Properties of DNA

Properties of DNA 34Ronnie Pedersen, Alexandria N. Marchi, Jacob Majikes,Jessica A. Nash, Nicole A. Estrich, David S. Courson, Carol K. Hall,Stephen L. Craig, and Thomas H. LaBean

Keywords

DNA • Deoxyribonucleic acid • Properties • Structural DNA nanotechnology

• Self-assembly

Introduction

This chapter is targeted towards researchers both inside and outside the field of

nanomaterials. For those without experience in DNA-based nanotechnology, it will

serve as an introduction to important concepts, background, and properties of DNA

as a biopolymer, a chemical, a material, and a medium for nanofabrication and

molecular computation. For specialists in DNA nanotech, this chapter will serve as

a review and reminder of the most important properties of DNA, hopefully provid-

ing fresh insights to enhance problem solving and new viewpoints leading to novel

research directions.

R. Pedersen

Duke University, Durham, NC, USA

A.N. Marchi

Department of Biomedical Engineering, Duke University, Durham, NC, USA

J. Majikes • J.A. Nash • N.A. Estrich • D.S. Courson • T.H. LaBean (*)

Department of Materials Science and Engineering, North Carolina State University,

Raleigh, NC, USA

e-mail: [email protected]

C.K. Hall

Department of Chemical and Biomolecular Engineering , North Carolina State University,

Raleigh, NC, USA

S.L. Craig

Chemistry Department, Duke University, Durham, NC, USA

B. Bhushan et al. (eds.), Handbook of Nanomaterials Properties,DOI 10.1007/978-3-642-31107-9_10, # Springer-Verlag Berlin Heidelberg 2014

1125

mailto:[email protected]

The use of DNA as a nanoscale construction material has come to be known as

structural DNA nanotechnology – in order to differentiate it from the field of

biotechnology, in which DNA is used to encode genetic information for altering

living cells, for example, by reprogramming them to produce novel or transplanted

proteins. The primary goal of structural DNA nanotechnology is to exploit com-

plementary base pairing in order to program the self-assembly of molecules into

supramolecular complexes with desired properties. In separate sections, we will

examine the properties of DNA from the following points of view: chemical,

mechanical, biological, optical, electrical, informational, and structural.

Chemical Properties of DNA

The importance of DNA for biological processes cannot be overstated. It is

a biological polymer with a simple, yet robust information-encoding system.

DNA’s fundamental structure leads to efficient replication and transmission of

encoded genetic information. DNA has also been recognized as a unique material

for various nanotechnology applications (see section ‘DNA as a Self-Assembling

Construction Material’ below). A fundamental understanding of DNA starts at the

level of chemical structure and properties.

Basic Structure

Most people are familiar with the double-helical model of a double-stranded DNA

molecular complex (dsDNA). In this chapter, unless otherwise stated, we will

discuss this canonical DNA structure (right-handed, B-form DNA). Specific dimen-

sions and feature sizes of B-form dsDNA as well as ssDNA are summarized in

Table 34.1. The other common helix forms include A- and Z-form (see Fig. 34.1).

A-form dsDNA resembles the double-helical form of RNA, is often seen in

dehydrated samples of DNA, and has a shorter, more-compact helix than B-form

dsDNA. Z-form dsDNA has a left-handed helix and is promoted under solution

Table 34.1 Dimensions

of DNAdsDNA (B form) [1, 2] ssDNA [3]

Pitch (nm) 3.36

Repeat length: BP/turn 10.5

Helix width (nm) 2.2–2.6

Major groove width (nm) 1.17

Major groove depth (nm) 0.87

Minor groove width (nm) 0.57

Minor groove depth (nm) 0.75

Rise/BP (nm) 0.33 0.6

Charge/length (e�/nm) 6 1.66

Persistence length (nm) 50 1.5–3

1126 R. Pedersen et al.

conditions involving certain salts (especially hexamminecobalt chloride) or by high

torsional strain of the helix [1].

The fundamental unit of the DNA polymer is the nucleotide monomer.

It consists of three covalently linked chemical motifs: an aromatic nucleobase,

a deoxyribose sugar, and a phosphate group. Nucleobases are divided into two

classes: pyrimidines and purines (Fig. 34.2). The canonical purines are adenine

(A) and guanine (G) and the canonical pyrimidines are cytosine (C) and thymine

(T). Numerous other nucleobases, including uracil (U) which replaces thymine in

RNA, are found in natural and synthetic systems but they will not be addressed

here. In dsDNA, nucleobases on opposite antiparallel backbone strands interact with

one another to form ‘Watson-Crick’ hydrogen bonding base pairs. Adenine forms

two stable hydrogen bonds with thymine, and guanine forms three stable hydrogen

bonds with cytosine. The base-pairing hydrogen bonds provide the specificity for

strand-strand hybridization, while the hydrophobic (p-p) base stacking provides the

Fig. 34.1 The three most common types of DNA helices. B-form DNA is right-handed, with 10.5

base pairs per helical turn. It is the normal, expected form of dsDNA under physiological-like

solution conditions. The narrower minor groove and wider major groove are indicated. A-form

DNA is also right-handed and has a shorter more-compact helix with 10 bases per turn. Z-DNA is

left-handed and slightly stretched with 12 bases per full turn of the helix

34 Properties of DNA 1127

energetic driving force [1, 4]. Although G-C pairs contribute greater stabilization

free energy to dsDNA than do A-T pairs, this is due to their base stacking

interactions rather than to the additional hydrogen bond in a G-C pair, as often

mistakenly stated. The strength of base stacking interactions is evidenced by the

tendency of assembled DNA structures consisting of multiple, blunt-ended helices

to stack their ends and cluster next to one another on surfaces [5–7].

The DNA backbone consists of alternating ribose sugars and phosphate groups

(see Fig. 34.3). The ribose sugar is connected to the nucleobase at the 10 carbonposition of the sugar; the phosphate bridges the 50 and 30 positions of alternatingsugars. Directionality of the sugar-phosphate backbone in ssDNA stems from this

asymmetry in bonding; thus, the molecule will have distinct termini, noted as the

50 and 30 ends. Formation of a dsDNA complex from two ssDNA molecules

requires alignment of two strands with (mostly) complementary sequences in an

antiparallel orientation with respect to their backbones. Parallel backbone orienta-

tion prevents proper hydrogen bonding between the bases as well as significant loss

of aromatic base stacking that is seen in well-ordered B-form dsDNA [1].

Fig. 34.2 The nucleobases on the left are purines; those on the right are pyrimidines. The dottedlines indicate Watson-Crick hydrogen bonding pairs. DNA helices have major and minor grooves;

the latter is defined as the side of the helix the sugars are bound on, the lower half in this figure


Noncanonical Hydrogen Bonding

Despite the normal base-pairing rules stated above, nucleic acids are capable of

forming noncanonical hydrogen bonding arrangements, with both parallel and

antiparallel backbone orientations. The majority of these, however, cannot hydro-

gen bond along a continuous helix. Ionization of functional groups in nucleobases

can provide further opportunities for noncanonical bonding; at low or high pH,

the N, NH, OH, and CO groups which participate in hydrogen bonding can become

protonated or deprotonated, allowing for a variety of hydrogen bonding pairs. These

arrangements are relatively rare in physiological pH ranges (between 4 and 9) and

are less stable than well-formed duplex. Two of these structures which are of

particular interest due to their stability and use in self-assembling DNA

nanomaterials are the G-quartet and the I-motif. The G-quartet motif consists of

four guanine nucleobases as depicted in Fig. 34.4. G-quartets were discovered with

parallel chain directions but can form in a variety of parallel and antiparallel

configurations [8, 9]. Multiple, stacked, G-quartets are often referred to as

G-quadruplexes.

