The composition of hereditary material from deoxyribonucleic acid (DNA) was discovered in 1944 [17]. Its double-helical structure and complementary base-pairing was proposed in 1953, by James Watson and Francis Crick [18]. Today, the human genome has been almost completely mapped, with the DNA sequence identified for 99 % of the gene-containing part of human DNA [1]. The next step is the evaluation of how differences in the DNA sequences of individuals of the same organism affect size, appearance, ability and predisposition to disease.
2.2 The Principles of DNA Structure 10 Duplex DNA is a double-helical molecule consisting of two anti-parallel polydeoxynucleotide chains, held together by the hydrogen bonding of nucleic acid base pairs. The fundamental building blocks of DNA are the four nucleic acids adenine (A), thymine (T), guanine (G), and cytosine (C) (see Figure 2.1). These nucleic acids are covalently attached to the C10
carbon of the sugar in the sugar phosphate backbone (Figure 2.2(b)).
Figure 2.1: DNA bases and complementary base pairs; Cytosine-Guanine (top-row) and Thymine- Adenine (bottom-row) (Figure adapted from Wolfram Saenger,‘Principles of Nucleic Acid Struc- ture’, Springer-Verlag, 1984 [19])
The bonding of C ≡ G is stronger than between A = T, with the total binding energies between the base pairs being 70.3 kJ/mole and 29.3 kJ/mole respectively [19]. This is known as Watson-Crick complementary base pairing.
2.2.1
DNA Hybridisation and Melting
Hybridisation occurs when two different single stranded complementary DNA sequences self- assemble by hydrogen bonding interactions between the nucleic acid molecules by Watson- Crick base-pairing to yield the duplex DNA double helix (see Figure 2.2(a)). DNA melting is
(a) Duplex DNA helix (b) Base pairing of complementary DNA strands
Figure 2.2: The duplex DNA molecule. 2.2(a) The double-helix conformation of hybridised com- plementary DNA strands (Figure from [20]). 2.2(b) Schematic of nucleotide chain formation and hybridisation. Polynucleotide bonding forms the backbone of a DNA strand and hydrogen bonding between complementary base pairs in duplex DNA (Figure from [17]).
the thermal denaturation of the duplex DNA into two separate strands. The transformation from the double stranded DNA conformation to the single stranded DNA sequences can be detected by an increased UV light absorption at a wavelength of 260 nm, which is caused by reduced base stacking and π orbital overlap of the nucleic acid bases. The melting temperature is defined as the temperature (Tm) at which half of the DNA sample is single
stranded (see Figure 2.3). The process is reversible; when the DNA solution is cooled, annealing occurs and the sample returns to its double stranded form.
The melting temperature is dependent upon the G-C content, DNA strand length (number of bases,Nb) and salt concentration of the solution. The melting temperature can be estimated
and is dependent upon the DNA strand length of the hybrid region and sequence content [19]. For two oligonucleotide sequences shorter than 18 nucleotides (Nb):
2.2 The Principles of DNA Structure 12
Figure 2.3: Changes in UV absorption of duplex DNA with temperature. As the double helix uncoils and the double strand denatures (indicated schematically), absorption in the UV wavelengths increases (Figure from D L Hartl and E W Jones, Genetics: Principles and Analysis, Jones and Bartlett Publishers Inc, 1998 [21]).
For longer hybrid sequences, with chain length (Nb) up to around 70 nucleotides long:
Tm = 81.5 + 16.6 log10(Na +
Molar concentration) + 0.41(G+C fraction)−600/Nb
When large numbers of short oligonucleotide sequences that are complementary to a region in the DNA sequence of interest are added to a melting DNA sample, these will effectively compete for hybridisation because of both the kinetic advantage (smaller sequences will diffuse quicker) and the higher number density that can be added.
The length of a DNA sequence necessary to ensure it is a statistically unique sequence can be estimated as follows [22]; the probability of finding any given base (A, C, G, or T) in a DNA sequence is 1/4. The probability of matching two bases in a row is 1/4×1/4 = 1/16; the probability of matchingNb bases in a row is:
1 4Nb
Consequently, whenNb = 16, the probability of matching the sequence is approximately once
in every 4 billion bases, which is roughly the length of the human genome (3.08×109 base
it is a unique sequence is therefore chosen as at least 17 base pairs. This is simply an estimate of the minimum probe length. Certain sequences of DNA contain repetitive sequences of around 10− 100 base pairs, and so longer oligonucleotide probes would be required [24]. When DNA probes are required to decode the amino acid sequences of proteins, 18−20 base pairs is often quoted as a minimum length for a probe to ensure specificity, since amino acids correspond to codons of 3 nucleotides and so probes are prepared based on 6 amino acids or more to ensure specificity [25]. For certain sequences of DNA a complementary probe cannot be prepared as the probe sequence could form hairpins (5−10 bases apart) or stem-loops (more than 50 bases apart), hybridising with itself [25].
2.2.2
DNA-Templated Assembly
Figure 2.4: Schematic diagrams of DNA strands forming double crossover molecules with anti- parallel helices, with even (DAE) and odd (DAO) number of helical half-turns between crossover points (Figure from [26]).
DNA’s molecular recognition properties have been exploited to assemble materials in a con- trolled fashion [27, 28]. Initial development in this field by Seeman and co-workers, exploited DNA’s molecular recognition properties to develop structures created solely from DNA. They created branched DNA junctions featuring three to six arms, geometrical objects (e.g. cubes), DNA knots and catenanes [26, 29]. However, these structures are not rigid, so they rely more on topology than fixed geometry due to flexible junctions and linkers. They also created double-crossover (DX) molecules of DNA (Figure 2.4). These consist of two, four-arm branched DNA molecules that contain two crossover sites between each helical domain [30]. It was found that DX molecules with antiparallel helical domains have a rigidity comparable to linear duplex DNA, making them useful for construction of DNA-based materials.