O
nly
a
sm
a
ll fr
ac
tion
ca
n surviv
e
this
e
n
d
osom
a
l
de
gr
ada
tion
.
O
n
ce
r
e
l
ea
s
ed
into th
e c
ytopl
a
sm
,
th
e
mol
ec
ul
e
tr
a
v
e
ls to th
e
nu
c
l
e
us
,
d
iffus
e
s
through th
e
m
e
m
b
r
a
n
e
por
e
s
,
a
n
d
lo
ca
t
e
s its t
a
rg
e
t within th
e
nu
c
l
e
us
.
T
h
e
r
a
py is mor
e
str
a
ightforw
a
r
d
if
a
ll g
e
n
e
s or m
e
ss
e
ng
e
rs in th
e
nu
c
l
e
us
a
r
e
unprot
ec
t
ed
or lin
ea
r
.
H
ow
e
v
e
r
,
in r
ea
lity
,
b
oth
DNA a
n
d
RNA a
r
e
intri
ca
t
e
ly fol
ded a
n
d c
ov
e
r
ed b
y prot
e
ins
.
T
his issu
e
is
p
a
rti
c
ul
a
rly
ac
ut
e
for
RNA d
u
e
to th
e
limit
ed
un
de
rst
a
n
d
ing of its
stru
c
tur
e
within living
ce
lls
.
Ac
hi
e
ving th
e
r
a
p
e
uti
c
go
a
ls oft
e
n involv
e
s
a
tri
a
l
-
a
n
d
-
e
rror
a
ppro
ac
h
:
a
s
e
ri
e
s of
e
xp
e
rim
e
nts with
RNA be
gin
a
t
th
e
t
a
rg
e
t sit
e
for tr
a
ns
c
ription or tr
a
nsl
a
tion
,
hoping th
a
t th
e
sit
e
is
r
e
l
a
tiv
e
ly
acce
ssi
b
l
e
.
T
h
e
n
e
xt
c
h
a
ll
e
ng
e
is un
de
rst
a
n
d
ing how th
e
int
e
r
ac
tion
be
tw
ee
n th
e
r
a
py
a
n
d
t
a
rg
e
t
ca
n l
ead
to
a
st
ab
l
e
hy
b
ri
d
.
G
u
a
nin
e
(
G
)
a
n
d c
ytosin
e
(
C
)
a
r
e c
onn
ec
t
ed b
y thr
ee
hy
d
rog
e
n
b
on
d
s
,
m
a
king this
b
on
d
mor
e
st
ab
l
e
th
a
n th
e
two hy
d
rog
e
n
b
on
d
s
be
tw
ee
n
ade
nin
e
(
A
)
a
n
d
thymin
e
(
T
).
T
h
e
minimum l
e
ngth for
de
signing
a
n
RNA
str
a
n
d
is
influ
e
n
ced b
y th
e
g
e
nom
e
siz
e
.
I
n th
e
hum
a
n g
e
nom
e
,
RNA
mol
ec
ul
e
s
short
e
r th
a
n
12
-
15
ba
s
e
s t
e
n
d
to un
de
rgo
d
upli
ca
tion
,
whi
c
h
ca
n
pot
e
nti
a
lly
d
isrupt in
c
orr
ec
t g
e
n
e
s or m
e
ss
e
ng
e
rs
.
C
ons
e
qu
e
ntly
,
for
th
e
th
e
r
a
py to
be b
oth st
ab
l
e a
n
d e
ff
ec
tiv
e
,
th
e
nu
c
l
e
oti
de ba
s
e
l
e
ngth
shoul
d
r
a
ng
e
from
13
to
20
ba
s
e
s
.
O
n
ce a
hy
b
ri
d
is
c
r
ea
t
ed
,
th
e
su
b
s
e
qu
e
nt st
e
p involv
e
s t
a
rg
e
ting
a
n
d da
m
a
ging th
e
int
e
n
ded
m
e
ss
e
ng
e
r
.
T
h
e e
ff
ec
tiv
e
n
e
ss of
a
ntis
e
ns
e
de
sign
ed
for m
RNA
r
e
li
e
s on th
e
pr
e
s
e
n
ce
of th
e RNa
s
e H e
nzym
e
,
whi
c
h
c
l
ea
v
e
s th
e
m
e
ss
e
ng
e
r
RNA
.
I
f th
e a
ntis
e
ns
e c
onsists of
a
singl
e
DNA
str
a
n
d
,
it
d
ir
ec
tly
c
ontri
b
ut
e
s to th
e de
stru
c
tion of th
e
m
e
ss
e
ng
e
r
RNA
.
