Remote Homology Detection of Beta-Structural Motifs Using Random Fields
Practical applications of three-way junction structural motifs
description
Transcript of Practical applications of three-way junction structural motifs
Practical applications of three-way junction structural motifs
with (mostly) Steven Taylor, Renjun Pei
CT A C
GG
CG
AT
T AG C
TACG
S1
S2
S3
More on junctions:
Kallenbach & Seeman, 1990s’, Kato’s group 1999
CT A C
GG
CG
AT
T AG C
TACG
S1
S2
S3
CA
TC
GG
AT
CG
TA
GC
TA
AG
ATTG
GCG
CAT
CG
CG
GA
A
TT T
C
AT
C
GGA
T
CG
TA
GC
TA
A
GA
TT
G
GC
GC
AT
CG
CG
GA
A
TT T
d1d2
Cocaine Corticosterone Cholic acid
+ +
- + +
CT AC
GG
AG
AT
T AG C
TA
CGS1
S2
S3
CT A C
GG
CG
AT
T AG C
TACG
S1
S2
S3
-
What do we do with crossreactive junctions?
Stojanovic et al, JACS, 2003
specif icaptamerfor cocaine
specif icaptamerfor cholatecholate
cocaine
fully matched
aptamer
junction
In a way, two sensors together are more powerful:
… and even more sensors make cross-reactive array
How able are cross-reactive arrays?
N
N
O OOH
OOO
Vindoline
NH
N
HO
H
N
N
OOH
O
OO
O
O OVinblastine
NH
N
HO
H
N
N
O OOH
OO O
O O
OVincristine
NO
NHHHN
Methylergonovine
N
H
H
N
O
HHO
Quinine Quinidine
N
H
H
N
O
HOH
Cinchonine Cinchonidine
N
H
H
N
HHO
N
H
H
N
HOH
O
O
NO
O
(-)-Cocaine (+)-Cocaine
O
O
NO
N
O
O
HH
NH
Strychnine
N
O
OH
H
NH
O
O HH
Brucine
O1 23 4
5 6 7 8
10 11
12
9
O
OH
OH
MeMe
OH
OOHMe
Me
O
Deoxycorticosterone --21 glucoside
Deoxycholic acid
MeMe
HO
Dehydroisoandrosterone --3-sulfate
OSulfate
Glucoside
13 14 15
0.7-12 M 0.7-12 M
5-190 M5-190 M
5-100 M 5-100 M
5-100 M
5-200 M
50-750 M50-750 M 50-750 M
50-750 M 50-750 M
10-200 M 30-500 M
How to make high resolution crossreactive array?
TAGAGC
GCA
T
GC
GC
TA C
G
TAGAGC
GCA
T
GC
GC
TA C
G
TAGAGC
GCA
T
GC
GC
TA C
Ga) b) c)
N
O
OH
H
NH
O
O HH
N
O
O
HH
NH
N
O
O
O
NO2
T GTAGAGC
GCA
T
GC
GC
AT
TA
TTT
CG G
C TA C
G CG C
G
GT
CG
TC
CC
GC A
T GC A
T TA
GA
A GT
CG
TC
CC
GA
GA
T
+
Stem 1
Stem 2
Stem 3T G
TAGAGC
GCA
T
GC
GC
AT
TA
TTT
CG G
C TA C
G CG C
GC
TC
TA
GA
A
GA G
CG G
C
CG
5'
QF F
Q
Yingfu Li, JACS 2003.
