Download - SELF-ASSEMBLED MONOLAYERS OF PEPTIDE NUCLEIC ACIDS … · self-assembled monolayers of peptide nucleic acids (pna): from the molecular structure to biosensor applications. c. briones

SELF-ASSEMBLED MONOLAYERS OF PEPTIDE NUCLEIC ACIDS (PNA):

FROM THE MOLECULAR STRUCTURE TO BIOSENSOR APPLICATIONS.

C. Briones , E. Mateo-Martí , V. Parro, C. RogeroCentro de Astrobiología (CSIC-INTA). Madrid, Spain

J.A. Martín-Gago, C. Gómez-Navarro (UAM) , J. Mendez, E. Román, Instituto de Ciencia de Materiales de Madrid (CSIC). Madrid, Spain

Victor Fernandez, Marcos PitaInstituto de Catalisis (CSIC). Madrid, Spain

http://www.icmm.csic.es/esisna/

Interdisciplinary work: biology, chemistry, Physics

Motivation of the research project:

To investigate the interaction of nucleic acids with solid surfaces

Origin of life on earth could be a catalytic processMetallic oxides, pyrite, clay minerals, silicates

Surface science can model all these interactions

XPS,AFM, NEXAFS, IRSGold, pyrite, silicon, nanoparticles

1.- Nanoscience:

To build new biosensors from the bases

3SSpecificSensitiveSimple

2.-Nanotechnology:

ssDNA: (single stranded) single chain

dsDNA double stranded in double chain (helicoidal :Every nucleo-base recognaice by H-bridges a complementary one

Sugar(deoxyribose)

Phosphategroup

Nitrogeneous bases(nucleo-bases)

DNA (deoxyribonucleic acid): polynucleotide

nucleotide

Rapid bio-background for physicists

backbone

?? ??labelling

Two unknownnucleotidesWhich is the sequence

DNA- microaarrays, how does it work?

AG AT AC

GT GC TC

Robot imprinting

Inkjet or piezo actuation technology. Spots of 50-150 μm in diameter, and low-density microarrays with no more than 5,000 spots/cm2

Target

Probe

TC

?? ??labelling

Robot imprinting

?? = AG

Fluoresce Scanner

DNA- microaarrays, how does it work?Two unknownnucleotides

In every spot a sequence

DNA

Spacer

Surface

DNA- microaarrays, how does it work?

DNA-chip pattern

Drawbacks of the current technology:Nanotechnology comes to help…

1.- Fluorescence molecular label of the target

2.- Expensive equipments

3.- No quantitative information,lack of specificity, not very sensitive

-Alkanethiol layers have been extensively studied due to their important technological properties and their outstanding capability to form self-

assembled monolayers (SAMs).

AFM From E.Barrena and C.Ocal

Alkanethiols self-assembled monolayers

gold

S-head

Alkyl chain

Functional group

IDEA!!!

Let us add a S atom at the end of a DNA to recover the alkanethiol ideas and made a solid-state biosensor

Clean Au Immobilized DNA Hybridized DNA

More details:E. Casero, M. Darder, D. J. Díaz, F. Pariente, J. A.Martín-Gago,H. Abruña and E. Lorenzo

Lamgmuir 2003,19, 6230-6235

strong surface-molecule molecule-molecule interactions

-Thiolated DNA also immobilized on gold surfaces, but most of theresults have been disappointing: DNA fold into itself leading to a formless globular structure, with a very reduced bioactivity.

PNA is an achiral and uncharged DNA mimic

the sugar-phosphatebackbone has been replaced with a peptide-

like N-(2-aminoethyl)glycine polyamide structure. The nucleobases are connected by methylenecarbonyl linkages.