T
his
de
stru
c
tion is in
deed
th
e
go
a
l
,
b
ut ov
e
r tim
e
,
this hy
b
ri
d ca
n
l
ead
to th
e
pro
d
u
c
tion of g
e
n
e
ti
c
instru
c
tions th
a
t tr
a
nsl
a
t
e
into
prot
e
ins
a
sso
c
i
a
t
ed
with
d
is
ea
s
e
s
,
a
pro
ce
ss f
ac
ilit
a
t
ed b
y ri
b
osom
e
s
whi
c
h int
e
rpr
e
t
a
n
d
r
ead
th
e
g
e
n
e
ti
c
inform
a
tion
.
T
o pr
e
v
e
nt this
,
it is
e
ss
e
nti
a
l to
de
sign
a
ntis
e
ns
e
with
a
ro
b
ust
b
on
d
.
DNA
/
RNA d
upl
e
x
e
s
a
r
e
l
e
ss st
ab
l
e c
omp
a
r
ed
to
RNA
/
DNA d
upl
e
x
e
s
,
prompting ongoing
e
fforts to
de
v
e
lop
DNA
th
a
t mimi
c
s
RNA
.
RNA
int
e
rf
e
r
e
n
ce
(
RNA
i
)
is r
ec
ogniz
ed
for its high sp
ec
ifi
c
ity in
t
e
rms of
e
ff
ec
tiv
e
n
e
ss
.
N
on
e
th
e
l
e
ss
,
lik
e
oth
e
r sm
a
ll mol
ec
ul
e
s
,
it f
ace
s
pot
e
nti
a
l issu
e
s in
c
lini
ca
l us
e
.
P
ossi
b
l
e
si
de e
ff
ec
ts in
c
lu
de
unint
e
n
ded
suppr
e
ssion of non
-
t
a
rg
e
t g
e
n
e
s
,
whi
c
h
ca
n r
e
sult from m
RNA
de
gr
ada
tion
,
tr
a
nsl
a
tion inhi
b
ition
,
or glo
ba
l g
e
n
e
suppr
e
ssion
trigg
e
r
ed b
y
a
n int
e
rf
e
ron r
e
spons
e
,
p
a
rti
c
ul
a
rly wh
e
n si
RNA
is
de
liv
e
r
ed
vi
a a
vir
a
l v
ec
tor
.
IV
.
2
R
i
b
osom
a
l
RNA
R
i
b
osom
e
s
a
r
e
sm
a
ll
,
de
ns
e
org
a
n
e
ll
e
s within
ce
lls th
a
t fun
c
tion
a
s sit
e
s for prot
e
in synth
e
sis
.
Mea
suring
ab
out
20
nm in
d
i
a
m
e
t
e
r
,
ri
b
osom
e
s
a
r
e c
ompos
ed
of
65
%
ri
b
osom
a
l
RNA
(
r
RNA
)
a
n
d
35
%
ri
b
osom
a
l prot
e
ins
,
m
a
king th
e
m
a
typ
e
of ri
b
onu
c
l
e
oprot
e
in
(
RNP
).
T
h
e
s
e
org
a
n
e
ll
e
s tr
a
nsl
a
t
e
m
RNA
into polyp
e
pti
de c
h
a
ins
,
whi
c
h
a
r
e
ultim
a
t
e
ly prot
e
ins
,
b
y using
a
mino
ac
i
d
s
b
rought
b
y t
RNA d
uring
tr
a
nsl
a
tion
.
R
i
b
osom
e
s
ca
n
be
foun
d e
ith
e
r susp
e
n
ded
in th
e c
ytosol
,
a
tt
ac
h
ed
to th
e
rough
e
n
d
opl
a
smi
c
r
e
ti
c
ulum
,
or on th
e
nu
c
l
ea
r
m
e
m
b
r
a
n
e
.
T
h
e
y
a
r
e c
ru
c
i
a
l for tr
a
nsl
a
ting th
e
g
e
n
e
ti
c c
o
de
from
m
RNA
into prot
e
ins
,
following th
e ce
ntr
a
l
d
ogm
a
of
b
iology wh
e
r
e
DNA
is tr
a
ns
c
ri
bed
into
RNA
,
whi
c
h is th
e
n tr
a
nsl
a
t
ed
into prot
e
ins
.
D
uring tr
a
nsl
a
tion
,
ri
b
osom
e
s r
ead
th
e
m
RNA
s
e
qu
e
n
ce a
n
d
us
e
it to
a
ss
e
m
b
l
e a
mino
ac
i
d
s into th
e c
orr
ec
t s
e
qu
e
n
ce
to form prot
e
ins
.
