Chapter 1





Synthesis and Characterization of Self-Assembled DNA Nanostructures

Chenxiang Lin, Yonggang Ke, Rahul Chhabra, Jaswinder Sharma, Yan Liu, and Hao Yan

Abstract



The past decade witnessed the fast evolvement of structural DNA nanotechnology, which uses DNA as blueprint and building material to construct arti.cial nanostructures. Using branched DNA as the main building block (also known as a “tile”) and cohesive single-stranded DNA (ssDNA) ends to designate the pairing strategy for tile–tile recognition, one can rationally design and assemble complicated nanoarchi-tectures from speci.cally designed DNA oligonucleotides. Objects in both two- and three-dimensions with a large variety of geometries and topologies have been built from DNA with excellent yield; this development enables the construction of DNA-based nanodevices and DNA-template directed organiza-tion of other molecular species. The construction of such nanoscale objects constitutes the basis of DNA nanotechnology. This chapter describes the protocol for the preparation of ssDNA as starting material, the self-assembly of DNA nanostructures, and some of the most commonly used methods to characterize the self-assembled DNA nanostructures.

Key words: DNA nanotechnology, Self-assembly, Electrophoresis , Atomic force microscopy

1. Introduction



The notion that DNA is merely the gene encoder of living systems has been eclipsed by the successful development of DNA nano-technology. DNA is an excellent nanoconstruction material because of its inherent merits: First, the rigorous Watson-Crick base-pairing makes the hybridization between DNA strands highly predictable. Second, the structure of the B-form DNA double helix is well-understood; its diameter and helical repeat have been determined to be ~2 and ~3.4 nm (i.e., ~10.5 bases), respectively, which facilitates the modeling of even the most com-plicated DNA nanostructures. Third, DNA possesses combined

Giampaolo Zuccheri and Bruno Samorì (eds.), DNA Nanotechnology: Methods and Protocols,

Methods in Molecular Biology, vol. 749, DOI 10.1007/978-1-61779-142-0_1, . Springer Science+Business Media, LLC 2011





2. Material

2.1. Denaturing Polyacrylamide Gel Electrophoresis for the Puri. cation of Synthetic Single-Stranded DNA



structural stiffness and .exibility. The rigid DNA double helixes can be linked by relatively .exible single-stranded DNA (ssDNA) to build stable motifs with desired geometry. Fourth, modern organic chemistry and molecular biology have created a rich tool-box to readily synthesize, modify, and replicate DNA molecules. Finally, DNA is a biocompatible material, making it suitable for the construction of multicomponent nanostructures made from hetero-biomaterials.

The .eld of structural DNA nanotechnology began with Nadrian Seeman’s vision of combining branched DNA molecules bearing complementary sticky-ends to construct two-dimensional (2D) arrays ( 1 ) and his experimental construction of a DNA object topologically equal to a cube ( 2 ) . Today, DNA self-assembly has matured with such vigor that it is currently possible to build micro- or even millimeter-sized nanoarrays with desired tile geometry and periodicity as well as any discrete 2D or 3D nano-structures we could imagine ( 3– 8 ) . Modi.ed by functional groups, those DNA nanostructures can serve as scaffolds to con-trol the positioning of other molecular species ( 9– 21 ) , which opens opportunities to study intermolecular synergies, such as protein–protein interactions, as well as to build arti. cial multi-component nanomachines ( 22– 24 ) .

Generally speaking, the creation of a novel DNA motif usu-ally requires the following steps: (1) Structural modeling: physical and/or graphic models are used to help the design of a new DNA motif; (2) Sequence design: in this step, speci.c sequences are assigned to all ssDNA molecules in the model; (3) Experimental synthesis of the DNA nanostructure; and (4) Characterization of the DNA nanostructure. The .rst two steps are crucial to pro-gram the outcome of self-assembly and assisted by computer soft-ware ( 25– 30 ) . In this chapter, we are going to describe the experimental protocols involved in steps 3 and 4 .

All chemicals are purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. All buffer solutions are .ltered and stored at room temperature unless otherwise noted.

1.

Synthetic ssDNA (Integrated DNA Techonologies, Coralville, IA) with designated sequences.



2.

TBE buffer (1×): 89 mM Tris–boric acid, pH 8.0, 2 mM eth-ylenediaminetetraacetic acid disodium salt (EDTA-Na 2).



3.

20% urea-acrylamide Mix: 20% acrylamide (19:1 acrylamide:bis, Bio-Rad Laboratories, Hercules, CA), 8.3 M urea in 1× TBE buffer.







