Switching with nucleic acid hybridization

Response

→ Light→ Light→ Protein activity

DNA/RNAProteinDNA/RNA

Who

pushsthe

button?

Trigger: Nucleic Acid ; Response: Light

DNA-drivenlight switch

Yurke, Nature 2000

Molecular

Beacons

Tyagi, Kramer, Nat. Biotechnol. 1996

Molecular Beacons: Enhanced specificity through constraint

constraining element

DNA duplex

Tyagi, Kramer, 1996

homo-DNA duplex

Leumann, 2005

LNA

Benner, Tan, 2005

hydrophobic probe

Seitz / Frank-Kamenetskii, 2000

DNA quadruplex

Jullien, Mergny, 2006

?

Molecular Beacons: Enhanced specificity through constraint

constraining element

DNA duplex

Tyagi, Kramer, 1996

homo-DNA duplex

Leumann, 2005

LNA

Benner, Tan, 2005

hydrophobic probe

Seitz / Frank-Kamenetskii, 2000

DNA quadruplex

Jullien, Mergny, 2006

DNA2

·PNA triplex

Grossmann, Röglin, Seitz,Angew. Chem. Int. Ed. 2007,46, 5223

Modular system:

one dual labeled oligonucleotide

tuning of restoring force throughunlabeled stem forming strand

High fidelity probes

Aim:

Improve

target

specificity

1)

improve

hybridization

selectivity

of probes

2)

add

another

sequence

discriminating

event

FIT probes

match

mismatchFIT probe

target

Forced

intercalation

(FIT): fluorescent

dye

serves

as base

surrogate

responds

to perturbations

Temperature dependence of fluorescence increase

Dilip Jarikote

matchedmismatcheddiscrimination

Τ � ��

Fluo

resc

ence

incr

ease

0

5

10

15

20

25

30

25 30 35 40 45 50 55 60 65 70 75 80 85

X=C

X=T

X=G

5´-CGGCT XT TACGGC-3´C-Gly-gccga tO atgccg-N

FIT PNA

Q

D

QD

hybridization

match

mis-match

noDNA

MolecularBeacon

Tyagi, Kramer, Nat. Biotechnol. 1996, 14, 303

Detection of Her2-neu

FIT probe:

AcNHgtactg Orn(TO) aagcct-GlyCONH2target DNA :

3’-CATGAC T TTCGGA- 5’

FIT probe SYBR-Gold

Linear range: 7 orders

of magnitudeSensitivity:

102

copiesLinear range: 5 orders

of magnitudeSensitivity:

105

copies

Improved

sensitivity

compared

to DNA-stain

Socher, Jarikote, Knoll, Röglin, Burmeister, Seitz, Anal. Biochem. 2008, 375, 2, 318-330

50

150

250

350

10 20 30 40

Rel

ativ

e Fl

uore

scen

ce [a

.u.]

10% match8% match6% match5% match3% match2% match1% match0% match

-100

400

900

1400

1900

2400

2900

0 20 40

Rel

ativ

e Fl

uore

scen

ce [a

.u.] 100% match

75% match50% match25% match10% match8% match6% match5% match3% match2% match1% match0% match

Elke Socher

Single base mutation analysis with wildtype

background

Probe:

AcgccgtaTOatagccg; target:

3‘---CGGCATTT/AATCGGC---5‘

FIT Probes: High sequence

specifity(Fma

-F0

)/(Fmi

–F0

) ≤

10

at non-stringent

conditions(Fma

-F0

)/(Fmi

–F0

) ≤

200

at stringent conditions

High dynamic

range

of fluorescence

signalingFma

/F0

≤

30

Next

generation: Low

noise

stem-less

PNA beacons

Socher, Bethge, Knoll, Jungnick, Herrmann, Seitz, Angew. Chem. Int. Ed. 2008, 47, 9555-9559

Next

generation: Low

noise

stem-less

PNA beacons

low

→ twisting, FRET, contacthigh

low

→ contacthigh → FRET

Fluorescence

Single strand:Double strand:

Socher, Bethge, Knoll, Jungnick, Herrmann, Seitz, Angew. Chem. Int. Ed. 2008, 47, 9555-9559