R
i
b
osom
e
s
a
r
e
intri
ca
t
e
stru
c
tur
e
s
c
ompos
ed
of
RNA a
n
d
prot
e
ins
,
a
n
d
th
e
y
c
onsist of two su
b
units
:
a
sm
a
ll
e
r on
e
th
a
t
a
tt
ac
h
e
s
to m
RNA
,
a
n
d a
l
a
rg
e
r on
e
th
a
t int
e
r
ac
ts with t
RNA a
n
d a
mino
ac
i
d
s
.
U
pon
c
ompl
e
ting th
e
m
RNA
tr
a
nsl
a
tion
,
th
e
su
b
units s
e
p
a
r
a
t
e
.
R
i
b
osom
e
s
a
r
e ca
t
e
goriz
ed a
s ri
b
ozym
e
s
beca
us
e
th
e
ri
b
osom
a
l
RNA
pl
a
ys
a c
ru
c
i
a
l rol
e
in th
e
p
e
pti
d
yl tr
a
nsf
e
r
a
s
e ac
tivity th
a
t joins
a
mino
ac
i
d
s
.
T
h
e
r
e a
r
e
not
ab
l
e
stru
c
tur
a
l
a
n
d RNA
s
e
qu
e
n
ce d
iff
e
r
e
n
ce
s
a
mong ri
b
osom
e
s from
bac
t
e
ri
a
,
a
r
c
h
aea
,
a
n
d e
uk
a
ryot
e
s
,
whi
c
h
a
llows
ce
rt
a
in
a
nti
b
ioti
c
s to t
a
rg
e
t
bac
t
e
ri
a
l ri
b
osom
e
s without
a
ff
ec
ting thos
e
in hum
a
ns
.
E
uk
a
ryoti
c
mito
c
hon
d
ri
a
l ri
b
osom
e
s
r
e
s
e
m
b
l
e bac
t
e
ri
a
l on
e
s
,
in
d
i
ca
ting
a
n
e
volution
a
ry
c
onn
ec
tion
.
R
i
b
osom
e
s l
ac
k m
e
m
b
r
a
n
e
s
beca
us
e
th
e
y
a
r
e
th
e
sm
a
ll
e
st org
a
n
e
ll
e
s
a
n
d a
r
e c
ompos
ed
only of prot
e
ins
,
not lipi
d
s
.
A
lt
e
rn
a
tiv
e
mol
ec
ul
a
r
m
e
tho
d
s lik
e
polym
e
r
a
s
e c
h
a
in r
eac
tion
(
PCR
)
ca
n
de
t
ec
t
e
ntir
e
bac
t
e
ri
a
l
c
ommuniti
e
s without
c
ulturing
b
y
a
mplifying
a
n
d
s
e
qu
e
n
c
ing
th
e
16
S
r
RNA
r
e
gion with univ
e
rs
a
l
bac
t
e
ri
a
l prim
e
rs
.
T
h
e
d
iff
e
r
e
nti
a
tion of
bac
t
e
ri
a
l sp
ec
i
e
s is
ba
s
ed
on th
e
ir migr
a
tion p
a
tt
e
rns
in g
e
ls
,
whi
c
h
de
p
e
n
d
on th
e
gu
a
nin
e
plus
c
ytosin
e
(
G
+
C
)
c
ont
e
nt
a
n
d
m
e
lting prop
e
rti
e
s
,
c
r
ea
ting
a ba
r
-
c
o
de
-
lik
e
profil
e
wh
e
r
e eac
h
ba
n
d
m
a
y r
e
pr
e
s
e
nt
a d
istin
c
t mi
c
roorg
a
nism
.
T
h
e PCR
m
e
tho
d
is
a
t
ec
hniqu
e
us
ed
in mol
ec
ul
a
r
b
iology to
a
mplify
on
e
or mor
e c
opi
e
s of
a DNA
s
e
gm
e
nt multipl
e
tim
e
s
,
r
e
sulting in
thous
a
n
d
s to millions of
c
opi
e
s of
a
sp
ec
ifi
c DNA
s
e
qu
e
n
ce
.
