Nanoscale Mapping of Pax6 mRNA in a Tissue with Picoforce AFM Apr. - - PowerPoint PPT Presentation

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Nanoscale Mapping of Pax6 mRNA in a Tissue with Picoforce AFM Apr. 17, 2008 Joon Won Park Dept of Chemistry, Center for Integrated Molecular Systems Pohang University of Science and Technology Self-Assmebly of Dendron on Surface O O HN O

SLIDE 1

Joon Won Park Dept of Chemistry, Center for Integrated Molecular Systems Pohang University of Science and Technology

Apr. 17, 2008

Nanoscale Mapping of Pax6 mRNA in a Tissue with Picoforce AFM

SLIDE 2

1

Self-Assmebly of Dendron on Surface

O Si HO O Si O O Si O Si O Si O Si HO HO HO HO HO O OH O OH O OH O OH O OH O OH O Si HO O O Si O O Si O Si O Si O Si HO HO HO HO HO O OH O OH O OH O OH O OH O OH

O Si HO O Si O O Si O Si O Si O Si HO HO HO HO HO O O O O O O O O O OH O Si HO O O O O Si O O Si O Si O Si O Si HO HO HO HO HO O O O OH O O O OH O OH O OH O O HN O NH O O O O O O NH O O O O O O NH O O O O O O NH O O O O O O HO OH OH O OH

SLIDE 3

2

Immobilization of Gold Nanoparticles at the Dendron Surface

d: 1.4 nm

Park et al. Langmuir 2005, 4257

SLIDE 4

3

Spacing between Dendron Molecules on Surface

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 10 20 30 40 50 60 70 80

Population Distance (nm)

Average distance: 3.2 nm
Standard deviation: 0.4 nm
Particle number / area : 130 ea / 50 x 50 nm2
Density: 0.05 – 0.06 ea/nm2

SEM Image Distance between Dendron Molecules

SLIDE 5

4

Various Spacing up to 10 nm

O HN O HN O O O O O O HN O O O O O O OH OH OH HN O O O O O O HO HO OH NH O O O O O O HO HO HO O

N H O N H O O O O O O N H O O O O O O OH OH OH HN O O O O O O OH OH OH HN O O O O O O OH HO OH O O HN O NH O O O O O O HN O NH O O O O O O HN O O O O O O OH OH OH HN O O O O O O OH HO OH HN O O O O O O HO HO HO HN O H N O O O O O O HN O O O O O O HO HO HO HN O O O O O O HO HO HO HN O OO O O O HO HO HO HN O O O O O O HN O O O O O O OH OH OH H N O O O O O O OH OH OH N H O O O O O O HO HO HO O O HN O NH O O O O NH O O O O O O HN O O O O O O HO HO OH NH O O O O O O HO HO HO NH O O O O O O HO HO HO

[9]-acid [18]-acid [27]-acid, [81]-acid

~ 3 nm ~ 10 nm Spacing

SLIDE 6

DNA-DNA Interaction with 9-acid Modified AFM tips

In collaboration with School of Pharmacy, Nottingham University Yu Jin Jung and Joon Won Park

SLIDE 7

6

Proper Spacing Simplifies Force-Distance Curve

AFM Tip Retracting

Tip Tip

Joint US Patent Application (2005) Force-Distance Curve Single Dip : 100 %

SLIDE 8

7

Unprecedented Binding Event

Distance (nm) Force (pN) Force (pN) Counts

(A) (B) Park et al. J. Am. Chem. Soc., 2007, 9345

SLIDE 9

8

Resolved Binding Force Histogram

GC content = 60 %

Force (pN) Counts

SLIDE 10

9

Discrimination of Single Base Mismatched Pairs

(-2) (-2) (-7) (-9)

Force (pN) Counts

SLIDE 11

Yu Jin Jung and Joon Won Park Pohang University of Science and Technology

2-D Image of Pax6 mRNA Distribution with 27-acid modified AFM tips

In collaboration with Professor Hong Gil Nam and Dr. Yu Shin Park

SLIDE 12

11 11

Pax6 mRNA

The in situ hybridization analysis with probes for Pax6 (A and C) Development, 2002,129, 5041. Expression of Pax6(green), Tbr2, and Tbr1 protein in E14.5 cortex (coronal sections).

J. Neuroscience, 2005, 25, 247.

Pax6 mRNA plays a pivotal role in embryonic cell of mouse brain. Pax family consists of genes encoding transcription factors that regulate the development of the visual,

lfactory, central nervous systems, pituitary, and pancreas.

The essential role of Pax6 in early eye induction is conserved throughout the evolution of multicellular animals.