The I-motif is similar to the G-quartet and consists of C groups intercalating

and hydrogen bonding diagonally between strands as shown in Fig. 34.4 [10].

The I-motif is only stable at low pH when the cytosine nucleobases are protonated

[10, 11]. In contrast, G-quartets are as stable as dsDNA but with significantly slower

Fig. 34.3 A ribose sugar is

depicted bounded by two

phosphate groups. The uppergroup is bound to the 50

carbon and the lower to the 30

carbon. The 50 to 30 directionof the strand runs down the

page. The base is bound to the

10 carbon


unfolding kinetics [8, 12]. Both G-quartets and I-motifs have been exploited

for their potential in nanotechnology applications [11, 13–16].

Major and Minor Grooves

The rungs (bases) of the dsDNA ladder do not protrude from the rails (sugar-phosphate

backbone) and join at 180�. Rather, the rungs are bent at an angle of about 146� on oneside and 214� on the other, when looking down the helix axis. As shown in Fig. 34.2,the sides of the bases closest to the sugars form the minor groove, and the side farthest

from the sugar forms the major groove (see also Fig. 34.1 for wider view of the

two grooves). The major and minor grooves play significant roles in the behavior

of dsDNA. As the phosphate groups are slightly negatively charged, the minor

groove has a significantly higher negative charge density than the major groove.

This charge density encourages salts, and some positively charged proteins, to bind

to dsDNA along the minor groove without AT/GC sequence specificity [17, 18].

Fig. 34.4 Examples of noncanonical base pairing. (a) A G-quartet is depicted with hydrogen

bonding between four guanine nucleobases. An example of G-quadruplex is shown with a single

strand of DNA folding into the full structure. (b) An I-motif is depicted with two protonated

cytosine nucleobases. The cytosines intercalate vertically to maximize base stacking


Further, bending the helix increases the negative charge density at the bent region. As

the helices bend, intertwine, or simply enter into proximity, electrostatic screening is

necessary to accommodate the charges. This is particularly true for nanotechnology

purposes asmany helices must be in very close proximity for successful self-assembly

during annealing.

Thermal Melting and Annealing

Determination of the melting temperature (Tm) of a dsDNA complex into its constit-

uent ssDNA molecules requires experimental measurement via techniques such as

differential scanning calorimetry (DSC) or ultraviolet absorption spectroscopy

(UV–vis, discussed in optical properties section). An approximate melting tempera-

ture can be predicted based solely on the nucleobase sequences with a variety of

formulas. Most of these formulas assume specific conditions such as buffer compo-

sition and DNA concentration. Use of approximate melting temperatures instead of

direct empirical measurement can be useful when working with nanotechnology

applications where sample sizes may be exceedingly small or expensive.

Predictive equations for Tm are parameterized from experimental data and

typically are algebraic relationships addressing information such as AT/GC con-

tent, strand length, salt concentration, and pH [19, 20]. In general, A-T pairs

contribute less to the enthalpy of melting than do G-C pairs, ensuring that the Tm

changes with AT/GC content. Numerous programs and websites are freely avail-

able to calculate the melting temperature of dsDNA under typical solution condi-

tions and will often compare multiple predictive formulae [21]. Base pair

mismatches between strands lower stability and therefore the Tm, of the resulting

dsDNA. The degree of this change can vary depending on the type of mismatch and

local sequence but is typically between 0.6 �C and 1.5 �C for each 1 % sequence

mismatch [22]. Steric hindrance due to molecular crowding or surface interactions

may also affect Tm [23]. In nanotechnology applications, this can be especially

relevant, particularly in situations requiring dense strand packing on surfaces. Steric

concerns are often addressed via the addition of ssDNA spacers (consisting of only

T or A residues) between functional groups such as thiols or biotin moieties and the

sequence responsible for specific binding [24, 25].

Salt Effects

As mentioned above, the formation of dsDNA from ssDNA creates a region

of increased negative charge density around the minor groove. This increase in

charge density is unfavorable without the presence of counterions that screen the

backbone charges and improve the energetics of duplex formation [1, 26]. Coun-

terions are especially important for systems that rely on DNA for self-assembly

where many of the helices wind up being close together. Holliday junctions, which

are one of the most common motifs used in DNA nanotechnology (see section


‘Holliday Junctions’ below), prefer divalent ions [27]. It is for this reason that most

buffers used in DNA nanotechnology applications carefully control the concentra-

tion of divalent cations, such as Mg2+.

Monovalent cations such as Na+ typically increase the stability of B-DNA up to

a concentration of�1 M. The Tm generally increases linearly with log[Na+]. Above

1 M, the Tm of dsDNA is decreased by additional monovalent ions. Monovalent

cations typically coordinate with the phosphate of the DNA backbone [28]. Divalent

cations such as Mg2+ show evidence of site-selective binding to the negatively

charged phosphates along the DNA strand. However, unlike monovalent cations,

divalent cations can also bind to the nucleobases. This can result in destabilization

of the helix, and decreased Tm, at higher salt concentrations. Figure 34.5 shows the

change in DNA melting temperature that is brought about by varying the concen-

tration of several divalent cations [1, 29].

Solvent Effects

Typically, the addition of organic solvents to water reduces the stability of dsDNA

and decreases the Tm roughly linearly with solvent concentration [30]. In particular,

formamide has a fairly linear effect on Tm, which changes by�0.62–0.72 �C per %

formamide content [1, 31]. This decrease in Tm with formamide has been attributed

to increasingly favorable base/solvent interactions and has been used as a substitute

for thermal denaturation in the annealing of DNA nanostructures, allowing self-

assembly to occur without thermal cycling [32, 33]. Solvent effects are also

exploited during DNA purification. The most common method by far is ethanol

precipitation. Other methods exist, however, including the use of alkaline solutions

to separate circular dsDNA from linear, using cetyltrimethylammonium bromide

(CTAB) in low ionic strength solution [34, 35].

Fig. 34.5 Variations of Tm

for solutions of DNA as

a function of divalent metal

ion concentration [1]: This

figure illustrates the

importance of cation choice

for buffers; the binding

locations of divalent cations

are often dependent on

concentration and drastically

effect dsDNA stability


Changes in pH within the range 5–9 have a negligible effect on the melting/

annealing of dsDNA systems [36, 37]. Below pH 4, the nucleobases become

protonated, interfering with hydrogen bonding and making the unhybridized single

strand more hydrophilic. At significantly higher pH, the G and T bases become

deprotonated, thus preventing Watson-Crick hydrogen bonding. As such, high and

low pH can rapidly denature dsDNA.

