From Confocal Microscopy to Molecular Imagineering Dr. Michael L. Norton Department of Chemistry...
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Transcript of From Confocal Microscopy to Molecular Imagineering Dr. Michael L. Norton Department of Chemistry...
From Confocal Microscopy to Molecular Imagineering
Dr. Michael L. Norton Department of Chemistry
Marshall University
1:30 p.m.
Outline:
Applications:•Molecular and Biological Imaging Center •Leica Two-Photon Microscope
•Molecular Models/Mechanical Engineering•DNA Origami•Molecular Lego Parts•Enzymes, Structural Proteins, Toxins
•Molecular Models/Molecular Electronics
Molecular and Biological Imaging Center
Biotechnology Building
Leica Two Photon Microscope
Two-photon excitation versus one-photon excitation
543 nm excitation
1046 nm excitation
Dye solution, safranin O
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Cellular Imaging -
3% of the image
Christopher Cox, Collier Lab
How Small is a Micron100X a DNA Nanoarray
Space Filling Representation of 2D Crystal Tile (4 X 16 nm)
Introduction to DNA OrigamiFolding DNA to create nanoscale shapes and patterns
Rothemund, P. W. K. Nature 2006, 440, 297–302.
Folding Pattern
inSēquio by Parabon NanoLabsinSēquio by Parabon NanoLabs
The inSēquio editor provides CAD tools for specifying nanostructure designs. It then uses grid supercomputing capacity to search through the vast space of possible DNA sequence sets for the rare few able to self-assemble into the target design. Typical searches would take years to perform on a single computer, but require only hours on the Parabon Computation Grid.
The inSēquio editor provides CAD tools for specifying nanostructure designs. It then uses grid supercomputing capacity to search through the vast space of possible DNA sequence sets for the rare few able to self-assemble into the target design. Typical searches would take years to perform on a single computer, but require only hours on the Parabon Computation Grid.
Single Block Production AFM Image Of The Rectangular Origami with Biotinylated DNA staples, modified to prevent agglomeration
AFM Image Of The Rectangular Origami with Biotinylated DNA staples
AFM Image Of The Rectangular Origami with Biotinylated DNA staples after Protein Addition
The Rectangular Origami with Biotinylated Staples In 1D Array Before and After addition of Streptavidin
Sequential Assembly of Origami/Protein Complexes
Sequential Assembly of Origami/Protein Complexes via NTA Linkage
Ni
Shen, Norton, NTA Directed Protein Nanopatterning on DNA Origami Nanoconstructs, JACS 2009Polylysine binder to mica surface nitrilotriacetic acid (NTA)
Streptavidin-biotin-origami (SBO) construct
54nm
=Biotin
=Streptavidin
Streptavidin-biotin-origami reaction mixture was 5:1 streptavidin: DNA origami ratio. Sample 1: SBO mixture was incubated at RT for 2 hrs. keep at 4C Sample 2: SBO mixture was incubated at 4C
Neutravidin-Biotin Origami construct
54nm
=Biotin =Neutravidin
Neutravidin distance=56nmOrigami=109nm
AFM analysis of the Rectangular origami-Neutravidin-Ricin AB conjugate
Surface Immobilized Single Enzyme Oscillator
Distance between Leu864 and Asp812 calculated using: 27.550 Angstroms
• Chains A (blue), B (purple), and C (green) with helices in orange and Leu864A in red
4rnp T7-RNA Polymerase
Molecular Electronics
Conjugated Fluorescent Polymers Length ~ 15nm
Polyvinylidene
HOMO of 3mer
27
Central Ring UnsubstitutedCentral Ring substituted
LUMO of 3mer
28
Central Ring UnsubstitutedCentral Ring substituted
HOMO of 3mer
29 Central Ring Unsubstituted
LUMO of 3mer
HOMO of 3mer
30 Central Ring substituted
LUMO of 3mer
Schematic structure of the A and B tiles of the Cross-Origami and the their binding
orientation on 2D origami array
A
B AA
B
BB
A81nm30nm
AFM of Cross Origami (low resolution)
AFM of Cross Origami
AFM analysis: Origami Cross
AFM by D. Neff
Single Cross-origami study
AFM by D. Neff
Conclusions
The viz lab perfectly complements our current and futureimaging systems
Successful Protein engineering will likely require collaborative studies.
Molecular simulations are a necessary enhancement for design, because they provide our only way to “observe” the relationship between properties and structural perturbations
There are tremendous opportunities for engineering collaborations catalyzed by these infrastructure investments
Norton Group Spring 2011
N
Acknowledgments• Qrigami Design, Fabrication and Characterization
Hong Zhong Masudur Rahman Jacob Potter
• Nanoscale Optical Imaging Anuradha Rajulapati
• Micro-Fluidics System Nathaniel Crow • Origami Sequence Selection
Steven Armentrout, Parabon NanoLabs, DNA sequence design software
• e-beam LithographyAaron Gin, CINT, Center for Integrated Nanotechnologies
• Protein Design Ben Owen
• Protein-DNA ConstructsDawn Nicholas
Wanqiu Shen
• Imaging Support David Neff
• Computational Chemistry Jack Smith • Viz Lab Imaging Support
Justin Chapman
DNA Benchmarks/NanoFab AdviceNadrian Seeman, NYUErik Winfree, CaltechDavid Lederman, WVUMark Reed, YaleSteven Brueck, UNMChris Dwyer, Duke
Funding
Question/Comments ?