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.