Backbone Cleavage

The dsDNA backbone can be readily cleaved by hydrolysis of the phosphodiester

bond between the phosphate and ribose groups. The mechanism is that of an

addition-elimination reaction. During hydrolysis, the phosphorus is attacked and

becomes the center of a trigonal bipyramidal intermediate that subsequently

eliminates phosphate. In the hydrolysis, the oxygen attached to the 30 of the sugaris the leaving group, and it is in-line from the attacking group. Base catalysis

accelerates the addition step by virtue of increasing the concentration of the

attacking hydroxide ion. Acid catalysis accelerates the elimination step, during

which the leaving oxygen is protonated to become a hydroxyl. Metal catalysis

encourages cleavage by neutralizing the negative charge of the intermediate state.

The end result is a 30 hydroxyl group separated from a 50 phosphate. This, and the

requirement of many nucleases for divalent cations, is why, for long-term storage,

DNA should be stored in buffer that includes chelating agents like EDTA and be

buffered at neutral pH [1].

Chemical Cross-Linking and Modification of DNA

Many chemicals are available to chemically cross-link dsDNA so that single strands

become covalently linked. A common cross-linker is psoralen, a heterocyclic

compound which intercalates between two nucleobases. When hit with a photon of

300–400 nm, psoralen will form a covalent cross-link if the nucleobase pairs above

and below it have pyrimidines on opposite sides of the dsDNA [38]. Glutaraldehyde

and formaldehyde are used to form cross-links to lysine residues in proteins [1].

Unsaturated aldehydes [39], cisplatin, and nitrogen mustards [40] have also been

used to form interstrand chemical cross-links.

During chemical synthesis, DNA can also be chemically modified to provide

various capabilities such as additional chemical linkages, attachment of

fluorophores, or nuclease resistance. Chemical linkers are often placed at the

50 and 30 positions. Chemical linkers include thiols or amines for binding to

metals or other surfaces, alkynes and azides for click chemistry, as well as biotin

labels for strong, non-covalent binding with avidin protein. Nonnatural

nucleobase incorporation as well as other chemical modifications can be used

for a variety of purposes.


Synthesis

In recent years, a dramatic reduction in the price of custom synthetic DNA

oligonucleotides has made it feasible for researchers to buy specific DNA strands

for applications from nanotechnology to whole gene synthesis. The synthesis of

DNA is typically performed via solid-phase phosphoramidite chemistry. In this

method, the 30 terminal monomer is attached to a solid support (typically silica),

and then, the next monomer (with a protected 50 end) is attached to the 50 end of

the first monomer. The 50-OH of the first monomer and the 30-phosphoramidite

of the second monomer join to make a phosphite diester, which is then oxidized to a

phosphate diester, followed by deprotection of the 50 group of the second monomer.

The cycle is repeated until the desired molecule is synthesized. Many advances

have been made in this area, particularly in using ink-jet printers to perform the

reactions on micron-sized spots on glass or plastic surfaces for rapid, automated

synthesis [1, 41–44].

Mechanical Properties of DNA

DNA is a biological polymer that has many unique physical and chemical proper-

ties and serves a critical and central role in the function of all known life. For the

purposes of this discussion, it is important to remember that there are several

different types of molecular and atomic interactions that give DNA its properties

including, but not limited to, covalent bonding between atoms in a strand, hydrogen

bonding between bases on opposite strands, stacking interactions between bases on

the same strand, and solvent interactions. Measurements of DNA mechanical

properties must be interpreted with this diverse interaction set in mind.

Curvature and Flexibility

Despite typically being depicted as a rigid rod, DNA can be bent without breaking.

Indeed, in the cellular context, it is almost always found in tightly bent and wound

conformations. To illustrate this, the extended length of the DNA in a single human

cell is between 1.5 and 3.0 m [45], while a typical cell volume is between 200 and

2,730 mm3 [46]. For a cell volume of 200 mm3 and a DNA length of 3 m, assuming

cells are cubes with side �6 mm in length and the DNA is not limited to a smaller

nucleus, a single DNA would have to be bent 500,000 times. Another way of

approaching the problem is to consider the cylindrical volume of a DNA strand

3 m in length. If we assign B-form DNA a diameter of 2.0 nm [47], the volume of

the DNA alone would be �90 mm3. When one considers that the nucleus of a cell

typically occupies less than half the volume of the cell, and that much of the cellular

DNA must be accessible to the cell at all times, the compaction problem becomes

truly staggering. DNA achieves much of this compaction through wrapping around

protein oligomers called histones, which have diameters of 10 nm [48]. Despite the


large distortions due to twist and writhe as the DNA packs tightly against these

proteins, we are aware of no reports of DNA undergoing material failure as a result

of histone packaging.

Theoretical Models of DNA Elasticity and Force-Induced Transitions

Two widely used models that describe the solution structure and molecular

mechanics of ssDNA and dsDNA are the freely jointed chain (FJC) model and

the wormlike chain (WLC) model [49]. The simplest forms of both models do not

directly describe the details of the chain such as base sequence, hydrogen bonding,

and helical twist. For the FJC model, a polymer chain is approximated by stiff

monomers of a fixed length whose orientations are completely independent of one

another. In comparison, for the WLC model, the polymer is approximated as a rigid

rod that bends smoothly in response to thermal fluctuations [49, 50]. Due to p-pstacking of the nucleobases and electrostatic repulsions along the sugar-phosphate

backbone, dsDNA is a relatively stiff polymer [51]. Thus, the WLC model is better

suited to describe the flexibility and force extension behavior of dsDNA than the

FJC model.

Predictions of DNA stretching behavior based on these models can be compared

to experimental data from single-molecule force extension studies in which one end

of the DNA strand is fixed while the other end is pulled. At low forces, extension

and force scale linearly; the behavior is well described by both the FJC and WLC

models. Because of DNA’s stiffness, the forces needed to elongate a strand past its

linear regime are very low compared to conventional synthetic polymers [51].

At intermediate forces, the extension curve becomes nonlinear and the more

complicated WLC model predicts stretching. For an in-depth review of these

models, the reader is referred to the literature [49–52].

When end-to-end tensile forces reach 60–70 pN, dsDNA undergoes a force-

induced transition from B-form to the so-called S-form DNA. The generally

accepted structure of S-form DNA is based on computer simulations, although

the specific structure might depend on the specific attachment points [53]. The

B-to-S transition results in a roughly 2.2-fold extension in DNA contour length,

beyond which high-energy enthalpic distortions in bond angles are necessary for

further extension.

We point out that high forces are only achieved in long dsDNA helices, because

short oligomers will dissociate under force. This force-induced duplex melting is

a kinetic phenomenon, in which the applied force helps to surmount the activation

barrier for duplex dissociation. As such, the force at which dissociation occurs is

time scale dependent, but it also depends on dsDNA stability (and hence the same

factors of temperature, salt and salt concentration, and sequence discussed above).

Additional mechanical properties, such as the torsional stiffness of dsDNA and its

interplay with tensile properties, have been studied in recent years, and a full review

of dsDNA mechanics is outside the scope of this chapter. The interested reader

is referred to recent reviews on the topic [49]. Because nanotechnological


applications of DNA often involve a structural component, it is reasonable to

assume that these applications will involve mechanical forces.

