1

APTAMER BIOSENSORS AS DNA/RNA-BASED TOOLS FOR PROTEOMICS

S. Tombelli, M. MasciniUniversità degli Studi di Firenze, Italy

Department of Chemistry

Outline

• Introduction on aptamersdefinitionselection procedurestarget moleculescomparison with antibodies

• Aptamer based biosensorsacoustic sensorsmicromechanical sensorsoptical sensorsarrays

2

Aptamers are oligonucleotides (DNA or RNA molecules) that can bind with high affinity and specificity to a wide range of target molecules (proteins, peptides, drugs, vitamins and other organic or inorganic compounds).

Aptamers

They were “discovered” in 1990 by the development of an in vitro selection and amplification technique, known as SELEX (Systematic Evolution of Ligands by Exponential enrichment).

Their name is derived from the Latin word “aptus” which means “to fit” and the Greek suffix “-mer”.

T7 Constantregion

Constantregion

Randomsequence

5’ 3’

A library containing a 40-nucleotide random region is represented by 440 (~1024) individual sequences available for partitioning. Normally, the starting round contains no more than 1014-1015 individual sequences.

Cloning and sequencing

The SELEX process

Targetmolecule

Systematic Evolution of Ligands by Exponential enrichment

3

Target molecules

PROTEINSSyrian golden hamster prionEscherichia coli SelBL-selectinTyrosine phosphatase

Ff gene 5ThrombinHIV-1 TatHIV-1 Rev

Inosine monophosphate dehydrogenaseVascular endothelial growth factorBasic fibroblast growth factor

Human IgETaq DNA polymeraseIron regulatory proteinHuman oncostatin M

Human neutrophil elastaseHuman CD4

INORGANIC COMPOUNDSMalachite greenMg2+

ORGANIC COMPOUNDSATP

FMNTheophyllineOrganic dyes

Cocaine

VITAMINSCyanonobalaminBiotin

DRUGSNeomycin B

StretpomycinTobramycinTetracyclinKanamycine A

J.C. Cox, A.D. Ellington, Bioorg. Med. Chem. 9, 2525-2531, (2001)

• PhotoSELEX:modified ssDNA aptamers capable of photocross-linking the target molecule.The method is based on the incorporation of a modified nucleotide, 5-bromo-2’deoxyuridine (BrdU), activated by absorption of light, in place of a native base in the randomised oligonucleotide library. The aptamers selected with this method have the ability to form a photo-induced covalent bond with the target

Automation and modification of the SELEX process

M.C., Golden, B.D. Collins, M.C. Willis, T.H. Koch, J. Biotechnol. 81, 167-178, (2000)C. Bock et al., Proteomics, 4, 609-618, 2004

• Automated selection of aptamers

4

Applications based on molecular recognition:

Therapeutics: aptamers have been selected to disrupt the function of their targets and

to inhibit or modify the metabolism associated with that target

Diagnostics: the impressive discrimination between two molecules of very similar structure has suggested that aptamers can be potential diagnostic reagents

Analytical tools: flow cytometrycapillary electrophoresis and electrochromatographyaffinity chromatography

biocomponents in biosensors

Applications

Why aptamer-based biosensors for proteomics?

One major postgenomic goal is the analysis of a chosen cellular proteome

gene expression profiles based on nucleic acid detection

increasing need for rapid and sensitive techniques for direct analysis of proteins

Conventional ligands for non-nucleic acid targets are antibodies

Alternative: aptamersSuccess in the automation of aptamer selection suggests that selection of aptamers on a proteome scale will soon be possible

Profiling arrays based on aptamer biosensors specific for cancer-related molecules will help to profile, at the protein level, the differences between cancer and normal cell types

5

Why aptamers can rival antibodies?

• Overcoming of the use of animals for their productionThe immune response can fail when the target molecule, i.e. protein, has a structure similar to endogenous proteins and when the antigen consists of toxic or non-immunogeniccompounds

• After selection, aptamers are produced by chemical synthesis and purified to a very high degree by eliminating the batch-to-batch variation found when using antibodies

• By chemical synthesis, modifications in the aptamer can be introduced enhancing the stability, affinity and specificity of the molecules

• Higher temperature stability

• Because of their small size, denser receptor layers can begenerated

• Amplification by PCR

Transducers

• Acoustic sensors

• Cantilever-based sensors

• Optical sensors

• An almost unexplored area of aptamers is in sensors based on electrochemical detection.Aptamers, being polyanionic, may be attractive for sensing the changes in conductance in the presence, or absence of target binding*.