De
v
e
lop
ed
in
1983
,
this t
ec
hniqu
e
h
a
s
bec
om
e e
ss
e
nti
a
l in m
ed
i
ca
l
a
n
d b
iologi
ca
l
r
e
s
ea
r
c
h
,
s
e
rving v
a
rious purpos
e
s su
c
h
a
s
DNA c
loning for
s
e
qu
e
n
c
ing
,
fun
c
tion
a
l g
e
n
e a
n
a
lysis
,
d
i
a
gnosing inh
e
rit
ed d
is
ea
s
e
s
,
g
e
n
e
ti
c
fing
e
rprinting for for
e
nsi
c a
n
d
p
a
t
e
rnity t
e
sting
,
a
n
d de
t
ec
ting
inf
ec
tious
d
is
ea
s
e
s
.
PCR
involv
e
s th
e
rm
a
l
c
y
c
ling
,
whi
c
h
a
lt
e
rn
a
t
e
s
be
tw
ee
n h
ea
ting
a
n
d c
ooling to
de
n
a
tur
e DNA a
n
d
f
ac
ilit
a
t
e
r
e
pli
ca
tion
with th
e
h
e
lp of
DNA
polym
e
r
a
s
e a
n
d
short
DNA
prim
e
rs th
a
t
a
r
e
c
ompl
e
m
e
nt
a
ry to th
e
t
a
rg
e
t s
e
qu
e
n
ce
.
A
s th
e
pro
ce
ss
ad
v
a
n
ce
s
,
th
e
DNA
pro
d
u
ced ac
ts
a
s
a
t
e
mpl
a
t
e
for furth
e
r r
e
pli
ca
tion
,
a
mplifying th
e
DNA e
xpon
e
nti
a
lly
.
PCR
is highly
ada
pt
ab
l
e a
n
d ca
n
be
mo
d
ifi
ed
for
num
e
rous g
e
n
e
ti
c
m
a
nipul
a
tions
.
PCR
,
whi
c
h st
a
n
d
s for
P
olym
e
r
a
s
e C
h
a
in
Reac
tion
,
is
a
l
ab
or
a
tory m
e
tho
d
us
ed
for
a
mplifying
DNA
.
T
his t
ec
hniqu
e e
n
ab
l
e
s
th
e
r
e
pli
ca
tion of sp
ec
ifi
c DNA
s
e
qu
e
n
ce
s in
a
t
e
st tu
be
without
r
e
quiring th
e DNA
to
be
ins
e
rt
ed
into living
ce
lls
.
T
h
e
pro
ce
ss of
PCR
r
e
li
e
s on th
e
fun
da
m
e
nt
a
l stru
c
tur
e
of
DNA
.
DNA
in its n
a
tur
a
l form is
a
d
ou
b
l
e
h
e
lix
c
ompos
ed
of two
a
ntip
a
r
a
ll
e
l str
a
n
d
s h
e
l
d
tog
e
th
e
r
b
y
hy
d
rog
e
n
b
on
d
s
.
T
h
e
s
e
hy
d
rog
e
n
b
on
d
s form
be
tw
ee
n
c
ompl
e
m
e
nt
a
ry
ba
s
e
p
a
irs
:
Ade
nin
e
(
A
)
p
a
irs with
T
hymin
e
(
T
),
a
n
d G
u
a
nin
e
(
G
)
p
a
irs
with
C
ytosin
e
(
C
).
T
h
e ba
s
e
s
a
r
e a
tt
ac
h
ed
to
de
oxyri
b
os
e
sug
a
r
mol
ec
ul
e
s
,
a
n
d eac
h sug
a
r mol
ec
ul
e
is
c
onn
ec
t
ed
to th
e
n
e
xt
b
y
phosph
a
t
e b
on
d
s
.
A
.
PCR
st
a
g
e
s
Eac
h
c
y
c
l
e c
onsists of thr
ee
prim
a
ry st
a
g
e
s
.
D
uring
de
n
a
tur
a
tion
,
th
e d
ou
b
l
e
-
str
a
n
ded DNA
s
e
p
a
r
a
t
e
s into singl
e
str
a
n
d
s
d
u
e
to high
t
e
mp
e
r
a
tur
e
s
b
r
ea
king th
e
hy
d
rog
e
n
b
on
d
s
be
tw
ee
n
c
ompl
e
m
e
nt
a
ry
ba
s
e
s
,
pr
e
v
e
nting
a
ny
e
nzym
a
ti
c
r
eac
tions
,
su
c
h
a
s
polym
e
riz
a
tion
,
from o
cc
urring
.
I
n th
e a
nn
ea
ling st
a
g
e
,
prim
e
rs
a
tt
ac
h to sp
ec
ifi
c
r
e
gions of th
e DNA
th
a
t
a
r
e c
ompl
e
m
e
nt
a
ry to
th
e
m
,
forming hy
d
rog
e
n
b
on
d
s
.
