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 Micron 100X a DNA Nanoarray

Space Filling Representation of 2D Crystal Tile (4 X 16 nm)

Introduction to DNA Origami Folding DNA to create nanoscale shapes and patterns Rothemund, P. W. K. Nature 2006, 440, 297–302. Folding Pattern

inSēquio 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.

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 2009 Polylysine binder to mica surfacenitrilotriacetic 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 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=56nm Origami=109nm

AFM analysis of the Rectangular origami- Neutravidin-Ricin AB conjugate

Surface Immobilized Single Enzyme Oscillator

Distance between Leu864 and Asp812 calculated using: 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 A A B B B A 81nm 30nm

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 future imaging 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 Lithography Aaron Gin, CINT, Center for Integrated Nanotechnologies Protein Design Ben Owen Protein-DNA Constructs Dawn Nicholas Wanqiu Shen Imaging Support David Neff Computational Chemistry Jack Smith Viz Lab Imaging Support Justin Chapman DNA Benchmarks/NanoFab Advice Nadrian Seeman, NYU Erik Winfree, Caltech David Lederman, WVU Mark Reed, Yale Steven Brueck, UNM Chris Dwyer, Duke

Funding

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