Computational Methods to Describe Nucleic Acids

Computational methods are useful for understanding complex behavior of macro-

molecules such as their structures and dynamics in solution. The type of informa-

tion desired will dictate the method used. Molecular dynamic (MD) simulations

allow for dynamic properties and interactions within systems to be studied. Other

methods, such as Monte Carlo, may be used for structure prediction [54].

For MD simulations, software programs such as AMBER (Assisted Model

Building with Energy Refinement) [55] have sets of force fields for the simulation

of biomolecules, particularly proteins and nucleic acids. MD may be performed at

the atomic level. However, these simulations are computationally expensive; there-

fore, large systems are often coarse grained. This means that components of the

molecule are replaced with less complicated but approximately equivalent models.

For example, a DNA base may be replaced by a single pseudo-atom that mimics the

behavior of that base. However, it must be assured that the coarse graining

accurately represents the system. For an overview of this topic, the reader is

referred to the review listed in [54].

A notable example of a computational program relevant to DNA nanotechnol-

ogy is the program CanDo (computer-aided engineering for DNA origami)

[56]. CanDo uses a finite element method to predict the solution structure of

DNA origami assemblies (see section ‘DNA Scaffolded Origami’ below).

Double-stranded DNA is approximated as a homogenous elastic rod with mechan-

ical parameters taken from experimental measurements. DNA origami structures

are modeled as bundles of rods that are rigidly constrained to their nearest neighbor

at specific crossover points. This program currently accounts for bend, twist, and

stretch stiffness of DNA. CanDo accurately predicts flexibility and solution shape

for many DNA origami nanostructues. However, the model’s predictive ability is

currently limited to designs on a square or honeycomb lattice and does not consider

sequence effects.

Biological Properties of DNA

DNA is the primary information carrier in biology. Its role can be summed up in the

central dogma which states that DNA codes for DNA and for RNA and RNA codes

for protein. That DNA codes for DNA is evident in the way that hereditary

information is passed on from parent to progeny. To understand what the rest of

the dogma means, we have to know that RNA (ribonucleic acid) is a molecule

structurally similar to DNA except it has a hydroxyl group at the 20 ribose position.A given DNA sequence will thus have a complementary RNA sequence to which it

can anneal. In this way, a piece of DNA sequence – such as a gene – can be


transcribed into a complementary RNA sequence, which can then be translated into

a specific protein. Proteins are biological polymers made from 20 types of amino

acids, and for any triplet of nucleobases (a codon), the genetic code specifies

a specific amino acid. Proteins are the structural and catalytic ‘machines’ of living

cells and thus determine the actions and makeup of the cell. In the following, we

will look at how the properties of DNA are intimately tied to its role as the

information carrier in living cells.

DNA in Cells and in Molecular Cloning

The central dogma is a universal characteristic of all life as we know it. However,

when considering the biological properties of DNA in more detail, we have to

differentiate between the major classifications of life. Biologists group different

forms of life based on their similarity and relationship. Humans are distinct from

apes but more closely related to them than they are to dogs. All three are distinct

from plants and again these four share characteristics distinct from an Escherichiacoli bacterium. At the very root of the classification of life, we find three so-called

domains. These are Archaea, Bacteria, and Eucarya with members of Eucarya often

referred to as eukaryotes [57]. Members of all three domains have the cell as the

basic unit of life. A cell is a lipid membrane encapsulating the molecular machinery

required for executing the central dogma. One defining feature of eukaryotes is that

they have an additional internal lipid compartment, the nucleus, encapsulating their

DNA. This is distinct from Archaea and Bacteria, and for this reason and others,

these two domains have often been grouped together as prokaryotes [58]. In the

following, we will be primarily concerned with common features and will only

distinguish between eukaryotes and Bacteria, disregarding the interesting but less

studied Archaea.

In Bacteria, the entire genomic sequence is usually a single circular dsDNA

condensed in one area of the cell. For E. coli, the size of this molecule is 4.6 Mb

(million base pairs). In addition, Bacteria often have smaller auxiliary pieces of

dsDNA called plasmids. These are smaller circular molecules that can be replicated

independently and transferred between different Bacteria and thus can be used to

transfer genetic traits such as antibiotic resistance. This is at the heart of molecular

cloning and biotechnology. By inserting a gene of interest in a plasmid and

‘transfecting’ it into a bacterium, the gene will then be replicated as the cell divides.

This technique is made possible by the use of restriction endonucleases which

are a class of enzymes that cleave DNA at specific sequences. Type II endonucle-

ases typically cleave palindromic sequences, meaning sequences that read the same

backward and forward. In the context of DNA, this is understood in the way that the

sequence that reads 50 to 30 on one strand reads the same in the 50 to 30 direction on

the complementary strand. By convention, genetic sequences are written 50 to 30

(see section ‘Chemical Properties of DNA’ above for a description of DNA chain

directionality). For example, the restriction endonuclease EcoRI recognizes the

sequence 50-G · AATTC-30 and cleaves the backbone of both the written strand


and the identical complementary strand (between the G and A). This creates

a duplex with a short overhang (4 bases, in this case) called a sticky end. If

a genetic sequence is cut from a genome with a certain restriction enzyme and

mixed with purified plasmids cut with the same enzyme, the sticky ends will be

complementary and can bridge the cut plasmid by hybridizing sticky ends to

sticky ends.

Endonucleases typically hydrolyze the oxygen-phosphorus bond on the 50 side ofthe phosphorus, meaning that the 30 end of the product will have a hydroxyl group

and the 50 end will terminate at the phosphate group. This configuration allows for

ligase enzyme to covalently ligate the backbone once the sticky ends have annealed.

Ligation can be prevented by removing the phosphate on the 50 end with phospha-

tase enzyme.

Eukaryotic cells have larger genomes than Bacteria, from tens of millions of

base pairs (bp) to more than 100 billion bp, distributed on several different DNA

molecules termed chromosomes. These excessively long DNA molecules are

organized on several different hierarchical levels during the cell cycle with the

most compacted form visible through light microscopy during S phase as irregular

X shapes. The first step of organization is achieved by spooling DNA onto alkaline

proteins called histones. This creates a unit called a ‘nucleosome’ containing 146 bp

that is visible in electron microscopy as beads on a string.

Compaction of DNA by histones plays an important role in regulating

gene expression as more compact DNA are less available for RNA polymerases

to bind. Another level of transcriptional control observed in some vertebrates – but

not all eukaryotes – is based on methylation of cytosine on carbon 5. This

chemical modification of DNA generally serves to repress genetic expression.

Both histone-mediated compaction and methylation can change during

a cell’s life cycle but can also be passed on through several generations. Thus, the

effective genetic expression is altered without altering the underlying DNA

sequence.

DNA Replication

The most important biological aspect of DNA’s structure is arguably the ability to

accurately transmit genetic information to offspring. As noted by Watson and Crick

in their seminal paper from 1953, the double-helical structure immediately suggests

a mechanism for DNA replication. This mechanism is based on a semiconservative

scheme where unwinding of the double helix allows each single strand to function

as a template for the synthesis of new strands. Completion of a round of replication

thus results in two helices, each containing one strand carried over from the parent

duplex and one newly synthesized strand. If we consider this scheme, we can

identify three processes that need to occur. The duplex needs to unwind. The

resulting supercoil needs to be alleviated. And finally, new strands need to be

synthesized using the parent strands as templates. Each of these processes is

catalyzed by a different group of enzymes.