Next

generation: Low

noise

stem-less

PNA beacons

detection

modesdonor

fluorescenceacceptor

fluorescenceFRET

detection

modesdonor

fluorescence

Stem-less

PNA beacons

provide

for

high sensitivity

5´-CGGCTATTTAGGC-3´C-gccgataOatgccg-NIR

0

100

200

300

400

500

600

700

800

500 550 600 650 700

ds: 26ss: 1

Ex: 485 nm

5´-CGGCTATTTAGGC-3´C-gccgataOatgccg-C

0

20

40

60

80

100

120

500 550 600 650 700 750 800 850

ds: 28ss: 1

ds: 108ss: 1

Ex: 485 nm

λ � �� λ � ��

Socher, Bethge, Knoll, Jungnick, Herrmann, Seitz, Angew. Chem. Int. Ed. 2008, 47, 9555-9559

RNA Detection

Compared to FIT-probe

Short synthetic RNA

In vitro transcribed 650 nt RNA

• 1µM probes and 2µM RNA

NO

N

S

NO

N

O

TO YO

• 30 nM

probes and 300 nM

RNA

DNA-catalyzed transfer

FAM TAMRA

Dabcyl

Großmann, Seitz, J. Am. Chem. Soc. 2006, 128, 15596-15597

The chemistry

Transfer: 3 3Entropy

TM

= 38°CAffinity TM

= 38°C ?

PNA-HN COOH

S

O

H2NSH

PNA

O+

PNA-HN COOH

SHH2N

SPNA

O+

O

PNA-HN COOH

SH

HNSH

PNA

O

+O

Grossmann, Seitz, J. Am. Chem. Soc. 2006, 128, 15596-15597

DNA-catalyzed transfer

no DNA

37 °C, pH

7.0, 0.2 µM probes,10 mM

PO4

, 200 mM

NaCl,0.2 mM

TCEP, 0.1 mg/mL RBR

1 eq. DNA

0.1 eq. DNA

65 TO (3.5)

215 TO

402 TO

Grossmann, Seitz, J. Am. Chem. Soc. 2006, 128, 15596-15597

Amplified detection of DNA and RNA

Grossmann, Röglin, Seitz, Angew.Chem. Int. Ed. 2008, 47, 7119

Amplified detection of DNA and RNA

after

addition

of 1M H2

SO4

:

no RNA

50 fmol RNA

Grossmann, Röglin, Seitz, Angew.Chem. Int. Ed. 2008, 47, 7119

Switching with nucleic acid hybridization

Response

→ Light→ Light→ Protein activity

DNA/RNAProteinDNA/RNA

Who

pushsthe

button?

Hairpin Peptide Beacons

Peptide Beacons

AA2

AA6

AA3

AA5

AA

4

AA1

AA7AA1 AA2 AA3 AA4 AA5 AA6 AA7

ProteinProtein

AA1 AA2 AA3 AA4

AA5AA6

AA

7

Hairpin

Peptide Beacons

AA2

AA6

AA3

AA5

AA

4

AA1

AA7

AA1 AA2 AA3 AA4 AA5 AA6 AA7

ProteinProtein

PNA peptide

Thurley, Röglin, Seitz, J. Am. Chem. Soc. 2007, 129, 12693-12695

Target: SH2 domain of Src

kinase

Src-SH2 binds

peptide

in extended

conformation

ProGlu Pro

IleGlu

GluTyrGln

PO32

t at ag cg c

Lys Lys

O

=design,synthesis

Thurley, Röglin, Seitz, J. Am. Chem. Soc. 2007, 129, 12693-12695

addition

of Src-SH2

addition

of Grb2-SH2

F

t / min

addition

of Src-SH2

0

2

4

6

8

10

12

0 2 4 6 8 10 12 14 16 18 20

addition

of Grb2-SH2

HPB PBF

0

2

4

6

8

10

12

t / min0 2 4 6 8 10 12 14 16 18 20

Target: active site of protease renin

Renin

bindspeptide

inextended

conformation N N

NIR-664

N N CO

N

DabcylO

O3S

HisPro Ile

ValStat

HisPhePro

g cg ct at a

Lys Lys-GlyCONH2Ac

=

=

Stat

=NH

OH O

Reversible binding, not

cleaved

⇒ increase

and decrease

of protein

activity

can

be

measured

Thurley, Röglin, Seitz, J. Am. Chem. Soc. 2007, 129, 12693-12695

Switching with nucleic acid hybridization

Response

→ Light→ Light→ Protein activity

DNA/RNAProteinDNA/RNA

Who

pushsthe

button?