T
his st
e
p
a
llows
DNA
polym
e
r
a
s
e
to
b
in
d e
ff
ec
tiv
e
ly
,
e
nsuring th
a
t th
e b
on
d
s r
e
m
a
in st
ab
l
e
for
su
b
s
e
qu
e
nt polym
e
riz
a
tion r
eac
tions
,
typi
ca
lly
c
on
d
u
c
t
ed a
t
72
°
C
.
F
in
a
lly
,
in th
e
polym
e
riz
a
tion or
e
xt
e
nsion st
a
g
e
,
whi
c
h usu
a
lly
o
cc
urs
a
t
72
°
C
,
th
e
prim
e
r
e
xt
e
n
d
s with
c
ompl
e
m
e
nt
a
ry
dNTP
s on
th
e
3
'
e
n
d
.
C
ons
e
qu
e
ntly
,
st
a
rting from
a
singl
e
g
e
n
e c
opy
,
th
e
a
mount
d
ou
b
l
e
s
eac
h
c
y
c
l
e
,
l
ead
ing to
e
xpon
e
nti
a
l growth with
eac
h su
b
s
e
qu
e
nt
c
y
c
l
e
—
2
c
opi
e
s
a
ft
e
r on
e c
y
c
l
e
,
4
a
ft
e
r two
c
y
c
l
e
s
,
8
a
ft
e
r thr
ee c
y
c
l
e
s
,
a
n
d
so on
.
B
.
PCR c
ompon
e
nts
a
.
DNA P
olym
e
r
a
s
e E
nzym
e
H
istori
ca
lly
,
PCR
w
a
s p
e
rform
ed
using th
e K
l
e
now fr
a
gm
e
nt of
DNA
polym
e
r
a
s
e I d
uring th
e
polym
e
riz
a
tion ph
a
s
e
.
T
his
e
nzym
e
prov
ed
to
be
th
e
rm
a
lly in
ac
tiv
e d
uring th
e de
n
a
tur
a
tion st
e
p
,
r
e
quiring r
e
s
ea
r
c
h
e
rs to
add
fr
e
sh
e
nzym
e
in
eac
h
c
y
c
l
e
.
Add
ition
a
lly
,
th
e K
l
e
now fr
a
gm
e
nt
c
oul
d
only
e
xt
e
n
d DNA
up to
200
ba
s
e
p
a
irs
a
n
d
work
ed
optim
a
lly
a
t
a
low t
e
mp
e
r
a
tur
e
of
37
°
C
,
l
ead
ing to l
e
ss sp
ec
ifi
c
ity
.
T
o
add
r
e
ss th
e
s
e
limit
a
tions
,
Ta
q
polym
e
r
a
s
e
,
whi
c
h r
e
m
a
ins
ac
tiv
e a
t high
e
r t
e
mp
e
r
a
tur
e
s
,
w
a
s
intro
d
u
ced
.
T
his
ad
v
a
n
ce
m
e
nt
e
limin
a
t
ed
th
e
n
eed
for
e
nzym
e
add
ition in
e
v
e
ry
c
y
c
l
e a
n
d a
llow
ed PCR
to
be c
on
d
u
c
t
ed
in
a
singl
e
m
ac
hin
e
.
H
ow
e
v
e
r
,
using
a
n
e
x
ce
ssiv
e a
mount of
Ta
q
polym
e
r
a
s
e ca
n l
ead
to non
-
sp
ec
ifi
c bac
kgroun
d
pro
d
u
c
ts
,
whil
e
insuffi
c
i
e
nt
a
mounts
ca
n r
e
sult in in
e
ffi
c
i
e
nt
a
mplifi
ca
tion
a
n
d
low
e
r yi
e
l
d
.
b
.
P
rim
a
ry
Idea
lly
,
th
e
s
e
l
ec
t
ed
prim
e
r shoul
d
h
a
v
e a G
+
C c
ont
e
nt of
a
pproxim
a
t
e
ly
50
%
.
I
t is
a
lso import
a
nt to
a
voi
d
prim
e
rs with
polypurin
e
s or polypyrimi
d
in
e
s
.
Add
ition
a
lly
,
s
ec
on
da
ry stru
c
tur
e
s
a
n
d c
ompl
e
m
e
nt
a
rity
be
tw
ee
n prim
e
r
-
d
im
e
rs shoul
d be
minimiz
ed
.
T
h
e
prim
e
r shoul
d
h
a
v
e a
m
e
lting t
e
mp
e
r
a
tur
e
(
T
m
)
gr
ea
t
e
r th
a
n
55
°
C
.
