Key Experimental Approaches in DNA Nanotechnology

Nadrian C. Seeman1

1 New York University, New York, New York
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 12.1
DOI:  10.1002/0471142700.nc1201s09
Online Posting Date:  August, 2002
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DNA nanotechnology combines unusual DNA motifs with sticky‐ended cohesion to build polyhedral objects, topological targets, nanomechanical devices, and both crystalline and aperiodic arrays. The goal of DNA nanotechnology is control of the structure of macroscopic matter on the finest possible scale. Applications are expected to arise in the areas of X‐ray crystallography, nanoelectronics, nanorobotics, and DNA‐based computation. DNA and its close molecular relatives appear extremely well suited for these goals. This overview covers the generation of new DNA motifs, construction methods (synthesis, hybridization, phosphorylation, ligation), and a variety of methods for characterization of motifs, devices, and arrays. Finally, the use of DNA nanotechnology as a tool in biochemistry is discussed.

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Table of Contents

  • The Generation of New DNA Motifs
  • Construction Methods
  • Characterization in DNA Nanotechnology
  • Characterization of Devices
  • DNA Nanaotechnology as a Tool in Biochemistry
  • Conclusions
  • Literature Cited
  • Figures
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  •   FigureFigure 12.1.1 Formation of a two‐dimensional lattice from a four‐arm junction with sticky ends. A sticky end and its complement are indicated by X and X′, respectively. The same relationship exists between Y and Y′. Four of the monomeric junctions on the left are complexed in parallel orientation to yield the structure on the right. DNA ligase can close the gaps left in the complex. Note that the complex has maintained open valences, so that it could be extended by the addition of more monomers.
  •   FigureFigure 12.1.2 Reciprocal exchange between DNA strands. (A) Reciprocal exchange between two juxtaposed helical half turns. A black and a gray hairpin are shown. The helix axis is horizontal in this view and the dyad axis is vertical. Arrowheads on strands indicate the 3′ ends. A negative node is formed in the rightward reaction, where the strands have retained their initial shading. (B,C) Reciprocal exchange generates a four‐arm junction from two double helices. Panel B shows exchange between strands of the same polarity, and panel C shows exchange between strands of opposite polarity. Symmetry elements are indicated by arrows in B.
  •   FigureFigure 12.1.3 The structural isomers of DNA double‐cross‐over (DX) molecules. DPE, DPOW, and DPON are the three parallel DX molecules. The DAE and DAO molecules are the antiparallel isomers. Symmetry in DAO is between the thick and thin lines. DAE+J is a molecule in which the cyclic strand of DAE is extended to form a three‐arm bulged junction. Arrowheads indicate 3′ ends. Symmetry elements are shown by arrows, and line thickness is related by symmetry in each drawing. In abbreviations, A is antiparallel, P is parallel, E and O refer to even and odd numbers of double‐helical half turns between cross‐overs, W and N indicate wide or narrow groove spacings for the odd half turn, and J is junction.
  •   FigureFigure 12.1.4 The design of a stable four‐arm branched junction. The junction shown is composed of four strands of DNA, as indicated by Arabic numerals. The 3′ end of each strand is indicated by a half arrow. Each strand is paired with two other strands to form double‐helical arms, labeled with Roman numerals. There is no homologous two‐fold sequence symmetry flanking the central branch point, thereby stabilizing its position. Tetrameric elements are boxed in black; trimeric elements are boxed in gray. The tetramers are all unique, and there is no complement to any tetramer flanking the junction. Competition with the target octamers can only occur from trimers, such as the ATG sequences.
  •   FigureFigure 12.1.5 Protocol for the solid‐support synthesis of a quadrilateral. Beginning with the support containing a closed strand, alternate cycles of restriction and ligation are performed, always at the position indicated as site 1. Selection of the target product (e.g., triangle, quadrilateral, pentalateral) is determined by the point at which one chooses to restrict at site 2, exposing a sticky end complementary to that exposed by restriction at site 1. Note that the final closure converts a simple cyclic molecule to a catenane.
  •   FigureFigure 12.1.6 The hydroxyl radical autofootprinting pattern of a DNA triple‐cross‐over (TX) molecule. The top portion of the figure contains densitometer scans of autoradiograms for each strand of the TX molecule. The data for each strand are shown twice, once for its 5′ end and once for its 3′ end, as indicated above the appropriate panel. Susceptibility to hydroxyl radical attack is compared for each strand when incorporated into the TX molecule (TX) and when paired with its traditional Watson‐Crick complement (DS). Nucleotide numbers are indicated above every tenth nucleotide. The two nucleotides flanking expected cross‐over positions are indicated by two Js. Note the correlation between the Js and protection in all cases. Additional protection is seen at further locations (arrows), indicating occlusion a turn away from the cross‐over points on the cross‐over strands, and about four nucleotides 3′ to the cross‐overs on the helical strands, as noted previously. The data are summarized on a molecular drawing below the scans. Sites of protection are indicated by triangles pointing towards the protected nucleotide; the extent of protection is indicated qualitatively by the sizes of the triangles. Asterisks indicate labeled strands.
  •   FigureFigure 12.1.7 Ligation products of antiparallel double‐cross‐over molecules DAE, DAO, and DAE+J. One domain has been capped by hairpins. Ligation of the DAE molecule leads to a reporter strand, which is drawn more darkly. Ligation of the DAE+J molecule also leads to a reporter strand, similar to the one in DAE. However, ligation of the DAO molecule produces a polycatenated structure.
  •   FigureFigure 12.1.8 Restriction analysis of a DNA cube. The cube is drawn with strands of three different thicknesses, with the darkest in front and the lightest at the rear. The linear triple catenane shown at the center was the starting material for the last step of the construction. It corresponds to the left, front, and right sides of the cube. Its removal from the cube by restricting at the left‐front and right‐front edges leaves the top‐back‐bottom linear triple catenane as a product. Each edge of the cube contains a unique restriction site.


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