In bacterial cells, replication is initiated at a specific site on the genome called

the origin. Starting at this site, helicase enzymes hydrolyze adenosine triphosphate

(ATP) in order to peel the single strands apart, while single-stranded DNA-binding

proteins bind the single strands and help prevent reannealing. The bubble created

this way has two junctions called replication forks, and helicases run processively

along the DNA in both directions extending the loop (see Fig. 34.6). In both circular

prokaryotic and large eukaryotic genomes, this process results in supercoiling

which is alleviated by topoisomerase enzymes.

In both prokaryotes and eukaryotes, topoisomerase enzymes are divided into

type I and type II. Type I works by cleaving just one of the two strands creating

what is called a nick in the backbone (a single-strand break). This is accomplished

by a nucleophilic attack from a tyrosine hydroxyl group on a phosphorus atom in

the sugar-phosphate backbone creating a transient covalent bond between enzyme

and DNA. With the backbone nicked, either the duplex can now perform

a controlled rotation or the intact single strand can be passed through the nick

depending on the enzyme. Type II topoisomerases cleave both DNA strands of

a duplex using two tyrosine residues. By holding the ends in place, a different part

of the duplex is allowed to pass through the break, thus releasing or introducing

supercoil. This reaction cycle is completed with ATP hydrolysis.

DNA polymerase is the primary enzyme responsible for DNA replication;

however, it only catalyzes the addition of deoxynucleoside triphosphates (dNTP)

to the 30 end of a growing strand using exposed single strand as a template for dNTP

selection. This has two implications. The first is that a short primer strand is needed

for DNA polymerase to work. A primer is synthesized by a primase enzyme and

consists of RNA which is later excised and replaced with DNA. The second

Fig. 34.6 Schematic representation of replication fork. The DNA duplex is unwound by

a helicase enzyme that moves along the lagging strand. Single-stranded DNA-binding proteins

bind to the newly unwound duplex preventing reannealing. Two core polymerases associate in

a large complex consisting of several different subunits (not shown). The polymerases can only

synthesize in the 50 to 30 direction by adding nucleotides to a 30 end but both move in the same

direction along the parent duplex. This is accomplished by looping the lagging strand and

synthesizing it in the fragments. Each fragment is initiated with an RNA primer that is subse-

quently replaced with DNA (Adapted from [59])


implication is that DNA polymerase only works in the 50 to 30 direction as the 30-OHgroup makes a nucleophilic attack on the a-phosphate of the incoming dNTP. This

results in a semidiscontinuous replication scheme where the two strands are repli-

cated in different ways. One strand, called the leading strand, is initiated with one

primer and extends continuously as the parent duplex is unwound. The other strand,

called the lagging strand, can only be synthesized in short fragments as the

synthesis is in the opposite direction of the moving replication fork. These short

fragments are called Okazaki fragments and are 1,000–2,000 nucleotides long in

E. coli and 100–200 fragments long in eukaryotes.

Besides catalyzing addition of dNTPs, all DNA polymerases in E. coli also have30 to 50 exonuclease activity meaning that they can excise nucleotides in this

direction. This is done when a noncomplementary nucleotide is erroneously

added to the growing chain, thus improving the fidelity with which replication

occurs. In E. coli, the specific polymerase responsible for substituting the RNA

primers with DNA is called polymerase I. This enzyme also contains 50 ! 30

exonuclease activity enabling it to extend from a nick by excising nucleotides on

the 50 side as it moves along. It can thereby remove the RNA primers, and

subsequently, ligase can covalently link the fragments.

Polymerase Chain Reaction

A synthetic form of DNA replication that has been of paramount importance is the

polymerase chain reaction (PCR) technique. This method reliably amplifies minis-

cule amounts of DNA in a test tube within hours. This is accomplished by using

a heat-stable polymerase and adding a large excess of both forward and reverse

primers to a DNA target. Cycling between melting the duplex DNA, annealing

primers, and letting the polymerase extend results in an exponential replication of

the sequence bordered by the primers (see Fig. 34.7). (1) In the first step, a mixture

of target DNA, nucleotides, primers, and heat-stable polymerase is heated to 94 �C in

order to melt the target duplex. Heat-stable polymerase – such as Taq polymerase

from the bacterium Thermus aquaticus – prevents the enzyme from denaturing during

this step. (2) In the second step, the temperature is lowered to allow the primers to

anneal to the target single-stranded DNA. The forward primer anneals to the 30 end ofthe template strand and the reverse primer anneals to the 30 end of the complementary

strand. The temperature chosen may depend on the melting temperature of the

primers but will typically be around 55 �C. The high concentration of primers

prevents the target DNA from reforming a duplex. (3) In the third step, the temper-

ature is raised to the optimumworking temperature of the polymerase. For Taq, this is72 �C. The polymerase then extends the primers in the 50 to 30 direction.

The first round of replication results in the new strands that extend beyond

the region bordered by the primers in the 50 direction. Subsequent rounds of

replication from newly synthesized strands generate a sequence defined by primer

location.


Transcription and Translation

Transcription is the process of synthesizing messenger RNA (mRNA) molecules

based on the sequence of genes on a DNA molecule. Analogous to DNA replica-

tion, this is accomplished by an RNA polymerase that reads the template sequence

in the 30 to 50 direction and synthesizes an mRNA molecule in the 50 to 30 direction.One of the sharp differences between prokaryotes and eukaryotes is the fact that

there is only one known RNA polymerase in prokaryotes but three distinct RNA

polymerases are known in eukaryotes. This is mirrored in the fact that eukaryotes

have more complex RNA-mediated control of expression.

In E. coli, RNA polymerase associates with transcription factor proteins to

initiate transcription of a gene. For this to occur, the transcription complex

recognizes a specific sequence by binding non-covalently to the individual

nucleobases. Both strands in a genome contain genetic information. In the context

of a specific gene, the strand that is being ‘read’ by the polymerase – the com-

plementary strand – is called the noncoding strand or the antisense strand. The

strand that is identical in sequence to the resulting mRNA is called the coding

strand or the sense strand.

Transcription is terminated when reaching a specific sequence, sometimes

through the association of additional protein factors. The mRNA is processed

by ribosomes where amino acids linked to transfer RNAs (tRNA) complementary

to specific codons (nucleotide triplets) are transferred to a growing polypeptide

chain. The final product is a protein specified by the original gene sequence in the

DNA molecule.

Fig. 34.7 Polymerase chain reaction consists of several repeating cycles, all producing a set of

copies of a desired sequence. All cycles consist of 3 steps: denaturing, annealing, and extension.

The figure illustrates the 3 steps in the first cycles. 1 The sample is denatured at a high temperature.