Crosslinking

signal

transduction

pathways

mRNA

transducer

Bioactive

peptide

conformation

of SH2 ligands

Src

SH2 bindsextended conformation

Ala-Gln-pTyr-Glu-Glu-Ile-Pro-Gly-Tyr-Leu

Wakesman et al, Cell 1993, 72, 779

inactive

active

Pawson et al, TICB 2001, 11, 504

Intermolecular

hybridization

PNA PNAPeptide

RNADNA

PNA

Hybridization

triggers

inhibition

no inhibition

96% inhibition

Conditions:1.25 µM 1, complexed with 2 µM 5a in buffercontaining 20 nM 6 and 700 nM GST-Src-SH2,addition of 10 µM 4g after 2.25 minutes.

L. Röglin, R.M. Ahmadian, O. Seitz,

Angew. Chem. 2007, 2759-2763

IC50

= 6.9

μM IC50

= 0.5

μM

Repeated

switching

A G C G C A T A T G A C T A G G T T A T G CG CT C G C G T A T A C T G A T C C A A T A C GC C

T C G C G T A T A C T G A T C C A A T A C GC C

A G C G C A T A T G A C T A G G T T A T G CG C

Lars Röglin

RNA-induced

activation

of Src-kinase

p-Tyr

Tyr-p

act. RNA

deact. DNA

+

0

20

40

60

80

100

Src

reac

tivat

ion

/ %

+ act. RNA Chimera•

deact. DNA

Chimera•

deact. DNA

+act. RNA

O O

PO4

O O

O HO

O O

phosphorylatedSrc-kinase+ substrate

Pyruvat-kinase Lactat-gehydrogenase

ADP ATP NADH NAD+

ATP change ofUV-Absorption

inactive

active

IC50

= 6.9

μM

IC50

= 0.5

μMRöglin, Altenbrunn, Seitz,

ChemBioChem 2009, 10, 758

Overview

Nucleic

acid

detection

Protein/nucleotide

chemistry

COOHCOOHHS X Y H2NCONH2H2N

SH3COH

HN

H2NNHN

NH OH

Bergmann et al.Angew. Chem. 1999, 111, Mende et al. Angew. Chem. 2007, 119, 4661,Haase et al. Angew. Chem. 2008, 120, 1575,Haase et al. Angew. Chem. 2008, 120, 6912Hainke

et al. J. Org. Chem. 2007, 72, 8811

Seitz Angew. Chem. 2000, 112, 3389,Mattes et al. Angew. Chem. 2001, 113, 3277,Dose et al. Angew. Chem. 2006, 118, 5495,Grossmann et al. Angew. Chem. 2007, 119, 5315,Grossmann et al. Angew. Chem. 2008, 120, 7228,Socher

et al, Angew. Chem. 2008, 120, 9697Köhler et al. ChemBioChem 2005, 6, 69,Ficht et al. J. Am. Chem. Soc. 2004, 126, 9970,Grossmann et al. J. Am. Chem. Soc. 2006, 128, 15596.

Protein-protein/DNA interactions

Beuck

et al. Angew. Chem. 2003, 115, 4088,Röglin

et al. Angew. Chem. 2007, 119, 2759,Thurley

et al. J. Am. Chem. Soc. 2007, 129, 12693.100

500

900

1300

10 20 30 40Cycle

Rel

ativ

e Fl

uore

scen

ce [a

.u.]

1 ng0,1 ng0,01 ng0,001 ng0,0001 ng0,00001 ng0,000001 ngNTCTreshold

DNA/RNA

AA1 A

A5

AA2 AA4

AA3

Protein

AcknowledgementsThe

Group:

Frank AbendrothFrank AltenbrunnLucas BethgeFranziska DiezmannAnne ErbenMelanie FischbachChristian HaaseSven HainkeHendrik EberhardtDr. Rikard

LarsonDr. Andrea KnollFranziska MendeUlrike LauferJulia MichaelisBrigitte RedlichHeike RhodeAlexander RoloffChristian ScheibeJosephine SchmalischElke SocherChristian StutzStefanie ThurleyTanja Westphalen

Dr. Tom GrossmannDr. Lars Röglin

The

Collaborators:Amadian

(MPI Dortmund)Weinhold (RWTH Aachen)Ernsting

(HU Berlin)Röder (HU Berlin)Herrmann (HU Berlin)

The

Money:Fonds der Chemischen IndustrieDeutsche ForschungsgemeinschaftVolkswagenstiftungDeutscher Akademischer AustauschdienstNRW (Bennigsen-Foerder-Preis)Roche-DiagnosticsBayer Technology ServicesBayer-ScheringQiagen