*K.M. You, S.H. Lee, A. Im, S.B. Lee, Biotechnol. Bioproc. Eng. 8, 64-75, (2003)

6

Love-wave biosensor

• Target molecule: Thrombin and Rev peptide• DNA aptamer• Transducer: SAW Love-wave sensor • Immobilisation of the aptamer on the sensor surface:

M.D. Schlensog, T.M.A Gronewold, M. Tewes, M. Famulok, E. Quandt, Sensors Act. B, 101, 308-315, (2004)

s s s s s s s s

Love-wave sensors: highly sensitive analyte detection can be achieved in parallel fashion opening up the possibility of using the sensor-principle in an arrayformat

Love-wave biosensor

• Detection limit72±11 pg/cm2 (thrombin)77±36 pg/cm2 (Rev peptide)

• Dynamic range low nM- low µM

• Affinity-like constant K=500 nM

7

Quartz crystal biosensor 1

M. Liss, B. Petersen, H. Wolf, E. Prohaska, Anal. Chem., 74, 4488-4495, (2002)

• Target molecule: human IgE• DNA aptamer compared with anti-IgE antibody• Transducer: quartz crystal microbalance • Immobilisation of the aptamer on the sensor surface: 5’ biotinylated aptamer immobilised on streptavidin fixed on the gold surface with DSP.

Biosensor to detect concentrations and ligand affinity parameters of free unlabeled proteins in real time

• Detection limit 100 µg/L (Ab and aptamer)

• Linear range 0.1-1 mg/L (Ab)0.1-10 mg/L (aptamer)

• Affinity Kd= 1.9 nM (Ab)Kd= 3.6 nM (aptamer)

• Stability crystals modified with aptamers could be stored for several weeks

Quartz crystal biosensor 1

8

• Immobilisation of the aptamer on the transducer surface (gold)

Biotinylatedaptamer

(B)

Freeaptamer

Regeneration

(A)

RU

TimeInjection of protein

Washing

Free aptamerTime (sec)

0 500 1000 1500 2000 2500

Freq

uenc

y sh

ift (H

z)

-300

-200

-100

0

100

200

300

400

500

Buffer

Sample

Regeneration

Washing BufferA

B

Quartz crystal biosensor 2Surface Plasmon Resonance biosensor

M. Minunni, S. Tombelli, A. Gullotto, E. Luzi, M. Mascini, Biosens. Bioelectron, in press

HIV-1 Tat protein

HIV-1 replication cycle is controlled by the viral trans-activator of transcription protein Tat (Transcription Trans-Activator).The basic role of Tat is to promote effective elongation of viral mRNA during transcription.

Tat is a small polypeptide of 86-102 amino acids comprising a few functional regions

Acid region Cysteine-richregion

Arginine-richregion

Core region

Glutamine-richregion

1 22 37 48 57 77 101

The arginine-rich region (49-57) of Tat is involved in binding the RNA trans-activation response element (TAR).

TAR is a 59-base hairpin-bulge structure located at the 5’ end of all viral mRNAs.

TAR

9

Aptamer for Tat protein

SELEX has been used to isolate an RNA aptamer with high affinity for Tat protein.

The selected aptamer had two repeats of the TAR core binding elements and bound Tat peptide 133 times more efficiently than TAR.*

* R. Yamamoto et al., Genes to Cells (2000) 5, 371.

The selected aptamer RNAtat can be useful for inhibiting the Tat function (as a “decoy”) in vivo and as a diagnostic reagent for the detection of Tat.**

** R. Yamamoto et al., Genes to Cells (2000) 5, 389.

Piezoelectric biosensor results

0

20

40

60

80

100

120

0 0.5 1 1.5 2 2.5 3

conc TAT (ppm)

Shif

t (H

z)

Thermally treated aptamer

Aptamer

Improvements in reproducibility

Non-treated aptamer: CV%=16% (n=3 for each concentration) (1 crystal);CV%=21% (8 crystals)

Thermally treated aptamer: CV%=6% (n=3 for each concentration) (1 crystal); CV%=8% (8 crystals)

10

Specificity

-5

0

5

10

15

20

25

freq

uenc

y sh

ift (

Hz)

TAT 0.65 ppmBCL-2 0.65 ppmhIgG 0.65 ppmREV 0.65 ppm

“Blanks”: BSA 0.1% in binding buffer0.2M KCl + 5 mM glutathione (at different dilutions)

