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

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 OH NH O O O O O O O O O O O O O O O O O NH NH HO O HO HO HO HO HO O HO HO HO HO HO HO NH O O O OH O O O O O O O O O O O O O O O O O O O O O O OH HO O O O O O O O OH O OH O O O OH O OH O OH OH Si Si Si Si Si Si Si Si Si Si Si Si O O O O O O O O O O O O HO HO HO HO HO HO HO HO HO HO HO HO O O O O O O O O O O O O O O O O O Si Si Si Si Si Si Si Si Si Si Si Si 1

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 � SEM Image � Distance between Dendron Molecules 80 70 60 Population 50 40 30 20 10 0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 Distance (nm) • Particle number / area : 130 ea / 50 x 50 nm 2 • Average distance: 3.2 nm • Density: 0.05 – 0.06 ea/nm 2 • Standard deviation: 0.4 nm 3

Various Spacing up to 10 nm O O O O O O HN HN HN O O HO OH O O HO O HN OH O O OO NH O O O O O O O O NH O HO OH O O O N O HN O O N H O O HN O O O O H O H O NH O HN O N O N O O O HO H O O O O O O O O HN O O O O OH OH HN O O O O HO O HO O HO HN O O O OH O O O O O O O NH HN O O O O HN O NH HO HN OH HO O O HN O OH HO HN O O O O O O OH O O OH O O OH O O O HO O O O O O O HO O O O O O OH O O O O O O NH O NH O O O H HO N O OH OH N HO O O O O HN H O HO O O O O O OH HO O HO HO O O HO O O HN O O O HN O O O O O O O O OH O HO O HO O O O O OH HN O HO HO O O O O O OH HO O HO HO O O O O OH OH HO HO OH O HO O O O OH O O O OH O OH HO [9]-acid [18]-acid [27]-acid, [81]-acid Spacing ~ 10 nm ~ 3 nm 4

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 (A) (B) Force (pN) Counts Distance (nm) Force (pN) Park et al. J. Am. Chem. Soc ., 2007 , 9345 7

Resolved Binding Force Histogram Counts Force (pN) GC content = 60 % 8

Discrimination of Single Base Mismatched Pairs (-2) (-7) (-2) (-9) Counts 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 � 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, olfactory, central nervous systems, pituitary, and pancreas. � 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). The in situ hybridization analysis with probes for Pax6 (A and C) J. Neuroscience , 2005, 25 , 247. Development , 2002 , 129 , 5041. 11 11

Secondary Structure of pax6 RNA � 2,580 bases � in vitro transcript (802 bases) Free Energy Structure= -142.1 kcal/mol Free Energy Structure= -515 kcal/mol 12 12

Force Curve and Histogram for a Model System I Size DNA onto a tip RNA on a substrate GC % DNA30: 5’-NH 2 (CH 2 ) 6 -TGG RNA30: 5’-NH 2 (CH 2 ) 6 -ACA 30 [14/30 GCT GAC TGT TCA TGT UGC AAA CAC ACA UGA bases (47 %)] GTG TTT GCA TGT-3’ ACA GUC AGC CCA-3’ 13 13

Force Curve and Histogram for a Model System I � Binding Force Histogram � Unbinding Force Histogram 14 14

2D Image of mRNA with the Force Measurement 802 bases RNA DNA binding sequence 15 15

Interaction between Isolated 802 Bases and 30mer DNA � with non-complementary oligonucleotide � with complementary oligonucleotide 16 16

Microscope Images of the Mouse Embryonic Tissue � in situ hybridization with the digoxigenin-labeled antisense Pax6 RNA probe 17 17

Low Resolution Force Maps of Pax6 mRNA in a Mouse Embryonic Tissue Manuscript in preparation 18 18

Pohang is 50 min away from Seoul by Air Asia Japan Korea Seoul Pohang Use of KTX (Korea Train Express) is an alternative. 19 19

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