PEPTIDE NUCLEIC ACID (PNA)

Menchise et al., PNAS 2003

PNA recognizes complementary DNA with stronger affinity than DNA-DNA:

(5’)

(3’)

ON N

NH2

O

OO

OOO P

OO

OOO P

OO

OOO P

O

O

OOO P

N NH

O

O

NH2N

NN N

NH2

N

NN NH

O

(N)

O

N

NH

O

O

N

NH

O

O

N

NH

O

O

N

NH

O

(C)

NNH

O

O

N

NNN

H2N

NN

O

H2N

N

NH2N

HN

O

N

Can be used as probe for DNA biosensorsAbsence of P in the backbone:

Specific signature: no fluorescence

EXPERIMENTAL PROCEDURE

Cysteine

Spacer

ssPNA

Au

H2O H2OTarget NANA

Immobilization(22ºC; 4 h)

Washing(22ºC; 15 min)

Hybridization(53-58ºC; 1 h)

Washing(50-58ºC; 15 min)

XPS, AFM,XANES,IRS

ss-PNA Immobilized:Structural characterization

Ordered SAM

PNA-DNA duplexFunctional characterization

biosensor


XPS, AFM,XANES,IRS

H2O H2OTarget NANA





ss-PNA Immobilized:Structural characterization

Ordered SAM

PNA-DNA duplexFunctional characterization

biosensor


XPS, AFM,XANES,IRS XPS, AFM,XANES,IRS

49nm

50nm

RESULTS : Structure of the layers

AFM images: • ordered arrangement of the molecules, with reproducible aligned and meandering patterns.

• The ordered protrusions are 6 to 7 nm high from the bare surface.

• Width: 10 to 30 nm.

The ssPNA molecules stand-up on the surface with a small tilt.

RESULTS : Structure of the layers

- XANES spectra at the N-edge at grazing andnormal emission indicate a preferentialorientation of the molecule with the nucleo-bases nearly- paralel to the surface plane.

Inte

nsity

(arb

.uni

ts)

420415410405400Photon energy (eV)

Normal incidence

70º off normal incidence

Most of the π* orbitals lie along the backbone of the PNA and the σ* are parallel to the nucleobases plane,

49nm

Proposed Structural model

- Self-assembling of ssPNA molecules

promoted by non-complementary

H-bonding between nucleobases

RESULTS : Concentration dependence

41nm

[ssPNA] = 0.1μM

- Linear featuresfollowing crys-tallographic directions

- Height: ~ 1 nm- Length: ~ 10 nm- Individual mole-cules lying on the surface

200nm

[ssPNA] = 0.5μM

- Layer not com-plete

- Groups of mo-lecules standup as islands anchored to theupper part of the step edges

50nm

[ssPNA] ≥ 10μM

- Ordered featuresare lost

- Surface saturatedof amorphous groups of mole-cules

50nm

[ssPNA] = 1.0μM

- Aligned and meandering patterns

- Height: 6-7 nm- Width: 10-30 nm- Groups of mole-cules stand up on the whole surface

- Optimal coverage

With increasing PNA concentration

Advantage of ssPNA over ssDNAin the formation of bioSAMs

i) the lack of charged groups in the PNA backbone avoids electrostatic repulsions either among neighbouring molecules or among the solvent counterions.

ii) although relatively flexible, the PNA molecule is more rigid than DNA due to the planar amide

Now, let us see if they work as biosensors…

RESULTS : Concentration dependence

41nm

[ssPNA] = 0.1μM

200nm

[ssPNA] = 0.5μM

50nm

[ssPNA] ≥ 10μM[ssPNA] = 1.0μM

With increasing PNA concentration

PNA molecules standing up on the surface

PNAmolecules lying on

the surface

PNA molecules intermixed one with each other

RESULTS : Hybridization to DNA

The hybridization of complementary DNA is detected by XPS:- Increase of N1s/Au4f ratio in afactor of 2.8 to 3.

- Detection of aclear P2p signal.

130 132 134 136 138

396 398 400 402 404

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Inte

nsity

(arb

. Uni

ts)

600 500 400 300 200 100 0Binding energy (eV)

O 2p N 1s

C 1s Au 4f

Au

Immob. PNA

PNA/DNA

XPS overview

P 2p

RESULTS : Characterization of the biosensor

0,001

0,01

0,1

1

10

0,1 1 10 100

ImmobilizedHybridized

N1s

/Au4

f rat

io

ssPNA Concentration (μM)

Evolution of the normalized XPS intensity with the concentration of immobilized PNA:

I

II

III 50nm

I

II

III

AFM imaging:

0,001

0,01

0,1

1

10

0,1 1 10 100

Immobilized

Hybridized

N1s

/Au4

f rat

io

ssPNA Concentration ( μM)

50nm

NA concentrations higher than 5 μM are not useful since:1. Surface is saturated of NA, and the possibility of hybridization with target NA is strongly reduced2. During the hybridization incubation non-specifically bound NA is removed from the surface, and therefore N1s/Au4f decreases

41nm

[ssPNA] = 0.1μM

200nm

[ssPNA] = 0.5μM

50nm

[ssPNA] ≥ 10μM[ssPNA] = 1.0μM

Is that a general rule for nucleic acids?