T
h
e T
m of
a
prim
e
r
ca
n
be e
stim
a
t
ed
using th
e
formul
a
:
[(
sum of
A
+
T
)
x
2
°
C
] + [(
sum of
C
+
G
)
x
4
°
C
].
c
.
O
th
e
r r
ea
g
e
nts
I
n
add
ition to
e
nzym
e
s
a
n
d
prim
e
rs
,
th
e
su
cce
ss of
a PCR
r
eac
tion
is
a
lso influ
e
n
ced b
y oth
e
r f
ac
tors
.
T
h
e
s
e
in
c
lu
de dNTP
s n
ece
ss
a
ry
for polym
e
riz
a
tion
a
n
d a b
uff
e
r with
M
g
C
l
2
.
T
h
e c
on
ce
ntr
a
tion of
M
g
²
⁺
ions in th
e
r
eac
tion mixtur
e
is p
a
rti
c
ul
a
rly
c
ru
c
i
a
l
a
s it
a
ff
ec
ts
k
e
y pro
ce
ss
e
s su
c
h
a
s
a
nn
ea
ling
,
de
n
a
tur
a
tion
,
sp
ec
ifi
c
ity of th
e
pro
d
u
c
ts
,
e
nzym
e ac
tivity
,
a
n
d
ov
e
r
a
ll r
eac
tion
acc
ur
ac
y
.
He
n
ce
,
it
is
e
ss
e
nti
a
l to
ca
r
e
fully m
a
n
a
g
e
th
e add
ition of r
ea
g
e
nts to
a
voi
d
int
e
rf
e
r
e
n
ce
from oth
e
r ions or
c
h
e
l
a
ting
a
g
e
nts th
a
t
c
oul
d a
lt
e
r
th
e M
g
²
⁺
ion
c
on
ce
ntr
a
tion
.
T
ypi
ca
lly
,
th
e c
on
ce
ntr
a
tion of fr
ee
M
g
²
⁺
ions in th
e
solution shoul
d be a
pproxim
a
t
e
ly
2
m
M
.
I
n r
ece
nt
y
ea
rs
,
ad
v
a
n
ce
m
e
nts in mol
ec
ul
a
r
b
iology h
a
v
e
l
ed
to th
e
de
v
e
lopm
e
nt of s
e
v
e
r
a
l innov
a
tiv
e
t
ec
hniqu
e
s
,
in
c
lu
d
ing nu
c
l
e
i
c
ac
i
d a
n
a
lysis through polym
e
r
a
s
e c
h
a
in r
eac
tion
(
PCR
),
whi
c
h w
a
s
first intro
d
u
ced
in
1985
.
T
his t
ec
hniqu
e
involv
e
s th
e e
xpon
e
nti
a
l
a
mplifi
ca
tion of sp
ec
ifi
c DNA
s
e
gm
e
nts
,
r
e
sulting in millions of
c
opi
e
s th
a
t
ca
n
be
us
ed
for
a
r
a
ng
e
of
a
n
a
lyti
ca
l purpos
e
s
.
Add
ition
a
lly
,
PCR ca
n
be
pr
eceded b
y
a
r
e
v
e
rs
e
tr
a
ns
c
ription
(
RT
)
pro
ce
ss
,
known
a
s
RT
-
PCR
,
whi
c
h
c
onv
e
rts
RNA
into
DNA
,
a
llowing
for th
e e
v
a
lu
a
tion of g
e
n
e
tr
a
ns
c
ription in
ce
lls or tissu
e
s
.
C
.
Re
v
e
rs
e T
r
a
ns
c
ription
PCR
(
R T
-
PCR
)
RNA a
mplifi
ca
tion using
PCR
involv
e
s th
e
us
e
of prim
e
rs th
a
t
b
in
d
to th
e RNA
t
e
mpl
a
t
e
,
follow
ed b
y th
e
synth
e
sis of
c
ompl
e
m
e
nt
a
ry
DNA
(
cDNA
)
through th
e ac
tion of th
e
r
e
v
e
rs
e
tr
a
ns
c
ript
a
s
e
(
RT
)
e
nzym
e
,
a
n
d
th
e
n pro
ceed
ing with th
e PCR
pro
ce
ss
.
A
t this st
a
g
e
,
v
a
rious
DNA
polym
e
r
a
s
e
s
,
su
c
h
a
s
T
.
th
e
rmophilus
(
T
th
)
DNA
polym
e
r
a
s
e
,
ca
n
be
utiliz
ed
;
T
th
DNA
polym
e
r
a
s
e
,
in th
e
pr
e
s
e
n
ce
of
m
a
ng
a
n
e
s
e
(
M
n
),
is
ca
p
ab
l
e
of r
e
v
e
rs
e
tr
a
ns
c
ri
b
ing
RNA
.