50nm T25-1+150nm mns-1-Q
0
10000
20000
30000
40000
50000
60000
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
mM
Quinine
Quinidine
brucine
strychnine
cinchonine
cinchonidine
vinbla
vincris
vindol
ergo
Pei&Shen
But, first, we find suitable sensors:
High-resolution CRA for alkaloids (and steroids)
T GTAGNMC
GCA
T
GC
GC
AT
TA
TTT
CG G
C TA C
G CG C
G
GT
CG
TC
CC
GC A
T GC A
T TA
GA
A
GTC
GTC
C C GAG
AT
+
Stem 1
Stem 2
Stem 3
T GTAGNMC
GCA
T
GC
GC
AT
TA
TTT
CG G
C TA C
G CG C
GC
TC
TA
GA
A
5'
QF F
Q
E G J
I
II
III
IV
V
dA
N
NO2
OO
O
A
dC dC dC dC dA dA dA dA
dG dG dG dG dNI dG dG dG
dNI
dNI
dNIdNI
dNI
dNI
dNIdNI
dNI
dNIdNIdNI
B C D F H I K
dNI
dC
dG
I
V
IV III
a)
b)
c)
dNI
dG
dNI
dG
II
Pei&Shen, Olah
GAGC
GCA
T
GC T
A
GAGC
GCA
T
GC T
AN
GAGC
GCA
T
GC T
AN
NGAGC
GCA
T
GC T
AN
NN
GAGC
GCA
T
GC T
A
N
1
2
3
4
5
6
7
8
0.01 0.1 1
mM
F-U
0
1
2
3
4
5
6
7
8
0.01 0.1 1
mM
F-U
1
1.5
2
2.5
3
3.5
4
4.5
5
0.01 0.1 1mM
F-U
GAGC
GCA
T
GC T
A
NGCGC
GCA
T
GC T
A
GCGC
GCA
T
GC T
AN
N
H
H
N
O
HHO
1 - Quinine 2 - Quinidine
N
H
H
N
O
HOH
2
2 211
1
3 - Cinchonine 4 - Cinchonidine
N
H
H
N
HHO
N
H
H
N
HOH
0
2
4
6
8
10
12
0.01 0.1 1
mM
F-U
GAGC
GCA
T
GC T
A
1
1.5
2
2.5
3
3.5
4
4.5
5
0.01 0.1 1
mM
F-U
GAGC
GCA
T
GC T
A
L
1
1.5
2
2.5
3
3.5
4
4.5
0.01 0.1 1mM
F-U
GCGC
GCA
T
GC T
AN
33 34
4 4
Recognition of diastereomers:
Pei&Shen
Visualization and analysis
Pei&Shen, Olah
Practical applications: Detection of Inborn Errors in Steroid Metabolism
-e.g.: congenital adrenal hyperplasia (all types), errors in bile acid metabolism.
Taylor, Pei et al. Angew. Chem., 2009
We set up an assay:
B
N
O
HO
N
C
A
A G
CTC
G
C
GG
AT
ATT
T
T A G
TA
AG
AG
GCTC
G
C
G
GA T
A
T C
TTT
T A
GT A
AG
G T C C C G A G A 3'C GA TG CC GA TG CG CG CC GT AC GT AG CG CCT
G
CTA
AGAT3'Fluorescein
C GA TG CC GA TG CG CG CC GT AC GT AG 3'
GCTCTA
L2 L1
L1
IN SU LIN
GTCGTCCCGAGACCGAGAT3'Fluorescein
L1
IN SULIN
D
+D
MATRIX MATRIX
A
GG
CTC
G
C
G
GA
TA
TC
TTT
T A
GT AAG
GTCCCGAGA
3 '
C GA TG CC GA TG CG CG CC GT AC GT AG CG CCT
G
CTA
AGAT3'Fluorescein
L2
L1
INSU LIN
D
MATRIX
GT
TA C T T C
AC
C
C
A
GCAGGGCTCT
(i) (ii) (iii)
0 2 4 6 8 100
20000
40000
60000QuinineSulindocNiflumicNaproxenPiroxicanIbuprofenMeloxicamResveratrolNimesulideCocaine
[ ]/microM
Flu
ore
scen
ce I
nte
nsi
ty
B
N
O
HO
N
C
A
A G
CTC
G
C
GG
AT
ATT
T
T A G
TA
AG
AG
GCTC
G
C
G
GA T
A
T C
TTT
T A
GT A
AG
G T C C C G A G A 3'C GA TG CC GA TG CG CG CC GT AC GT AG CG CCT
G
CTA
AGAT3'Fluorescein
C GA TG CC GA TG CG CG CC GT AC GT AG 3'
GCTCTA
L2 L1
L1
IN SU LIN
GTCGTCCCGAGACCGAGAT3'Fluorescein
L1
IN SULIN
D
+D
MATRIX MATRIX
A
GG
CTC
G
C
G
GA
TA
TC
TTT
T A
GT AAG
GTCCCGAGA
3 '
C GA TG CC GA TG CG CG CC GT AC GT AG CG CCT
G
CTA
AGAT3'Fluorescein
L2
L1
INSU LIN
D
MATRIX
GT
TA C T T C
AC
C
C
A
GCAGGGCTCT
(i) (ii) (iii)
0 2 4 6 8 100
20000
40000
60000QuinineSulindocNiflumicNaproxenPiroxicanIbuprofenMeloxicamResveratrolNimesulideCocaine
[ ]/microM
Flu
ore
scen
ce I
nte
nsi
ty
T GTAGAGC
GCA
T
GC
GC
AT
TA
TTT
CG G
C TA C
G CG C
G
GT
CG
TC
CC
GC A
T GC A
T TA
GA
A GT
CG
TC
CC
GA
GA
T
+
Stem 1
Stem 2
Stem 3T G
TAGAGC
GCA
T
GC
GC
AT
TA
TTT
CG G
C TA C
G CG C
GC
TC
TA
GA
A
G A G
CG G
C
CG
5'
QF F
Q
What can we release from an aptamer with quinine?