2 Lowering of the temperature allows for the primers to anneal. 3 In the final step, the temperature

is raised enough to allow the heat-stable polymerase to function but not so much that the primers

denature. 4 The newly formed duplexes then function as substrates for subsequent cycles resulting

in an exponential increase of sequence copies


Genetic Recombination

As the information carrier of life, DNA need not only facilitate accurate

transmission of information but also allow for genetic diversity, the substrate

of evolution. One process attributing to this is homologous recombination which

is the recombination of two different DNA duplexes with a stretch of identical

sequence. In other words, two different DNA duplexes exchanged their

binding partners and then they are cleaved and the pieces recombined. Different

organisms have different enzymatic pathways facilitating this process but one

common feature is the formation of a Holliday junction (see section ‘DNA as

a Self-Assembling Construction Material’ and Fig. 34.9 below). This four-arm

junction illustrates the topological diversity of DNA that makes it

suitable not only as a carrier of information but also as a nanometer-scale building

material.

Optical Properties of DNA

DNA molecules typically interact with electromagnetic radiation typically through

the absorption of photons causing excitation of electrons from one state to another.

This absorbed energy is then lost either by emission of photons, dissipation as heat,

or alteration of chemical bonding.

Simple Absorbance

Different atomic structures preferentially absorb different wavelengths of radia-

tion. To understand the optical properties of DNA, first, one must be familiar with

the chemical structure (discussed in section ‘Chemical Properties of DNA’,

above). Different wavelengths are preferentially absorbed by the backbone phos-

phates (near IR), the conjugated electron systems that make up the

bases (260 nm), and other groups in other biological polymer systems such as

aromatic side chains in proteins (254–280 nm) and peptide bonds in polypeptide

backbones (190–230 nm). Investigators routinely use optical properties to gain

useful insight into the structure and concentration of the organic molecules being

observed.

The most widely exploited optical property of DNA is its characteristic absorp-

tion at 260 nm. Molecules change absorption based on sequence composition and

number of bases present. Any particular sequence will show a characteristic absor-

bance spectrum, making the assignment of an extinction coefficient simple. This

provides a means of determining DNA concentration via an absorption measure-

ment. Protein, one of the most common contaminants of DNA solutions, typically

displays significant absorption at 280 nm; therefore, the ratio of absorbance


at 260–280 nm is a crude measure of the purity of a DNA sample. Note that RNA

shows a similar absorbance profile to DNA, so RNA contamination is more

difficult to detect.

Hyper-/Hypochromicity

Interestingly, ssDNA and dsDNA have different characteristic absorptions. A dsDNA

helix shows less absorbance at 260 nm than does a sample containing the same

concentration of the two-component ssDNA strands. This property of increasing

absorbance during the transition from double-stranded to single-stranded forms is

called hyperchromicity. The inverse property of going from high to low absorbance

when reannealing ssDNA into dsDNA is termed hypochromicity. Because DNA can

be denatured via heat or solute conditions, this change in absorbance with respect to

base pairing has proved experimentally useful. The most common application of this

principle is seen in DNA melting studies, where a solution of DNA is heated steadily

while the absorbance is monitored. Asmolecules in duplex form begin to separate, the

absorbance starts to rise. When all the molecules have denatured, a maximum absor-

bance is reached. Further addition of heat or denaturant at this pointwill not change the

absorbance. Similarly, as a solution is cooled, the DNA molecules will rehybridize,

and generally, the population will retrace the absorbance versus temperature curve

created during heating, although some hysteresis may be observed.

Light-Induced Chemical Modification

When DNA is exposed to high-energy light, absorbed photons can cause chemical

modifications. The most well-documented and characterized chemical modifications

due to light are the formation of pyrimidine dimers, 6–4 photoproducts, and abasic

sites under UV exposure (UVB [315–280 nm], UVC [280–100 nm]). This is highly

relevant for biological processes, since UVB radiation from the sun penetrates the

ozone and causes 50–100 of these modifications per second per exposed skin cell

[60]. Thesemodifications must then be corrected by the cell’s DNA repair machinery,

or else modifications may be propagated and lead to disease including melanoma.

Interestingly, these modifications disrupt base pairing, leading to a localized decrease

in DNA backbone rigidity. It is this increased backbone flexibility that DNA repair

enzymes use to identify modified bases rather than directly assessing the condition of

the base [61]. These modifications are rarely intentionally exploited in the laboratory

setting; however, many laboratory techniques, including assessing concentration via

260 nm measurement and visualizing DNA bands in a gel on a UV box, will cause

these types of modifications. This is one of the many reasons that laboratory workers

are encouraged to routinely sequence their DNA stocks if they are performing

experiments in which specific sequence is critical.


Electrical Properties of DNA

In this section, we will discuss the electrical properties of DNA, including early

debate of whether DNA is an insulator, semiconductor, conductor, or even

a superconductor; the conclusion of this debate; and a discussion of possible

conduction mechanisms, as well as underlying measurement difficulties.

Investigation of the electrical properties of DNA began in the late 1950s, in

which experimenters tested the conductivity or lack thereof in DNA. While some

researchers incorrectly found DNA to be insulating, in 1962, Eley et al. reported

that bulk DNA samples act as a semiconductor when compressed between plat-

inum electrodes and resistance is measured under vacuum [62]. They theorized

that the base-pair units of DNA are ‘arranged like a pile of coins along the helix

axis’, supposing that ‘a DNA molecule might behave as a one-dimensional

aromatic crystal and show a n-electron conductivity down the axis’, and, further-

more, that the orbital overlap of the bases along the axis of the helix could be

sufficient to promote conductivity. More sophisticated experiments were

performed to further investigate the conductivity of DNA, in one case, even

claiming superconductivity [63].

More precise measurements of DNA conductivity have been made by producing

a small voltage across two well-separated parts of a DNA molecule and measuring

the electric current while under vacuum so as to avoid contact with anything other than

the sample. Although different experiments report different band gap values, these

variations in results are likely due to the quality of DNA and the types of electrodes

used. To circumvent these issues, chemists have focused on the photochemical aspects

of well-defined oligonucleotide assemblies in solution rather than physically measur-

ing resistance in dry samples [64].

In 1999, Fink noted that, while experiments conclusively show that DNA

molecules are molecular conductors, the mechanism that allows the transport of

charge is not well defined [65]. Eley presented a model for electron transfer through

DNA, which is based on overlap between the p-orbitals in adjacent base pairs

[61]. Because of imperfections or irregularities in base-pair sequences, localization

of charge carriers and the reduction of the transfer rate of electrons make measure-

ments of conductivity difficult [66].

A few models exist to describe the possible mechanisms for electron transport

through DNA. One model suggests electronic interactions between the bases in

the DNA molecule, leading to a molecular band where the electronic states are

delocalized over the entire length of the molecule. In this model, electrons can

tunnel between the bound donor and acceptor. This tunneling from electrode to

electrode is often ruled out, as separating the electrodes a distance larger than what

is probable for tunneling still yields large conductivity [64, 66–68]. Another model

suggests sequential hopping between localized states. This could involve hopping

between discrete base orbitals or hopping from domain to domain, skipping several

bases at a time (Fig. 34.8) [64, 66–68]. One example would be incoherent hopping

through low-potential regions of the stacked base pairs, for example, over guanines,

which are the most easily oxidized bases [69].


With these ideas as to how charge is transported, many underlying measurement

difficulties arise. For example, in the case where there are stacks of high-barrier,

flexible base pairs, such as long A-T tracts, the stacking dynamics become less

than ideal, as does the charge transport. Such a situation would require a buildup

of charge before the barrier can be surpassed. To avoid these situations,

one can replace these high-potential sites with lower-potential sites, like guanine,

as mentioned above [69, 70].