0

R2 = 0,997

R2 = 0,975

0

10

20

30

40

50

60

70

80

0 0.2 0.4 0.6 0.8 1 1.2 1.4

conc. TAT (ppm)

Shift

(H

z)

AntibodyAptamer

Comparison with the immunosensor

Anti-Tat antibody(monoclonal)

Y EDAC/NHS

Y

Y

YY

11

0

200

400

600

800

1000

1200

1400

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75

Tat conc. (ppm)

Shi

ft (

RU

)

SPR biosensor results (normal aptamer)

Reproducibility

CV%=7% (n=3 for each concentration) (1 crystal);

CV%=6% (8 crystals)

Immobilised AptamerImmobilised Aptamer: ACGAAGCUUGAUCCCGUUUGCCGGUCGAUCGCUUCGA

Selectivity

Response of Rev protein (1.25 ppm): 25% respect to Tat at the same conc.

BSA( 0.1, 0.5 ppm): 0 RU

0

500

1000

1500

2000

2500

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4Conc. Tat (ppm)

Shift

(RU

)

CV% = 3%

StreptavidinBiotin

PolyA(20) tail

aptamer

SPR biosensor results (aptamer with polyA tail)

D.I. Van Ryk, S. Venkatesan, J. of Biol. Chem, 274, 17452-17463

12

0

200

400

600

800

1000

1200

1400

1600

1800

Sh

ift (R

U)

Specificity

Tat1.25 ppm 0.125 ppm

Rev1.25 ppm 0.125 ppm Bcl-2

1.25 ppm

hIgG1.25 ppm BSA

1.25 ppm

Cantilever-based biosensor

• Target molecule: Taq DNA polymerase• DNA aptamer• Transducer: cantilever• Immobilisation of the aptamer on the sensor:5’ thiolated aptamer immobilised on gold

Cantilever-based biosensing:

Label-free detectionBatch-fabricatedSmall scale

Arrays can be used in parallel to detect various proteins simultaneously

• Affinity: Kd=15 pM

C.A. Savran, S.M. Knudsen, A.D. Ellington, S.R. Manalis, Anal. Chem. 76, 3194-3198, (2004)

13

Fiber-Optic Microarray

• Target molecule: Thrombin

• DNA aptamer• Transducer: optical imaging fibers (fluorescence measurements)

• Immobilisation of the aptamer on the sensor surface: aptamer modified at the 5’ end with an amino group with a spacer arm (C6). The aptamer was fixed onto silica microspheres ((3-aminopropyl)triethoxysilane, glutaraldehyde, polyethylenemine, SBB activated oligonucleotides)

M. Lee, D.R. Walt, Anal. Biochem. 282, 142-146, (2000)

Pivotal role in the blood coagulation and anticoagulation cascades:relevant target protein for drug discovery

• Competitive assay: detection limit of 1 nM

volume of sample 10 µl

dynamic range low nM- low µM

• Stability: the beads modified with aptamer were stable for over 3 months

no degradation in activity during 8 h experiments

Fiber-Optic Microarray

14

• Target molecules: thrombininosine monophosphate dehydrogenasevascular endothelial growth factorbasic fibroblast growth factor

Aptamer chip-based biosensor

Cancer-associatedproteins

• DNA and RNA aptamers• Transducer: fluorescence polarisation (anisotropy)• Immobilisation of the aptamer on the sensor surface: 5’ biotinylated aptamers immobilised on streptavidin-derivatised glass substrate.• Aptamers labelled with a single 3’

fluorescein group

T.G. McCauley, N. Hamaguchi, M. Stanton, Anal. Biochem. 319, 244-250, (2003)

• Affinity studies:

solution-phase experiments Kdthrombin= 26 nM

chip-based sensor Kdthrombin= 15 nM

Aptamer chip-based biosensor

15

Photoaptamers

C. Bock et al., Proteomics, 4, 609-618, 2004

• Target molecule: 17 target proteins• DNA photoaptamers• Immobilisation of the aptamer on the sensor surface: 5’-amine-photoaptamers spotted onto activated slides

PHOTO-aptamers on the chip

Injection of sample

Gentle washing

Aggressive washing

Conjugation with dye and imaging

Irradiation with UV light(308 nm laser)

Photoaptamers

16

• Automation of SELEX will provide the tools for rapid and cheap isolation of new aptamers

• Many details of the highly elaborate genome-based DNA microarray technology are transferred to aptamers chips

• Aptamers could become the perfect complement for proteome-based analyses

Conclusions