P-AKT
AKT
Insulin - - - - - - + + +Insulin-DNA - - - + + + - - -
Insulin - - - - - - + + +Insulin-DNA - - - + + + - - -
P-ERK1/2
ERK
(MAP-KinasePathway)
(Phosphatidyl-inositol 3-Kinase Pathway)
P-AKT
AKT
Insulin - - - - - - + + +Insulin-DNA - - - + + + - - -
Insulin - - - - - - + + +Insulin-DNA - - - + + + - - -
P-ERK1/2
ERK
(MAP-KinasePathway)
(Phosphatidyl-inositol 3-Kinase Pathway)
A
C
NH2NH
O O
NH
OOO
O ON
O
O
PO
Ooligonucleotide
S
PO
O T AGA GCC AGA GCC CTG CTG-5'-Fluorescein
SH
(ii) TFA/anisole/0 OC/Ar
HN
12O
N
O
O / DMF
NHNH2
NH2
OHN
12O
N
O
OO
NHNH2
NH2
OHN
12O
N
O
OO
(1 equivalent)
(0.1 equivalent)
56% / PBS, pH 6.8 / EDTA, overnight
(i)
29%(2) (4)
(5)
B
P-AKT
AKT
Insulin - - - - - - + + +Insulin-DNA - - - + + + - - -
Insulin - - - - - - + + +Insulin-DNA - - - + + + - - -
P-ERK1/2
ERK
(MAP-KinasePathway)
(Phosphatidyl-inositol 3-Kinase Pathway)
P-AKT
AKT
Insulin - - - - - - + + +Insulin-DNA - - - + + + - - -
Insulin - - - - - - + + +Insulin-DNA - - - + + + - - -
P-ERK1/2
ERK
(MAP-KinasePathway)
(Phosphatidyl-inositol 3-Kinase Pathway)
A
C
NH2NH
O O
NH
OOO
O ON
O
O
PO
Ooligonucleotide
S
PO
O T AGA GCC AGA GCC CTG CTG-5'-Fluorescein
SH
(ii) TFA/anisole/0 OC/Ar
HN
12O
N
O
O / DMF
NHNH2
NH2
OHN
12O
N
O
OO
NHNH2
NH2
OHN
12O
N
O
OO
(1 equivalent)
(0.1 equivalent)
56% / PBS, pH 6.8 / EDTA, overnight
(i)
29%(2) (4)
(5)
B
Taylor et al. Angew. Chem., 2009
Can we mimic the pattern of natural release?
A B
C D
0123456789
10
0 5 10 15 20
Time/hrs
Dfm
olm
m-2
/Dh
r 0, 0, 0 M
8, 8, 8 M 1, 2, 2 M
0
2
4
6
8
10
12
0 5 10 15 20 25
Time/hrs
Dfm
olm
m-2
/Dh
r 1.5 M 18 M 6 M
0
2
4
6
8
10
0 5 10 15 20
Time/hrs
Dfm
olm
m-2
/Dh
r 0, 0, 0 M 0, 0*, 0 M 0, 5*, 5 M
A B
C D
0123456789
10
0 5 10 15 20
Time/hrs
Dfm
olm
m-2
/Dh
r 0, 0, 0 M
8, 8, 8 M 1, 2, 2 M
0
2
4
6
8
10
12
0 5 10 15 20 25
Time/hrs
Dfm
olm
m-2
/Dh
r 1.5 M 18 M 6 M
0
2
4
6
8
10
0 5 10 15 20
Time/hrs
Dfm
olm
m-2
/Dh
r 0, 0, 0 M 0, 0*, 0 M 0, 5*, 5 M
Taylor et al. Angew. Chem.