SLIDE 13

12 12

Secondary Structure of pax6 RNA

2,580 bases in vitro transcript (802 bases)

Free Energy Structure= -515 kcal/mol Free Energy Structure= -142.1 kcal/mol

SLIDE 14

13 13

Force Curve and Histogram for a Model System I

Size DNA onto a tip RNA on a substrate GC % 30 bases DNA30: 5’-NH2(CH2)6-TGG GCT GAC TGT TCA TGT GTG TTT GCA TGT-3’ RNA30: 5’-NH2(CH2)6-ACA UGC AAA CAC ACA UGA ACA GUC AGC CCA-3’ [14/30 (47 %)]

SLIDE 15

14 14

Force Curve and Histogram for a Model System I

Binding Force Histogram Unbinding Force Histogram

SLIDE 16

15 15 DNA binding sequence

802 bases RNA

2D Image of mRNA with the Force Measurement

SLIDE 17

16 16

Interaction between Isolated 802 Bases and 30mer DNA

with non-complementary oligonucleotide with complementary oligonucleotide

SLIDE 18

17 17

Microscope Images of the Mouse Embryonic Tissue

in situ hybridization with the digoxigenin-labeled antisense Pax6 RNA probe

SLIDE 19

18 18

Low Resolution Force Maps of Pax6 mRNA in a Mouse Embryonic Tissue

Manuscript in preparation

SLIDE 20

19 19 Seoul Pohang Asia Korea

Japan

Pohang is 50 min away from Seoul by Air

Use of KTX (Korea Train Express) is an alternative.

SLIDE 21

20 20

1,638,135 m² Land area 325,362 m² Buildings area

Main Campus RIST* Student Dorm. & Married Student Apt. Biotech Center Digital Library International House**

Campus Aerial View

Synchrotron Facility

SLIDE 22

Joon Won Park Dept of Chemistry, Center for Integrated Molecular Systems Pohang University of Science and Technology

Nanoscale Mapping of Pax6 mRNA in a Tissue with Picoforce AFM

1

Self-Assmebly of Dendron on Surface

2

Immobilization of Gold Nanoparticles at the Dendron Surface

d: 1.4 nm

Park et al. Langmuir 2005, 4257

3

Spacing between Dendron Molecules on Surface

Population Distance (nm)

SEM Image Distance between Dendron Molecules

4

Various Spacing up to 10 nm

[9]-acid [18]-acid [27]-acid, [81]-acid

~ 3 nm ~ 10 nm Spacing

DNA-DNA Interaction with 9-acid Modified AFM tips

In collaboration with School of Pharmacy, Nottingham University Yu Jin Jung and Joon Won Park

6

Proper Spacing Simplifies Force-Distance Curve

AFM Tip Retracting

Tip Tip

Joint US Patent Application (2005) Force-Distance Curve Single Dip : 100 %

7

Unprecedented Binding Event

Distance (nm) Force (pN) Force (pN) Counts

(A) (B) Park et al. J. Am. Chem. Soc., 2007, 9345

8

Resolved Binding Force Histogram

GC content = 60 %

Force (pN) Counts

9

Discrimination of Single Base Mismatched Pairs

(-2) (-2) (-7) (-9)

Force (pN) Counts

Yu Jin Jung and Joon Won Park Pohang University of Science and Technology

2-D Image of Pax6 mRNA Distribution with 27-acid modified AFM tips

In collaboration with Professor Hong Gil Nam and Dr. Yu Shin Park

11 11

Pax6 mRNA

Pax6 mRNA plays a pivotal role in embryonic cell of mouse brain. Pax family consists of genes encoding transcription factors that regulate the development of the visual,

The essential role of Pax6 in early eye induction is conserved throughout the evolution of multicellular animals.

12 12

Secondary Structure of pax6 RNA

2,580 bases in vitro transcript (802 bases)

Free Energy Structure= -515 kcal/mol Free Energy Structure= -142.1 kcal/mol

13 13

Force Curve and Histogram for a Model System I

Size DNA onto a tip RNA on a substrate GC % 30 bases DNA30: 5’-NH2(CH2)6-TGG GCT GAC TGT TCA TGT GTG TTT GCA TGT-3’ RNA30: 5’-NH2(CH2)6-ACA UGC AAA CAC ACA UGA ACA GUC AGC CCA-3’ [14/30 (47 %)]

14 14

Force Curve and Histogram for a Model System I

Binding Force Histogram Unbinding Force Histogram

15 15 DNA binding sequence

802 bases RNA

2D Image of mRNA with the Force Measurement

16 16

Interaction between Isolated 802 Bases and 30mer DNA

with non-complementary oligonucleotide with complementary oligonucleotide

17 17

Microscope Images of the Mouse Embryonic Tissue

in situ hybridization with the digoxigenin-labeled antisense Pax6 RNA probe

18 18

Low Resolution Force Maps of Pax6 mRNA in a Mouse Embryonic Tissue

Manuscript in preparation

19 19 Seoul Pohang Asia Korea

Japan

Pohang is 50 min away from Seoul by Air

Use of KTX (Korea Train Express) is an alternative.

20 20

1,638,135 m² Land area 325,362 m² Buildings area

Main Campus RIST* Student Dorm. & Married Student Apt. Biotech Center Digital Library International House**

Campus Aerial View

Synchrotron Facility

21 21

Postech Biotech Center and a Newly Dedicated Library