# Nucleic acid structure and base pairing > How the molecular structure of DNA and RNA governs their chemical properties, stability, recognition and biological function, from canonical base pairing to modified nucleobases, sugars, backbones and higher-order structures. With the full slide deck. Canonical URL: Published: 2026-09-13 Last updated: 2026-09-22 Category: Articles Tags: Presentations ## Nucleic acid structure and base pairing How the molecular structure of DNA and RNA governs their chemical properties, stability, recognition and biological function, from canonical base pairing to modified nucleobases, sugars, backbones and higher-order structures. This article, drawn from the slide deck, shows how the molecular structure of DNA and RNA governs their chemical properties, stability, recognition and biological function. It begins with the fundamental building blocks of nucleic acids and canonical base pairing, before examining DNA/RNA duplex structures, mismatches, replication and the molecular basis of genetic information. It then moves beyond natural nucleic acids to show how non-canonical base pairs, modified nucleobases, sugar and backbone modifications, and higher-order structures such as triplexes and G-quadruplexes can be used to alter nucleic-acid behaviour. These structural concepts provide the foundation for understanding and designing oligonucleotides for modern diagnostic and therapeutic applications, where precise target recognition, affinity, stability and structural control are critical. Overall, it highlights a central principle: small changes at the molecular level can produce significant changes in nucleic-acid structure and function, making a strong understanding of nucleic-acid chemistry essential for the rational design of next-generation nucleic-acid technologies. ***Ayan Dasgupta and Tom Brown*** The deoxyribose sugar exhibits dynamic mutarotation as a free monosaccharide, interconverting among furanose, acyclic, and pyranose forms; however, incorporation into DNA locks it into the furanoside form, providing the structural rigidity essential for nucleic acid architecture. A nucleoside is a base joined to a sugar; a nucleotide is a nucleoside carrying a phosphate. The natural nucleosides are β-anomers, and the base can sit anti or syn to the sugar. DNA is a salt of a phosphoric acid ester (a phosphodiester). It typically exists as a sodium or potassium salt and is soluble in aqueous buffers. DNA sequences are written by convention in the 5′- to 3′-direction. B-DNA is right-handed. The base pairs stack on each other, about 10.5 base pairs per helical turn, and each base pair is separated by 0.34 nm. The hydrophobic base pairs are on the inside; the hydrophilic sugar phosphate backbone is on the outside, exposed to water. The helix has a major groove and a minor groove. RNA duplexes form a right-handed antiparallel double helix in the A conformation. The major groove is very deep, the minor groove is very shallow, and the base pairs are pushed towards the outside of the helix. In the B form, the base pairs are closer to the centre of the helix compared to the A form. Watson-Crick base pairs are pseudo-symmetric and produce a regular double helix. All other combinations of DNA bases have different shapes. This process is controlled by Watson-Crick base pairing and by DNA polymerases. It does not work without DNA polymerase. Transcription, in which DNA codes for RNA, is a similar process catalysed by RNA polymerase enzymes. Mis-paired bases can also be incorporated at low level during replication. G–T and G–A mismatches are fairly stable base pairs. Purine:purine mismatches (A–A, A–G, G–G) rarely occur during replication as they are misshaped and some are very unstable, so the integrity of the genome is not significantly compromised. Purine:pyrimidine mismatches (for example A–C or G–T) are recognised by DNA polymerases and repair enzymes. They do not fit into a regular smooth double helix. The base from the daughter (unmethylated) DNA strand is removed by DNA repair enzymes. The parent strand has methylation at some cytosine residues, allowing it to be distinguished from the daughter strand. Pyrimidine:pyrimidine base pairs (C–C, C–T, T–T) are smaller than the Watson-Crick base pairs. They have poor base stacking and are therefore very unstable. Top: A–C\* and G–T\* against A–T. Bottom: A\*–C and G\*–T against G–C. The asterisk marks the minor tautomer. All base pairs in this figure have the same shape, so the A–C and G–T mismatches are excellent mimics of Watson-Crick base pairs. Around 1 in 10⁴ molecules of the canonical bases exist as minor tautomers at any time. The probability of a nucleobase being a minor tautomer during replication and also during proofreading is 10⁻⁴ × 10⁻⁴ = 10⁻⁸. This is the error rate during copying of the human genome. Evolution has 'chosen' DNA bases that have one tautomer that predominates. Each three-letter codon, drawn from four letters (A, G, C, U), corresponds to a specific amino acid. For example CCC = proline, UUU = phenylalanine, AGC = serine, CAG = glutamine. The natural four-letter genetic alphabet, AT and GC, is conserved throughout all life. Unnatural base pairs