PNA molecules standing up on the surface

PNAmolecules lying on

the surface

PNA molecules intermixed one with each other

Just when the molecules stand-up, do not interact with the surface and are isolated, are biosensitive

Let us see on pyrite: a more reactive, metallic, natural surface

Evolution of the SAM structure with PNA concentration

Absence of thiol Group: covalent bonding

PM-RAIRS RESULTS on goldE. Briand, C. M. PradierLab. De reactivité des surfacesCNRS-Jussieu

PM-RAIRS RESULTS on pyrite

PNA adsorbs on pyrite surface, however…

Presence of thiol group:

unspecific bonding

Wide peaksAll transition allowed

XANES at N1s on gold

Inte

nsity

(arb

.uni

ts)

420415410405400Photon energy (eV)

XANES at N1s on pyrite

Orientation of PNA on pyrite

PNA-DNA interaction on pyrite surface

SugarPhosphate

phosphate

PM-RAIRS RESULTS on pyrite

On Pyrite, ssPNA adsorbs strongly interacting with the surface,Without forming covalent bonding through the S atom

DNA is always on the surface, independently of the sequence:Unspecific bonding

Immobilization and hybridization of single-stranded PNA on aldehyde terminated monolayers prepared at Si (111) surfaces

CH3

SiSiSi

Si

CH3 CH3

OH

CH3CH3OH OH

SiSiSi

SiH|

H| H

|

H|

1

100 % C10CHO 0 % C11

50 % C10CHO 50 % C11

10 % C10CHO 90 % C11

1 % C10CHO 99 % C11

0 % C10CHO 100 % C11

0 % C10CHO 0 % C11 (clean H-Si(111)

Si(111) substrate

Col. B HorrowckUniversity New castle

400nm 400nm

2 x 2 µmContact Tapping

C11-Si(111)

N

NH

OO

Base

N

NH

OO

Base

N

NH

OO

Base

Immobilization and hybridization of single-stranded PNA on aldehyde terminated monolayers prepared at Si (111) surfaces

CH3

SiSiSi

Si

CH3 CH3

OH

CH3CH3OH OH

SiSiSi

SiH|

H| H

|

H|

CH3

SiSiSi

Si

CH3 CH3

OH

1 2

N

NH

OO

Base

N

NH

OO

Base

N

NH

OO

Base

3

CH3

SiSiSi

Si

CH3

CH3

O

OO O

O

OP

O

OO O

O

OPBase

BaseO

OO O

O

OPBase

100 % C10CHO 0 % C11

50 % C10CHO 50 % C11

10 % C10CHO 90 % C11

1 % C10CHO 99 % C11

0 % C10CHO 100 % C11

0 % C10CHO 0 % C11 (clean H-Si(111)

Si(111) substrate

CONCLUSIONS

- PNA molecules, in spite of theirlength of up to 7 nm, can self-assemble on gold surfaces similarly to short alkanethyol molecules.

- Two main reasons for the clear advantage of PNA over DNA:• lack of charged groups and intermolecular electrostatic repulsions• higher rigidity and restrited conformational flexibility

- Mechanism for the formation of SAMs of ssPNA:• at low coverage densities, molecules condensate on the surface andare absorbed as lying molecules

• at a certain coverage (corresponding to [PNA] ≈ 1 μM) the layer undergoes a phase transition: realignment of the molecule backboneperpendicular to the surface

- XPS can be used for the monitorization of the hybridization of complementary DNA, the characterization of a PNA-based biosensor, and its optimization to discriminate point mutations in target DNA.

