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 ?