have been developed. d5SICS–dNaM is equivalent to an additional base pair in DNA; when combined with AT and GC it provides a six-letter genetic alphabet. This modified DNA may be amplified by DNA polymerases with high efficiency and greater than 99.9% fidelity. The first organism that can grow and replicate with an unnatural base pair in its DNA, giving the cell six nucleotides instead of the usual four, has been created. Shape recognition of base pairs by DNA polymerases: hydrogen bonding is not necessary. Oligonucleotides are used extensively in diagnostic and therapeutic applications. They are required to recognise short complementary DNA or RNA sequences embedded in longer nucleic acid stretches. High selectivity for the correct target and tight binding are important, and this cannot always be achieved by unmodified oligonucleotides. Duplex stability can be increased by increasing the number of inter-base hydrogen bonds, by increasing base stacking, and by introducing positive charges into the nucleotides. Sometimes it is desirable to decrease duplex stability; this can be achieved by reducing the number of hydrogen bonds. G–C; the G-clamp base, with four hydrogen bonds and extra base stacking; G–5-propynyl-C, with three hydrogen bonds and extra base stacking; and deoxyinosine–C, with two hydrogen bonds, which is destabilising. Oligonucleotides containing 2-thio-T and 2-aminoadenine form stable duplexes with complementary unmodified DNA but do not form intramolecular structures that might adversely affect their performance. The 2-aminoadenine–2-thiothymine pair has a steric clash and only one hydrogen bond, so it is unstable; the adenine–2-thiothymine pair has two hydrogen bonds and is stable. These analogues are designed to bind to complementary DNA or RNA with high affinity. Examples are locked nucleic acid (LNA), which is conformationally locked in the A-form; hexitol nucleic acid (HNA); peptide nucleic acid (PNA), which is uncharged; and morpholino, which is uncharged or cationic. A–T, T–A, C–G and G–C are different in the major groove of DNA. The major groove provides an accessible surface for protein binding and recognition. This allows enzymes and other DNA-binding proteins to recognise specific DNA sequences through the major groove. A DNA duplex can bind a third strand of DNA. The third strand binds in the major groove of the DNA duplex, forming specific interactions with one strand of the duplex, giving T·A–T and C⁺·G–C base triads. In a parallel triplex the triplex-forming oligonucleotide (TFO) runs in the same direction as the purine strand of the duplex. Advantages: it recognises duplex sequence without denaturation, and is a means of recognising double-stranded genomic DNA. Disadvantages: pH dependence (the C⁺·G–C triad), low affinity and limited sequence recognition. A G-quartet, held by hydrogen bonding and base stacking, and a G-quadruplex folded from a single strand of DNA: ``` ...GGGTTAGGGTTAGGGTTA... ``` Single strands of DNA can fold into G-quadruplexes, which are stabilised by G-quartets. In the human genome these structures form and stabilise the ends of chromosomes. They also block gene expression in a reversible manner. The metal ion is essential: K⁺ is the best fit and the most stabilising, but Na⁺ is also possible. G-quadruplexes are very stable structures in the presence of K⁺, and can be very insoluble in aqueous media. Nucleic acids play a central role in modern diagnostics and therapeutics; therefore, understanding the basic structures of DNA and RNA is essential. The structure of a nucleic acid determines its chemical properties, stability and biological function. Understanding the basic components and structural features of DNA and RNA provides a foundation for studying more advanced nucleic-acid technologies. Small changes in nucleic-acid structure can have significant effects on their biological properties and applications. A strong understanding of nucleic-acid chemistry is therefore crucial for the rational design and development of nucleic-acid-based technologies. ## Works cited 1. Blackburn, G. M.; Gait, M. J.; Egli, M.; Watts, J. K. Nucleic Acids in Chemistry and Biology (The Royal Society of Chemistry, 2022). 2. Frank-Kamenetskii, M. D. Unraveling DNA (John Wiley & Sons, 1993). 3. Alberts, B. Molecular Biology of the Cell (W. W. Norton & Company, 2022). 4. Marky, L. A.; Lee, H.; Garcia, A. (2010). Watson–Crick Base Pairs and Nucleic Acids Stability. Encyclopedia of Life Sciences. [10.1002/9780470015902.a0003126.pub2](https://doi.org/10.1002%2F9780470015902.a0003126.pub2) 5. Topal, M. D.; Fresco, J. R. (1976). Base pairing and fidelity in codon–anticodon interaction. Nature. [10.1038/263289a0](https://doi.org/10.1038%2F263289a0) 6. Duca, M.; Vekhoff, P.; Oussedik, K.; Halby, L.; Arimondo, P. B. (2008). The triple helix: 50 years later, the outcome. Nucleic Acids Research. [10.1093/nar/gkn493](https://doi.org/10.1093%2Fnar%2Fgkn493) 7. Jonchhe, S.; Lahiri, S.; Rothenberg, E. (2025). DNA G-quadruplexes: Structural and functional insights. DNA Repair. [10.1016/j.dnarep.2025.103910](https://doi.org/10.1016%2Fj.dnarep.2025.103910) --- Website and LIMS by Alex Soldatkin alex.soldatkin@gmail.com