NUCLEIC ACIDS AND THEIR ANALOGUES AS NANOMATERIALS FOR BIOSENSOR DEVELOPMENTC. Briones, J.A. Martín-Gago . In press: current nanotechnology

E. Mateo-Martı´, C. Briones, E. Roma´n, E. Briand, C. M. Pradier, and J. A. Martín-GagoLangmuir 2005, 21, 9510-9517

Briones, C.; Mateo-Marti, E.; Gomez-Rodriguez, C.; Parro, V.Roman, E.; Martín-Gago, J. A.

Phys. Rev. Lett. 2004, 93, 208103

Understanding surface-molecule interactions could be of great help to design 3S biosensors

(specific, sensitive, simple)

More information:

No N1s signal is detected

108 106 104 102 100 98 96

s83 100% C10CHO s84 100% C10CHO PNA version 1

Si 2p

Binding energy (eV)108 106 104 102 100 98 96

s83 100% C10CHO

Si 2p

Binding energy (eV)

PNA immobilization:PG142- noE (AATCCCCGCAT).

• Version 1 (microarray-like): •drop of PNA solution (5 μM in milli-Q water) .•When the drop is dried,•30 μl of 5.0 M aqueous Sodium Cyanoborohydride (Sigma) is deposited on top. The solution is left to react for 18 h

• Version 2 : •drop of PNA solution (5 μM in milli-Q water) .•Immediately, a frop of 30 μl of 5.0 M aqueous Sodium Cyanoborohydride is added. The solution is left to react for 18 h

Not perfect. We damage the surface!!!!!

Surface completely oxidized

410 408 406 404 402 400 398 396 394 392 390

sample84 100% C10CHO PNA version 1 sample83 100% C10CHO

N 1s

Binding energy (eV)410 408 406 404 402 400 398 396 394 392 390

sample82 100% C10CHO PNA version 2 sample84 100% C10CHO PNA version 1 sample83 100% C10CHO

N 1s

Binding energy (eV)

108 106 104 102 100 98 96

s83 100% C10CHO

Si 2p

Binding energy (eV)

RESULTS : Identification of mutations in DNA

MISMATCH

SCOMP.

S

S

COMPLEMENTARYHYBRIDIZATION IS

MAINTAINED

S

WASHING

MISMATCHEDHYBRIDIZATION IS

REMOVED

Mutation screening experiment:160

140

120

100

80

60

40

20

0

Inte

nsity

(arb

. Uni

ts)

408406404402400398396

Binding Energy (eV)

Fully complementary target Mismatched target, after washing Immbilized NA

XPS result:

3,5

ssP

NA

N1s/Au4fP2p/Au4f

2,5

1,5

0,5

2,0

1,0

0ss

PN

A+

ssD

NA

(M)

ssP

NA

+ss

DN

A (M

M)

ssP

NA

ssP

NA

+ss

DN

A (M

)

ssP

NA

+ss

DN

A (M

M)

3, 0

Optimization of washing buffer:

DNART-PCR with

Cy5 -dUTP

STRAIN SELECTION

HYBRIDIZATION

MatchingProbe (M)

WASHING

M MM

SCANNING

M MM

RNA

Naturalsample

LabeledTarget DNA

MismatchingProbe (MM)

200nm

0 50 100 150 200 250 300

02

46

810

X[nm]

Z[nm]

100nm


Au

Target DNAH2O W.S.PNA





Immobilized PNA 1: HS-Cys-O-O-AATCCCCGCGTTarget- G142 (M): 5'-CCGCCAGTGCACGCGGGGATTTGGCTCACCT-3' Target- E142 (MM): 5'-CCGCCAGTGCACGCGAGGATTTGGCTCACCT-3'

Immobilized PNA 2: HS-Cys-O-O- GCCATCTCTTarget- M41 (M): 5'-GAAATTTGTACAGAGATGGAAAAGGAAGGGA-3' Target- L41 (MM): 5'-GAAATTTGTACAGAGTTGGAAAAGGAAGGGA-3'

We have Choosen sequencies of clinical relevanceAnd similar to the one used in microarray technoology

Table. Infrared assignment for the main frequencies of PNA on gold surface

PNA Concentration Assignment

2977

2931

2854

1250

1168

1086

933

2974

2928

2854

1240

1161

1084

2963

2931

2864

1736

1669

1613

1549

1250

1168

1100

933

2967

2935

2857

1680

1602

1235

1161

1083

933

0.01µM 1µM0.1µM 10µMνasym(CH3)

