Watson–Crick Base Pairs and Nucleic Acids Stability

Abstract

Watson–Crick base pairs are crucial in the formation of double‐helical deoxyribonucleic acid (DNA) and in the storage of genetic information. They comprise two of the most common geometries involving the pairing of deoxyadenosine (dA) with deoxythymidine (dT) and deoxyguanosine (dG) with deoxycytidine (dC) through the formation of two and three hydrogen bonds, respectively. The complementarity of the antiparallel strands forming a double helix is crucial in DNA replication since the complementary strand is synthesised by adding the Watson–Crick complementary bases of the parent strand. The hybridisation of DNA or DNA–RNA (ribonucleic acid) strands is widely used in biotechnology and molecular biology for multiple purposes. The stability of a nucleic acid helix depends on its sequence, conformation, hydration and solution conditions, and it is measured by temperature‐dependent techniques, such as UV (ultraviolet) melting. Examples of oligonucleotide helices with chemical modifications are provided to indicate how these modifications affect the overall stability of a DNA molecule.

Key Concepts:

  • Base pairs are two nucleotide bases interacting via hydrogen bonding.

  • Bulge is a nonpaired nucleotide base in a double‐stranded nucleic acid.

  • Hybridisation is a process where two complementary strands interact via base pairing.

  • Nucleic acid conformation is the three‐dimensional structural arrangement of a double‐helical nucleic acid.

  • Nucleic acid modification is the incorporation, removal or substitution of a chemical group on the natural occurring nucleic acids.

  • Melting curves are the optical changes of a nucleic acid or protein‐like molecules as a function of temperature.

  • Stability of nucleic acids is the resistance of nucleic acid duplex to undergo unfolding, proportional to the magnitude of ΔG.

  • Thermal stability is the ability of a nucleic acid duplex to resist temperature denaturation.

  • Unfolding thermodynamic profile is the free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) parameters associated with the unfolding of a nucleic acid or protein molecule.

  • Cooperativity refers to the number of base pairs that are broken simultaneously in the unfolding of a nucleic acid or protein.

Keywords: Watson–Crick base pairs; hybridisation; DNA melting curves; UV spectroscopy; circular dichroism spectroscopy; nucleic acid conformation; unfolding thermodynamic profiles; transition temperature (Tm); free energy and stability

Figure 1.

Structure of Watson–Crick A•T and G•C base pairs.

Figure 2.

Structure of the superimposed A•T and G•C base pairs with overlapping sugar–phosphate backbone.

Figure 3.

(a) UV spectra of calf thymus DNA at low and high temperatures. (b) The corresponding UV melting curve at 260 nm. These experiments were performed in 10 mmol L−1sodium phosphate buffer at pH 7.

Figure 4.

(a) CD spectra of calf thymus DNA at low and high temperatures. (b) The corresponding CD melting curve at 245 nm. These experiments were performed in 10 mmol L−1 mM sodium phosphate buffer at pH 7.

Figure 5.

UV melting curves of undecamer duplexes at 260 nm as a function of conformation: 5′−d(CCATCGCTACC)/5′−d(GGTAGCGATGG) (DNA, black), 5′−r(CCAU‐CGCUACC)/5′−r(GGUAGCGAUGG) (RNA, red), 5′−d(CCATCGCTACC)/5′−r(GGUAGCGAUG‐G)(Hyb‐1, blue) and r(CCAUCGCUACC)/5′−d(GGTAGCGATGG) (Hyb‐2, green). All experiments performed in 10 mmol L−1Hepes buffer, 100 mM NaCl, at pH 7.5, using an oligonucleotide concentration of 4×10−6 mol L−1 in total strands. Adapted from Kankia and Marky . With permission from the American Chemical Society.

Figure 6.

UV melting curves of DNA duplexes at 260 nm with and without bulges: 5′−d(CGCCTAATCG)/5′−d(CGATTAGGCG) (decamer, black), 5′−d(CGCCTATATCG)/5′−d(CGATATAGGCG) (undecamer, red), 5′−d(CGCCTATATCG)/5′−d(CGATTAGGCG) (T‐bulge, blue) and 5′−d(CGCCTAATCG)/5′−d(CGCCTAATCG) (A‐bulge, green). All experiments performed in 10 mmol L−1sodium phosphate buffer, 0.1 mM Na2EDTA, and 100 mmol L−1NaCl, at pH 7, using an oligonucleotide concentration of 2.5×10−4 mol L−1 in total strands.

Figure 7.

UV melting curves of DNA oligonucleotide duplexes at 260 nm with chemical modifications: (a) Containing dT→dU substitutions: 5′−d(CAAAGATTCCTC)/5′−d(GAGG‐AATCTTTG) (black, no substitutions), 5′−d(CAAAGAUUCCUC)/5′−d(GAGGAATCTTTG) (red, 3 substitutions) and 5′−d(CAAAGAUUCCUC)/5′−d(GAGGAAUCUUUG) (green, 7 substitutions). All experiments performed in 10 mM sodium cacodylate buffer, 100 mM NaCl, at pH 7, using a total strand concentration of 5×10−6 mol L−1. (b) Incorporation of an amino ethyl group at the italic thymine base of the bottom strand: 5′−d(GCACGACTACG)/5′−d(CGTAGTCGTGC), unmodified duplex (black) and modified duplex (red). All experiments were done in 10 mmol L−1Hepes buffer, 100 mM NaCl, at pH 7.5 and using a total strand concentration of 25×10−6 mol L−1(adapted from Soto et al. with permission from Oxford University Press). (c) UV melting curves of 5′−d(CCATCGCTACC)/5′−d(GGTAGCGATGG) duplex (5.6×10−6 mol L−1 in total strands) using a 10 mM sodium phosphate buffer, 34 mM NaCl at pH 7 and at the following ethylene glycol concentrations: 0 (black), 2.4 M (red) and 4 M (green).

Figure 8.

UV melting curves of the DNA duplex, 5′−d(CTCTGGTCTC)/5′−d(GAGACCAGAG), at 260 nm with the incorporation of cisplatin at the italic guanines of the upper strand (red) and without cisplatin (black). All experiments performed in 10 mmol L−1Hepes buffer, 100 mM NaCl, at pH 7.5, using a total strand concentration of 1×10−5 mol L−1. Adapted from Kankia et al..

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References

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Further Reading

Hilario E and Mackay J (2007) Protocols for Nucleic Acid Analysis by Nonradioactive Probes, 2nd edn. Totowa: Humana Press.

Lewin B (2006) Genes IV. Sudbury: Jones and Bartlett.

Marmur J and Doty P (1962) Determination of the base composition of deoxyribose nucleic acid from its thermal denaturation temperature. Journal of Molecular Biology 5: 109–118.

Watson JD and Crick FHC (1953) A structure for deoxyribose nucleic acid. Nature 171: 737–738.

Watson JD and Crick FHC (1953) Genetic implications of the structure of deoxyribose nucleic acid. Nature 171: 964–967.

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Marky, Luis A, Lee, Hui‐Ting, and Garcia, Angel(May 2010) Watson–Crick Base Pairs and Nucleic Acids Stability. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003126.pub2]