Furthermore, there are fundamental difficulties that must be considered, includ-

ing DNA as a biological sample – only conducting well in biologically relevant

conditions such as the presence of water and ions. As such, nanostructured devices

constructed with DNA will be fragile and easily perturbed [69, 70]. Also, Genereux

points out that charge transport across many DNA sequences occurs too fast for

measurement. In such situations, rapid charge recombination will occur. To prevent

this, an extended adenine tract can be added at the site of injection [69].

While it has become clear that DNA has electrical conduction properties ranging

between a semiconductor and conductor, it is apparent that the magnitude of the

Fig. 34.8 Representations

of three current opinions in

structural biology of the

mechanisms by which charge

transports through DNA.

(a) Charge tunnels all theway from the donor to the

acceptor. (b) Charge hoppingbetween discrete molecular

orbitals from the donor to the

acceptor. (c) Charge hoppingfrom domain to domain on its

way from the donor to the

acceptor. Broken lines

represent domains at which

electrons tend to collect

(Adapted from [64])


DNA conduction is dependent on many factors, including the quality of DNA,

environment in which conductance is measured, the length of DNA across which

voltage is applied, as well as the individual bases in a particular DNA strand. The

realization that DNA holds conductive electrical properties allows the possibility

of incorporating DNA into nanoelectronic devices.

Information Encoding in DNA

Since DNA is the genetic material of all known free-living organisms, and since

it is through this material that hereditary information is passed from generation

to generation, it is obvious that DNA is an excellent medium for the storage

and propagation of information. Separation of the complementary strands of the

double helix, followed by template-directed polymerization (as described in section

‘Biological Properties of DNA’, above), provides an elegant mechanism for making

copies of genetic information for passing to successive generations of cells and

organisms. In addition, it has been noted that nonbiological information can be

recorded in DNA, starting with the first demonstration of a DNA-based computer

[71] and leading to recent results showing efficient information propagation using

self-assembling DNA nanostructures as seed crystals [72].

This may lead one to inquire what amount of information can be encoded

within the nucleobase sequence of a DNA molecule. Information theory and

coding theory have established that the maximum amount of information (the

maximum number of bits per symbol) that a series of letters or symbols can

encode is equivalent to the base-two logarithm of the size of the alphabet

[73]. Therefore for DNA, with an alphabet of four bases, the maximum informa-

tion density is log2 4 ¼ 2 bits of information per nucleotide base in the sequence.

This is a maximum because less than two bits of information will be encoded if

the exact identity of all the bases is not required in order to specify the function of

the sequence, as, for example, the third position in the codons of protein-coding

genes since GGN codes for a glycine residue and CCN codes for proline (where

N can be any of the four bases), regardless of the identity of the base in the third

position. No information is recorded in this so-called wobble position of these

codons. Likewise, highly variable sites in protein or regulatory genes, as well

as sites that allow compensatory mutations in base-paired stem regions of self-

folding RNA structures, will each contain less than the maximum possible

information density.

A variety of methods have been used to estimate the information density within

different classes of biological sequences including protein-protein and protein-

DNA interaction motifs and secondary structure elements in RNA and protein

molecules [74, 75]. For example, a six-base recognition site for a restriction enzyme

that requires an exact match would be said to hold twelve bits of information.

Human-designed DNA sequence libraries for use in DNA-based computing have

been created with information densities on the order of 1 bit per 10 or 20 bases in

order to maximize the difference (Hamming distance) between neighboring


sequence words [76]. This provides sets of distinct words that can be reliably

annealed with their complements to form hydrogen-bonded, base-pairing couples

without significant probability of mispairing with incorrect words. We have there-

fore observed natural and artificial DNA sequences with information contents

between 2.0 and 0.05 bits per nucleotide residue.

DNA as a Self-Assembling Construction Material

The chemical, mechanical, and biological properties of dsDNA, as discussed in the

previous sections of this chapter, describe a stable and relatively stiff biopolymer

that is perfect for the self-assembly of functional architectures for bottom-up

fabrication at the nanometer scale. Nucleic acid molecules are readily programma-

ble and have predictable intermolecular interactions. Their extensive biological

study has led to marked advances in synthesis and modification methods. The

sequence of a DNA molecule can be read by other nucleic acids and proteins,

which leads to specific manipulation and modification by a large number of

enzymes. The objective of structural DNA nanotechnology is to take the unique

properties of DNA, which make it such a great molecule for genetic material, and

exploit them for the precise positioning of functional materials.

Holliday Junctions

In its natural, biological state, DNA is a double-helical, topologically linear

molecule that does not have the structural integrity for a basic unit of

a construction material. Instead, nanoscale materials and devices must be built

from a rigid unit capable of branching off into multiple directions. The most

biologically famous branched unit of DNA is the Holliday junction: an interme-

diate structure during genetic recombination where four strands of DNA associate

to form four double-helical arms [77]. The naturally occurring Holliday junction

is unstable. The homologous symmetry of the sequences involved in the arms

of the junction allows for branch migration, junction elimination, and formation

of two separate linear double-stranded complexes. In 1982, Nadrian Seeman

generated oligonucleotide sequences to form immobile junctions, incapable of

branch migration [78]. This development spurred the use of DNA as a structural

material for nanotechnology.

The structure of the four-armed Holliday junction in solution was a subject of

debate since its first discovery. Of the many isomeric conformations that the four arms

could adopt in solution, the junction strongly prefers a particular crossover configu-

ration where each pair of arms base-stacks to form two helical domains with a bias

towards these helices running antiparallel to each other [79]. Multi-arm junctions,

with 3–8 double-helical arms, form single tiles [78]. Tile arms may terminate with

several single-stranded residues (sticky ends) that can linkwith a neighboring tile with

complementary sticky ends viaWatson-Crick base pairing. High-fidelity sticky-ended


association and the long persistence length of double-stranded nucleic acids allow for

the prediction of intermolecular interactions between each tile component and of the

local structure of the hybridized product. These Holliday junction-based tiles

were expected to be a key unit for forming 2-dimensional periodic DNA lattices.

However, Holliday and multi-arm junctions are too flexible to produce large-scale

DNA networks [80]. Instead, the conformational flexibility of multi-arm tile

architectures has been exploited to produce three-dimensional objects including

cubes, truncated octahedra, octahedra, tetrahedra, dodecahedra, buckyballs, and

icosahedra [81–84].

Double-Crossover Constructs

Forming long-range DNA networks requires structurally rigid basic building

blocks. To generate stiffer DNA tiles, the so-called double-crossover motif was

fashioned. In this structure, two neighboring DNA helices are joined at two junction

sites where two strands are exchanged between the neighboring duplexes [85].

There are five strand-routing isomers of DNA double-crossover supramolecular

complexes that vary in their neighboring helix orientation (parallel or antiparallel),

the number of helical half-turns between each crossover point (odd or even), and,

for those with an odd number of half-turns, the excess groove between each

crossover point (major groove or minor groove) [86]. Of these five isomers,

DAE, the double-crossover complex with an antiparallel orientation and an even

number of helical half-turns between each crossover point, proved to be the most

stable and, thus, suitable for nanoconstruction [86]. Figure 34.9 illustrates the

differences between a single DNA helix, a Holliday junction between two helices,

and two helices connected by double crossovers.