S
in
ce T
th
DNA
polym
e
r
a
s
e ca
n us
e b
oth
DNA a
n
d RNA a
s t
e
mpl
a
t
e
s
,
th
e e
ntir
e
pro
ce
ss
ca
n
be c
on
d
u
c
t
ed
in
a
singl
e
tu
be
.
V
ir
a
l
RNA
t
e
mpl
a
t
e
s
,
in
c
lu
d
ing thos
e
from r
e
trovirus
e
s or poly
A RNA
,
ca
n
be a
mplifi
ed
using
e
ith
e
r r
a
n
d
om
a
ss
a
ys or sp
ec
ifi
c
prim
e
rs
.
RNA
(
RT
)
PCR
is
a
highly s
e
nsitiv
e
m
e
tho
d
for
a
n
a
lyzing g
e
n
e e
xpr
e
ssion
a
t th
e RNA
l
e
v
e
l
a
n
d
for m
ea
suring m
RNA
or vir
a
l
RNA
qu
a
ntiti
e
s
.
Re
v
e
rs
e
tr
a
ns
c
ript
a
s
e
is typi
ca
lly
e
mploy
ed
to
c
r
ea
t
e
th
e
first str
a
n
d
of
cDNA
from
RNA a
n
d
ca
n
be
o
b
t
a
in
ed
from v
a
rious sour
ce
s
,
su
c
h
a
s
a
vi
a
n my
e
lo
b
l
a
stosis
virus
(
AMV
)
a
n
d M
olon
e
y murin
e
l
e
uk
e
mi
a
virus
(
MMLV
).
AMV
r
e
v
e
rs
e
tr
a
ns
c
ript
a
s
e
,
a
n
RNA
-
de
p
e
n
de
nt
DNA
polym
e
r
a
s
e
,
utiliz
e
s singl
e
-
str
a
n
ded RNA a
s
a
t
e
mpl
a
t
e
to synth
e
siz
e cDNA
in th
e
5
'
→
3
'
d
ir
ec
tion
,
provi
ded a
prim
e
r is
a
v
a
il
ab
l
e
.
S
imil
a
r to
DNA
polym
e
r
a
s
e
,
this
e
nzym
e
a
lso
e
xhi
b
its ri
b
onu
c
l
ea
s
e H ac
tivity
,
whi
c
h is sp
ec
ifi
c
to
RNA
:
DNA
hy
b
ri
d
s
.
D
.
RNA e
xtr
ac
tion
T
h
e RNA e
xtr
ac
tion m
e
tho
d
p
a
r
a
ll
e
ls th
a
t of
DNA e
xtr
ac
tion
,
though
RNA
mol
ec
ul
e
s
a
r
e
short
e
r
a
n
d
l
e
ss pron
e
to
da
m
a
g
e
from
sh
ea
ring
,
a
llowing for mor
e
vigorous
ce
ll
d
isruption
.
De
spit
e
this
,
RNA
is highly sus
ce
pti
b
l
e
to
de
gr
ada
tion
b
y
RNa
s
e
,
whi
c
h is pr
e
s
e
nt in
v
a
rying l
e
v
e
ls within
ce
lls
a
n
d
on
e
xt
e
rn
a
l surf
ace
s lik
e
fing
e
rs
.
T
h
e
r
e
for
e
,
glov
e
s
a
r
e e
ss
e
nti
a
l
d
uring
RNA e
xtr
ac
tion
,
a
n
d
th
e
isol
a
tion
m
ed
ium must in
c
lu
de a
pot
e
nt
de
t
e
rg
e
nt to r
a
pi
d
ly in
ac
tiv
a
t
e RNa
s
e
.
Add
ition
a
lly
,
th
e de
prot
e
iniz
a
tion st
e
p must
be
mor
e
thorough
d
u
e
to
RNA
'
s strong
b
in
d
ing
a
ffinity with prot
e
ins
.
T
o
e
limin
a
t
e DNA
,
DNa
s
e
ca
n
be added
,
follow
ed b
y
e
th
a
nol pr
ec
ipit
a
tion of th
e RNA
.
G
u
a
ni
d
inium thio
c
y
a
n
a
t
e
,
a c
ommon r
ea
g
e
nt for
RNA e
xtr
ac
tion
,
s
e
rv
e
s
a
s
b
oth
a
pow
e
rful
RNa
s
e
inhi
b
itor
a
n
d a
prot
e
in
de
n
a
tur
a
nt
.
