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
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
NH
OH
O
O
HO
HO
HO
O
O
O
O
HO
O
O
HO
O
HO
HO
O
O
O
O
O
HO
HO
O
O
O
O
HO
O
O
HO
O
NH
Si
Si
Si
Si
Si
Si
Si
Si
Si
Si
NH
O
NH
O
O
O
O
O
OH
Si
O
O
HO
1
O
O
O
O
HO
Si
O
O
O
HO
O
O
O
O
O
HO
HO
HO
O
HO
O
OH
O
HO
HO
OH
O
OH
O
HO
HO
OH
O
O
O
O
O
O
O
O
O
O
OH
O
O
O
OH
O
O
O
O
O
HO
OH
O
HO
O
HO
O
O
O
O
O
O
O
O
O
O
O
O
Si
Si
Si
Si
Si
Si
Si
Si
Si
Si
Si
Si
Immobilization of Gold Nanoparticles
at the Dendron Surface
d: 1.4 nm
Park et al. Langmuir 2005, 4257
2
Spacing between Dendron Molecules on Surface
¾ SEM Image
¾ Distance between Dendron Molecules
80
70
Population
60
50
40
30
20
10
0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Distance (nm)
• Particle number / area : 130 ea / 50 x 50 nm2
• Average distance: 3.2 nm
• Density: 0.05 – 0.06 ea/nm2
• Standard deviation: 0.4 nm
3
5.5
6.0
Various Spacing up to 10 nm
O
O
O
HO
NH
O
O
HN
HN
O
O
HO
O HO
OH
OH
O
O
O
HO
O
O
HO
O
HO
O
O
O
O
O
O
O
O
OO
O
OHHO
O
O
OHO
O
O
HO
O HO
N
O
O
O
O
O
O
O
HN
O
O
O
O
NH
HN
O
O
OO
O
HO OHO O
OH
O
OH
O OH
O
OH
O
OH
OH
O
HO
HO
O
HO
O
O
O
O
O
HO
O
ON
H O
O
O
HO OHO
O
O
O
OO
NH
O
O
O
O
O
O
HO O
OO O
O
OH
HO
O
O
O
HN
O
OH
O
O
O
O
OH HO
O
OH
O
O
O
OH
O OH
O
[18]-acid
[27]-acid, [81]-acid
~ 3 nm
Spacing
~ 10 nm
O
O OH
OH
OH
O
O
O
[9]-acid
4
N
H
O OH
O
O
HN
O HN
HN
O
HO
N
H
O
O O
O
HO
O
O
O
HN O
O
O
O
O
HN
O
HN
HO O
HO O
HN
O
HN
O
O
O
NH
O
O
O
NH
O
O
HN
O
O
HO
NH
OH
O
OH
O
O
O
O
NH
O O
O
O
O
O
O
O
HO
O
NH
O
O
HO
HO O
HO
O
O
O
HO
O
O
O
HN
O
HN
HN
HN
O
O
O
HN
O
O
HN
O
O
O
OH
O
OH
OH
DNA-DNA Interaction with 9-acid Modified AFM tips
In collaboration with School of Pharmacy, Nottingham University
Yu Jin Jung and Joon Won Park
Proper Spacing Simplifies Force-Distance Curve
Tip
Tip
AFM Tip Retracting
Force-Distance Curve
Single Dip : 100 %
Joint US Patent Application (2005)
6
Unprecedented Binding Event
(B)
Counts
Force (pN)
(A)
Distance (nm)
Force (pN)
Park et al. J. Am. Chem. Soc., 2007, 9345
7
Counts
Resolved Binding Force Histogram
Force (pN)
GC content = 60 %
8
Discrimination of Single Base Mismatched Pairs
(-2)
(-7)
(-2)
Counts
(-9)
Force (pN)
9
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
Yu Jin Jung and Joon Won Park
Pohang University of Science and Technology
Pax6 mRNA
¾ Pax6 mRNA plays a pivotal role in embryonic cell of mouse brain.
¾ Pax family consists of genes encoding transcription factors that regulate the development of the visual,
olfactory, central nervous systems, pituitary, and pancreas.
¾ The essential role of Pax6 in early eye induction is conserved throughout the evolution of multicellular
animals.
Expression of Pax6(green), Tbr2, and Tbr1 protein in E14.5 cortex (coronal sections).
J. Neuroscience, 2005, 25, 247.
11
The in situ hybridization analysis with probes for Pax6 (A and C)
Development, 2002,129, 5041.
Secondary Structure of pax6 RNA
¾ 2,580 bases
¾ in vitro transcript (802 bases)
Free Energy Structure= -142.1 kcal/mol
Free Energy Structure= -515 kcal/mol
12
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 %)]
13
Force Curve and Histogram for a Model System I
¾ Binding Force Histogram
¾ Unbinding Force Histogram
14
2D Image of mRNA with the Force Measurement
802 bases RNA
DNA binding sequence
15
Interaction between Isolated 802 Bases and 30mer DNA
¾ with complementary oligonucleotide
¾ with non-complementary oligonucleotide
16
Microscope Images of the Mouse Embryonic Tissue
¾ in situ hybridization with the digoxigenin-labeled antisense Pax6 RNA probe
17
Low Resolution Force Maps of Pax6 mRNA
in a Mouse Embryonic Tissue
Manuscript in preparation
18
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