νasym(CH2)

νsym(CH2)

ν(C=O)

N-H

NH2 C=N

C=C, C=N

νasym(C-O-C)

ν(C-N,C-C),δ(C-H)

νsym(C-O-C)

ν(C-N)

δ(N-H)oop

Study of the N(1s) core level of PNA by means of XPS

N 1s Binding energy

Assignment % N Calculated % N Experimental

398.93 -N= 25 23.4

400.13 -NH- 59.4 62.2

401.10 NH2 15.6 14.4

Table. Assignment of N(1s) core level peak of PNA

XPS core-level peak of N (1s) for PNA adsorbed on Pyrite surface

XPS core-level peak of N (1s) for PNA-DNA adsorbed on Pyrite surface

Table . Experimental reported binding energy (eV) of of N(1s) core level peak for % of different chemical statesof nitrogen involved in the PNA chemical structure.

Specie 0.1μM PNA 1μM PNA 10μM PNA 1μM PNA-DNA

NH2 2.7 % 3.5% 11.3% 3.1%

-NH- 40.0% 30.1% 32.8% 21.1%

-N= 34.1% 44.1% 44.8% 42.6%

N-Fe 23.0% 22.3% 11.1% 33.2%

Specie 1μM PNA 1μM PNA-DNA

18.7% 20.2%

FeS2 (Cys) 55.1% 65.0%

Free-thiols SH 12.0% 8.3%

SO42- 14.2% 6.5%

Table . Experimental reported binding energy (eV) of of S(2p) core level peak for % of different chemical states of sulphur involved in the PNA-Pyrite interaction.

S (2p) Core Level of PNA S (2p) Core Level of PNA-DNA

XANES

XANES was used to corroborate the absence of any preferential orientation of PNA

adsorbed on pyrite surface.

AFM study

100nm 100nm100nm100nm

limpia 0,1μm 1μm 10 μm

120100806040200

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2

0

X [nm ]Z[

nm]

150100500

2.5

2

1.5

1

0.5

0

X[nm]

Z[nm

]

80706050403020100

1.5

1

0.5

0

X[nm]

Z[nm

]

100806040200

3.5

3

2.5

2

1.5

1

0.5

0

X[nm]

Z[nm

]

200nm 200nm200nm200nm

limpia 0,1μm 1μm 10 μm

Experiments realised on this year:

Nov 2004- Nov 2005

Si modified surfaces:

C11 C10CHO C11S C11NH2

Mixed layers:PNA immobilizedDNA hybridized

Mixed layers:Au nanoparticlePNA immobilizedDNA hybridized

For future PNA synthesis

Surface science techniques:

AFM (contact and tapping)XPS (home lab and synchrotron)XANES (normal and grazing emission)FTIR

OBJECTIVES

- Study the immobilization of thiol-modified ssPNA molecules on gold surfaces.

- Characterize the structure and ordering of the layers.

- Monitor the hybridization of complementary ssDNA to layers of immobilized ssPNA.

- Characterize the PNA-based biosensor: concentration, temperature,time, buffer composition.

- Determine the ability of the biosensor to detect point mutations(SNPs) in target DNA.

-Perform this structural and functional characterization by meansof powerful label-free techniques for surface characterization: atomic force microscopy (AFM), X-ray photoemission spectroscopy(XPS) and X-ray absorption near-edge spectroscopy (XANES)

Biosensors based on DNA-chips

DNA-chip pattern

Detection of hybridization is performed by using fluorescence molecules

- XPS Experiments: performed ex situ in an Ultra High Vacuumchamber (UHV: Pressure 10-9 mbar). The sample was transported inair for a few seconds. The chamber is equipped with a double-passcylinder mirror analyser (CMA) . A Mg anode in the X-ray source wasused for experiments (1253.6 eV photons).

- Synchrotron radiation experiments (XPS and XANES): performed atELETTRA-superESCA beam line. The overall resolution was around 80 meV.

- AFM images: recorded in air using a NANOTEC-AFM working intapping mode.

- Substrates used:• XPS: polycristaline Au layers evaporated on glass (Arrandee, Germany)

and single-crystal Au(111)• AFM: Au films Annealed to 600ºC for few minutes.


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