Development of Assay System for DNA-Binding

Development of Assay System for DNA-Binding Protein at Single-Molecular Level

Kobatake-Yanagida Laboratory Department of Biological Information Graduate School of Bioscience and Biotechnology Tokyo Institute of Technology 2002

Importance of Detecting DNA-Protein Interaction

Importance of Detecting DNA-Protein Interaction

Importance of Detecting DNA-Protein Interaction

Protein Binding-Site

mRNA

How to detect and identify Transcription Factor / Nuclear Receptor (DNA Binding Protein) activated by particular stimulations?

Methods for Detecting DNA-Protein Interaction Limited analysis for multiple Assay analytes

DNA Footprinting Assay Electrophoretic Mobility Shift (EMSA) Preparation is cumbersome Surface Plasmon Resonance (gel, gel banks, etc.) •Sample preparation is Quartz Crystal MicroIt Balance takes time for analyzing complicated to 1 hour for Electron Microscopy (e.g. 30 minutes •Analysis takes time gel running) Special techniques •Not suitable for manyare Limited analysis for X-ray Diffraction needed to arrange analytes multiple analytes capturing probes onto DNA Micro Array Sample amount ordered spots

Amount of DNA

AFM Method for Detecting DNA-Protein Interaction

AFM Image

Amount of DNA

DNA Size

DNA Size that makes bound

Purpose of This Study 1. Detection To establish a new method for detecting DNA-Protein Interaction

2. On-Chip Biosensing Using the new established method to propose a new method of on-chip biosensing for DNA-binding-protein

Main Principle Used for AFM Analysis Streptavidin 5‘

3‘

Protein Binding Site X nm

Y nm Total Length X+Y nm

Main Principle Used for AFM Analysis Results Possibilities from AFM Image No Binding Non Specific Binding X’≠X

X’=X

Y’≠Y

Y’=Y

Specific Binding

New Method for Detecting DNA-Protein Interaction

DNA probes with one specific protein binding site

New Method for Detecting DNA-Protein Interaction

DNA probes with different lengths each having multiple binding sites

Design and Production of ER-αDNA Probe Streptavidin

CAGGTCACAGTGACCTG

5‘

3‘

ER-α Binding Site

X= 103 nm (303-bp) Y= 73 nm (215-bp) Total Length 176 nm (518-bp)

pBS-ERE Plasmid & PCR Result for Probe Production 5‘ biotinylated primer Kpn I

pBS-ERE

Pst I 3‘ primer

518-bp ERE probe produced from PCR

PAGE Shift Assay of ERE-ERαBinding ER-α to ERE ratio

ERE only

1:1 2:1 4:1 8:1 16:1

ERE-ER-α complex band ERE only band at 518-bp

AFM Observation of ERE-ERαBinding Ni-treated mica substrate

AFM Observation of ERE-ERαBinding

ER-α only on substrate

AFM Observation of ERE-ERαBinding ERE-ER-α Complex

AFM Observation of ERE-ERαBinding ERE-ER-α Complex

ER-α Detection using AFM

5‘

+

3‘

Streptavidin

Streptavidin 5‘

3‘

ER-α Detection using AFM ERE probe to StAv molar ratio Marker 4

1:0 10:1 4:1 3:1 2:1 1:1 1:2 1:3 1:4 1:10

Marker 4

trimer dimer monomer

ER-α Detection using AFM StAv 5‘

3‘

Drop

Dry

+

ER-α

ER-α Detection using AFM

ER-α 0nM

ER-α Detection using AFM

ER-α 40nM Macro-scale View

ER-α Detection using AFM 9 8 7 6 5 4 3 2 1 0

ER-α 40nM Macro-scale View

011 5 -1 20 5 -2 30 5 -3 40 5 -4 50 5 -5 60 5 -6 70 5 -7 80 5 -8 90 5 10 -95 011 105 012 115 013 125 014 135 015 145 016 155 017 165 017 6

Amount (in number of molecules); N=24

Histogram of Complex Bounds

Class of Distance from 5'-StAv-Label to Binding Complex

Average Distance from 5’-StAv-Label to Binding Site: 100.14 + 8.67 nm (N=24)

ER-α Detection using AFM ER-α 40nM Zoom-in View

ER-α Detection using AFM ER-α 40nM Length Analysis Total Distance 102.983 nm

ER-α Detection using AFM ER-α 40nM Length Analysis Total Distance 71.918 nm

ER-α Detection using AFM ER-α 40nM Protein Size Analysis

ER-α Detection using AFM ER-α 40nM Zoom-in View

On-Chip Biosensing Scheme

Cell lysate

Solution containing target protein

On-Chip Biosensing Scheme

AFM Single-Molecular On-Chip Biosensing of ER-αProtein Mica DNA Chip

AFM Single-Molecular On-Chip Biosensing of ER-αProtein After 5nM ER-α sample

AFM Single-Molecular On-Chip Biosensing of ER-αProtein After 5nM ER-α sample

AFM Single-Molecular On-Chip Biosensing of ER-αProtein After 5nM ER-α sample

AFM Single-Molecular On-Chip Biosensing of ER-αProtein After 5nM ER-α sample

AFM Single-Molecular On-Chip Biosensing of ER-αProtein After 1 nM ER-α sample

AFM Single-Molecular On-Chip Biosensing of ER-αProtein After 1 nM ER-α sample

Conclusion 1. A DNA probe for ER-α protein that is modified with biotin at one end has been made and binds to ER-α as shown by PAGE shift assay and AFM imaging; 2. Further labeling of the probe with StAv was done, and the probe was successfully used to detect ER-α in solution, by using AFM single molecular analysis to show specific binding between the probe and ER-α protein (both through distance analysis between StAv label and binding site, and size comparison between StAv as the standard and the protein bound); 3. The possibility of using the StAv-labeled DNA probe in on-chip biosensing for ER-α protein on the surface of mica DNA chip has been shown too.

Further Works 1. To do further experiments to investigate the proper condition to utilize the StAv-labeled probe for on-chip biosensing; 2. To make stretching of the DNA probes to make AFM analysis easier; 3. To make probes with multiple binding sites for detecting several types of protein;

4. To make probes with different sizes, each having multiple binding sites for detecting even more kinds of proteins in a nanometer-scale DNA array system.

Regenerable Mica DNA Chip Cleaving for regeneration of mica DNA chip DNA Probe

Mica In-field or inlab AFM analysis

Mica DNA Chip In-field or inlab sampling