2.2. Self-Assembly of DNA Nanostructures

2.3. Non-denaturing PAGE for the Characterization of Self-Assembled DNA Nanostructures

2.4. Atomic Force Microscope Imaging of Self-Assembled DN

Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix



1 Synthesis and Characterization of Self-Assembled DNA Nanostructures . . . . . . . . 1

Chenxiang Lin, Yonggang Ke, Rahul Chhabra, Jaswinder Sharma,

Yan Liu, and Hao Yan

2 Protocols for Self-Assembly and Imaging of DNA Nanostructures . . . . . . . . . . . . 13

Thomas L. Sobey and Friedrich C. Simmel

3 Self-Assembly of Metal-DNA Triangles and DNA Nanotubes

with Synthetic Junctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Hua Yang, Pik Kwan Lo, Christopher K. McLaughlin, Graham D. Hamblin,

Faisal A. Aldaye, and Hanadi F. Sleiman

4 DNA-Templated Pd Conductive Metallic Nanowires . . . . . . . . . . . . . . . . . . . . . . 49

Khoa Nguyen, Stephane Campidelli, and Arianna Filoramo

5 A Method to Map Spatiotemporal pH Changes Inside Living Cells Using a

pH-Triggered DNA Nanoswitch. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Souvik Modi and Yamuna Krishnan

6 Control of Helical Handedness in DNA and PNA Nanostructures . . . . . . . . . . . . 79

Roberto Corradini, Tullia Tedeschi, Stefano Sforza, Mark M. Green,

and Rosangela Marchelli

7 G-Quartet, G-Quadruplex, and G-Wire Regulated by Chemical Stimuli . . . . . . . . 93

Daisuke Miyoshi and Naoki Sugimoto

8 Preparation and Atomic Force Microscopy of Quadruplex DNA . . . . . . . . . . . . . . 105

James Vesenka

9 Synthesis of Long DNA-Based Nanowires. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Alexander Kotlyar

10 G-Wire Synthesis and Modification with Gold Nanoparticle . . . . . . . . . . . . . . . . . 141

Christian Leiterer, Andrea Csaki, and Wolfgang Fritzsche

11 Preparation of DNA Nanostructures with Repetitive Binding Motifs

by Rolling Circle Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Edda Rei., Ralph H.lzel, and Frank F. Bier

12 Controlled Confinement of DNA at the Nanoscale: Nanofabrication and Surface

Bio-Functionalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Matteo Palma, Justin J. Abramson, Alon A. Gorodetsky, Colin Nuckolls,

Michael P. Sheetz, Shalom J. Wind, and James Hone

13 Templated Assembly of DNA Origami Gold Nanoparticle Arrays

on Lithographically Patterned Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Albert M. Hung and Jennifer N. Cha

14 DNA-Modified Single Crystal and Nanoporous Silicon. . . . . . . . . . . . . . . . . . . . . 199

Andrew Houlton, Bernard A. Connolly, Andrew R. Pike,

and Benjamin R. Horrocks



15 The Atomic Force Microscopy as a Lithographic Tool: Nanografting of DNA Nanostructures for Biosensing Applications . . . . . . . . . . . . . . . . . . . . . . . Matteo Castronovo and Denis Scaini 209

16 Trapping and Immobilization of DNA Molecules Between Nanoelectrodes. . . . . . Anton Kuzyk, J. Jussi Toppari, and P.ivi T.rm. 223

17 DNA Contour Length Measurements as a Tool for the Structural Analysis of DNA and Nucleoprotein Complexes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Claudio Rivetti 235

18 DNA Molecular Handles for Single-Molecule Protein-Folding Studies by Optical Tweezers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ciro Cecconi, Elizabeth A. Shank, Susan Marqusee, and Carlos Bustamante 255

19 Optimal Practices for Surface-Tethered Single Molecule Total Internal Reflection Fluorescence Resonance Energy Transfer Analysis. . . . . . . . . . . . . . . . . Matt V. Fagerburg and Sanford H. Leuba 273

20 Engineering Mononucleosomes for Single-Pair FRET Experiments. . . . . . . . . . . . Wiepke J.A. Koopmans, Ruth Buning, and John van Noort 291

21 Measuring DNA–Protein Binding Affinity on a Single Molecule Using Optical Tweezers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Micah J. McCauley and Mark C. Williams 305

22 Modeling Nanopores for Sequencing DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jeffrey R. Comer, David B. Wells, and Aleksei Aksimentiev 317

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359