RNA
int
e
grity is
a
ss
e
ss
ed
vi
a e
l
ec
trophor
e
sis on
a
n
a
g
a
ros
e
g
e
l
,
wh
e
r
e
promin
e
nt r
RNA ba
n
d
s su
c
h
a
s
23
S a
n
d
16
S
in prok
a
ryot
e
s or
18
S a
n
d
28
S
in
e
uk
a
ryot
e
s in
d
i
ca
t
e
th
a
t th
e RNA
is int
ac
t
.
T
his pro
ced
ur
e
is
typi
ca
lly p
e
rform
ed
un
de
r
de
n
a
turing
c
on
d
itions to
a
voi
d
s
ec
on
da
ry
stru
c
tur
e
form
a
tion in th
e RNA
.
RNA
is highly sus
ce
pti
b
l
e
to nu
c
l
ea
s
e
s
d
u
e
to its r
eac
tiv
e
2
-
hy
d
roxyl groups on
a
ll
ba
s
e
s
,
whi
c
h
ca
n
ea
sily p
a
rti
c
ip
a
t
e
in
c
h
e
mi
ca
l
r
eac
tions th
a
t l
ead
to w
a
t
e
r pro
d
u
c
tion
a
n
d da
m
a
g
e
to th
e
sug
a
r
bac
k
b
on
e
.
I
t
'
s import
a
nt to not
e
th
a
t nu
c
l
ea
s
e
s
a
r
e
r
e
l
a
tiv
e
ly st
ab
l
e
in
th
e e
nvironm
e
nt
a
n
d ca
n som
e
tim
e
s surviv
e
h
ea
t
de
n
a
tur
a
tion
a
n
d
ph
e
nol
e
xtr
ac
tion
.
T
o minimiz
e
nu
c
l
ea
s
e ac
tivity
d
uring nu
c
l
e
i
c ac
i
d
e
xtr
ac
tion
,
s
e
v
e
r
a
l m
ea
sur
e
s
ca
n
be
t
a
k
e
n
:
k
ee
ping in
c
u
ba
tion
a
n
d
ce
ntrifug
a
tion t
e
mp
e
r
a
tur
e
s
be
low th
e
optim
a
l nu
c
l
e
i
c ac
i
d
t
e
mp
e
r
a
tur
e
(
37
°
C
),
su
c
h
a
s
b
y m
a
int
a
ining th
e e
xtr
ac
t solution on i
ce
or
a
t
4
°
C
;
deac
tiv
a
ting nu
c
l
ea
s
e
s on surf
ace
s of gl
a
ss
,
w
a
t
e
r
,
a
n
d
c
onsum
ab
l
e
s with
c
h
e
mi
ca
ls lik
e d
i
e
thylpyro
ca
r
b
on
a
t
e
(
DEPC
);
using
c
h
e
mi
ca
l in
ac
tiv
a
tion or inhi
b
ition with su
b
st
a
n
ce
s lik
e
ph
e
nol or
gu
a
ni
d
inium s
a
lts
;
a
n
d
r
e
moving
c
o
-
f
ac
tor m
e
t
a
l ions for nu
c
l
ea
s
e
s
using
c
h
e
l
a
ting
a
g
e
nts
.
T
o isol
a
t
e e
uk
a
ryoti
c
m
RNA
(
whi
c
h is only
2
-
5
%
of
ce
llul
a
r
RNA
)
from
a
mixtur
e
of tot
a
l
RNA
mol
ec
ul
e
s
,
a
ffinity
c
hrom
a
togr
a
phy on
a
n
oligo
(
dT
)-
ce
llulos
e c
olumn
ca
n
be
isol
a
t
ed
.
A
t high s
a
lt
c
on
ce
ntr
a
tions
,
m
RNA c
ont
a
ining poly
(
A
)
t
a
ils will
b
in
d
to
c
ompl
e
m
e
nt
a
ry oligo
(
dT
)
mol
ec
ul
e
s in th
e a
ffinity
c
olumn
,
so th
a
t th
e
m
RNA
r
e
m
a
ins
be
hin
d
,
whil
e
oth
e
r
RNA
mol
ec
ul
e
s
ca
n
be
w
a
sh
ed c
l
ea
n from th
e c
olumn
using
a
high s
a
lt solution
.
Ne
xt
,
th
e b
oun
d
m
RNA ca
n
be d
issolv
ed
with
a
low
c
on
ce
ntr
a
tion of s
a
lt
.