Tile and Lattice Assemblies

The rigid antiparallel double-crossover motif (DAE) was a breakthrough achieve-

ment for the production of large lattices. DAE tiles self-assemble into periodic

networks via sticky-end sequence recognition. The rigid DAE motif is used exten-

sively to form 2-dimensional lattices and 3-dimensional objects [87–89]. Different

variations of DAE tiles form distinct semi-infinite two-dimensional periodic lat-

tices. Rigid tile assemblies including triple-crossover complexes, paranemic cross-

over molecules, bulged 3-arm triangular constructions, tensegrity designs, and

other geometries have been employed for structure formation [27, 90–96]. Addi-

tionally, surface-assisted anneals have proven to stabilize more flexible systems

that would otherwise be incapable of forming larger lattices in solution [97, 98].

Two tile systems are shown in Fig. 34.10 with their corresponding lattices.

The tile systems described above are engineered to propagate endlessly with

dimensions predicated on the thermodynamics of the annealed system. To this end,

DNA tile systems that self-assemble into finite-sized arrays have been established.


Some of these finite-sized nanoarrays use hierarchical assembly methods (anneals

set up to progressively build upon single tiles to the desired size) to promote

cost-efficiency and full addressability [99–102]. The ultimate network topology of

the nanoarrays is predictable based on sequence specificity, local geometry, and

flexibility. Thus, flexible tiles can create lattices with inherent curvature that can

roll up into DNA nanotubes [103–108]. DNA-based networks are useful for the site-

specific organization of inorganic nanomaterials (such as nanoparticles, nanorods, and

nanowires) and organic molecules (such as proteins, dyes, aptamers, and antibodies)

for electronic, photonic, chemical, and biomedical applications [109–112].

DNA Scaffolded Origami

The previously reviewed tile-based method was made possible by the rigidity of the

antiparallel double-crossover motif. This construct places two helices almost par-

allel to each other. In 2006, Paul Rothemund expanded this motif by specifically

positioning many helices with multiple double crossovers [5]. Each helix is com-

prised of a long, biologically derived single strand of DNA, termed the ‘scaffold’

strand (e.g., M13mp18 ssDNA), bound to numerous, short, chemically synthesized

ssDNA molecules, termed ‘staple’ strands, designed to tether neighboring helices

(Fig. 34.11a, b). Rothemund named this method DNA scaffolded origami since the

staple strands fold the scaffold strand into a desired shape.

Fig. 34.9 Basic DNA constructions used for DNA structural nanotechnology. The double-helical

structure is displayed terminating with and without unpaired regions (sticky ends). A DNA

Holliday junction structure is shown in two and three dimensions to exhibit the flexibility about

the crossover section. The double-crossover construction fixes the position of the two helices to be

parallel, as shown in the three-dimensional representation


The DNA origami scheme allows for the realization of more sophisticated

constructions while simplifying structure formation. Origami is formed by mixing

a scaffold strand with a molar excess (over 5 times more than scaffold) of staple

strands. Since its basis is a long scaffold strand, origami eliminates the necessity for

exact stoichiometry between all staple strands involved in the structure. In

Rothemund’s original origami designs, staple strands form crossovers every

32 bases along each helix. This spacing enforces three helical turns between

crossovers, which corresponds to an average twist density of 10.67 base pairs per

turn. Varying the number of bases between crossovers imposes torque along the

helix-parallel axis. Placing crossovers in precise positions between helices controls

the global curvature of two- and three-dimensional objects (Fig. 34.11f) [113–115].

With the goals of increasing the dimensions of objects and expanding the surface

area available for functionalization, methods to increase the size of origami

structures have been explored. Since the length of the scaffold strand ultimately

determines the total size of DNA origami structures, the first inclination is to

increase the size of the scaffold. However, it is difficult to develop long, single

strands of DNA biologically. Synthetic PCR techniques have been attempted, but

Fig. 34.10 Example of tile constructions and AFM of corresponding lattice formation.

(a) The triple-crossover tile further illustrates the use of double crossovers to impose rigidity.

(b) The six-arm, or six-point-star, tile forms 2D arrays with sticky-ended motifs


structure formation yield is low [116]. Methods to fold origami from double-

stranded scaffold sources have been achieved by the addition of chemical denatur-

ing agents (Fig. 34.11g) [32, 43, 117, 118]. Other approaches for scaling-up

origami include linking origami structures together via staple sequence recognition

along edges, shape complementarity, base stacking interactions at helix ends,

and predefined lattices with sticky-end interactions (Fig. 34.11c) [6, 119–125].

Analogous two-dimensional molecular canvases have also been created from

single-stranded tiles (Fig. 34.11h). Structures are built from 3 � 7 nm tiles made

from 42-base synthetic DNA strands that organize to form repetitive half-crossovers.

Fig. 34.11 DNA scaffolded origami. (a) Schematic shows how DNA origami is constructed by

folding a long scaffold strand with hundreds of short oligonucleotides, whose sequences determine

the final structure. (b) The same scaffold strand can be folded into many different two-dimensional

shapes. (c) DNA origami tiles are patterned into crystalline two-dimensional arrays. (d, e) Three-dimensional constructions are also possible. (f) By manipulating the DNA helical twist, structures

can exhibit global curvature and twisting. (g) In an effort to increase the functional surface area

of origami, double-stranded scaffolds can also be used to form (i) two separate shapes,

(ii) heterodimeric shapes, and (iii) one unified shape. (h) The complete elimination of

the scaffold strand is achieved by utilizing single strands of DNA forming tiles with half-

crossovers


This method eliminates the requirement for a single-stranded scaffold, while

maintaining the ability to construct nanostructures with complex two-dimensional

shapes.

The advent of origami has simplified structure formation based on DNA

[126, 127]. Even novices of the DNA nanotechnology field can easily design two-

or three-dimensional objects (Fig. 34.11d, e) [128]. The practicality of this method

makes DNA origami readily utilizable for applications in many fields including

supramolecular assembly, biomedical engineering, and nanofabrication [129–134].

Structure Design Programs

As structural DNA nanotechnology progresses, designs become larger and more

elaborate. The original tiled structures were designed by hand or with the assistance

of short, quick computational scripts that were not generalized for wider design

problems [78]. The drive towards greater complexity has compelled researchers to

develop simple programs where users may define shapes and sequences, and appro-

priate complementary sequences are produced [77–80]. With the rising interest in

DNA origami, software has been developed to simplify the design of two- and three-

dimensional structures following the origami architecture constraints [135–137].

Also, computational models (see section ‘Computational Methods to Describe

Nucleic Acids’ above) can predict origami shapes and their flexibilities in solution

based on the mechanical properties of DNA and crossover formation [56].

Conclusion

In this chapter, we have presented information on the chemical,

mechanical, biological, optical, electrical, informational, and structural properties

of DNA. We have also introduced the reader to the use of DNA as a nanoscale

construction material within the field of structural DNA nanotechnology. While these

topics are large and the space here is brief, we hope that this introduction will help

direct interested readers to further resources within the scientific literature.

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