8.1: Nucleic Acids - Structure and Function

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

DNA Structure: Primary, Secondary, and Alternative Helical Forms

• Describe the monomer building blocks of nucleic acids — nucleosides (sugar + nitrogenous base at N1 of pyrimidine or N9 of purine), nucleoside mono/di/triphosphates — and explain why DNA polymerization uses nucleoside triphosphates as substrates, how the 3'-OH of the growing chain attacks the α-phosphate of the incoming NTP in a nucleophilic substitution reaction releasing pyrophosphate, why the subsequent hydrolysis of pyrophosphate (ΔG = −7 kcal/mol) makes the overall reaction thermodynamically irreversible (preventing pyrophosphorolysis), and why synthesis is strictly 5'→3'.
• Compare the structural features of A-DNA (right-handed, 11 bp/turn, dehydrated conditions, found in RNA-DNA hybrids), B-DNA (right-handed, 10 bp/turn, the predominant in vivo form), and Z-DNA (left-handed, 12 bp/turn, zigzag phosphate backbone, found at alternating pyrimidine-purine sequences under high ionic strength) — explaining how the 2'-OH of RNA sterically prevents RNA-DNA hybrids from adopting the B-form, and describe the major and minor grooves of B-DNA in terms of the differential pattern of hydrogen bond donors and acceptors they present for sequence-specific protein recognition.
• Explain the primary stabilizing forces in double-stranded DNA — distinguishing the contributions of Watson-Crick hydrogen bonding (2 bonds for A·T, 3 for G·C) from base stacking (π-π hydrophobic interactions in the anhydrous core, ~−9.8 kcal/mol per stack for both AT and GC pairs), explaining why base stacking is the dominant contributor to thermodynamic stability, why GC-rich sequences have higher Tm despite equal stacking energies (due to one additional H-bond per GC pair), and connecting marginal stability to the biological necessity of local strand separation during replication, transcription, and repair.

Noncanonical Base Pairing and Higher-Order DNA Structures

• Distinguish Watson-Crick base pairing from four types of noncanonical base pairing — reverse Watson-Crick (pyrimidine rotated 180° giving antiparallel glycosidic bond arrangement), wobble base pairs (G·T/U from keto-enol tautomerism; A·C from amino-imino tautomerism; I·U, I·A, I·C from inosine in tRNA anticodons enabling third-codon-position degeneracy), Hoogsteen base pairing (purine rotated ~180° around the N-glycosidic bond, requiring protonation of cytosine N3 for G·C+ Hoogsteen pairs), and reverse Hoogsteen pairs — and explain how each influences DNA-protein recognition, translation fidelity, and DNA conformation.
• Describe the structural features and biological significance of three higher-order DNA structures — G-quadruplexes (four non-contiguous G residues per layer hydrogen-bonded in a tetrad stabilized by a central monovalent cation K⁺, formed from GmXnGmXoGmXpGm sequences as in telomeric TTAGGG repeats, serving as recognition sites for telomerase), triple helices (a third strand binding in the major groove through Hoogsteen/reverse Hoogsteen base pairing, requiring mirror-repeat homopurine-homopyrimidine sequences, stabilized by Mg²⁺ and polyamines, capable of inhibiting transcription), and four-way junctions (cloverleaf-like structures with coaxial stacking and G-quadruplex formation, exemplified by "lettuce" ssDNA that binds GFP-derived fluorophores through π-stacking, stabilized by K⁺ and Mg²⁺) — connecting each structure to its potential biological role in chromosome maintenance, gene regulation, or molecular recognition.

 

Introduction to Nucleic Acids

Alongside proteins, lipids, and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life. The nucleic acids consist of two major macromolecules, Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), that carry the genetic instructions for the development, functioning, growth, and reproduction of all known organisms and viruses. Both consist of polymers of a sugar-phosphate-sugar backbone with organic heterocyclic bases attached to the sugars. The sugar in DNA is deoxyribose, while in RNA it is ribose. DNA contains four bases, cytosine and thymine (pyrimidine bases) and guanine and adenine (purine bases). In vivo, DNA consists of two antiparallel strands intertwined to form the iconic double-stranded helix. RNA is single-stranded but may adopt many secondary and tertiary conformations not unlike those of a protein. Figure \(\PageIndex{1}\) shows a low-resolution comparison of the structure of DNA and RNA.

Figure \(\PageIndex{1}\): Low-resolution comparison of the structure of DNA and RNA. https://commons.wikimedia.org/wiki/F...DNA_RNA-EN.svg. Creative Commons Attribution-Share Alike 3.0 Unported license.

The biological function of DNA is quite simple: to carry and protect the genetic code. Its structure serves that purpose well. In the next section, we will study the functions of RNA, which are much more numerous and complicated. The structure of RNA has evolved to serve those added functions.

The core structure of a nucleic acid monomer is the nucleoside, which consists of a sugar residue + a nitrogenous base that is attached to the sugar residue at the 1′ position, as shown in Figure \(\PageIndex{2}\). The sugar utilized for RNA monomers is ribose, whereas DNA monomers utilize deoxyribose, which has lost the 2′-hydroxyl group of ribose. DNA contains four nitrogenous bases: the Purines Adenine (A) and Guanine (G), and the Pyrimidines Cytosine (C) and Thymine (T). RNA uses the same nitrogenous bases as DNA, except for Thymine. Thymine is replaced with Uracil (U) in the RNA structure.

When one or more phosphate groups are attached to a nucleoside at the 5′ position of the sugar residue, it is called a nucleotide. Nucleotides come in three flavors depending on how many phosphates are included: the incorporation of one phosphate forms a nucleoside monophosphate, the incorporation of two phosphates forms a nucleoside diphosphate, and the incorporation of three phosphates forms a nucleoside triphosphate, as shown in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): The Monomer Building Blocks of Nucleic Acids. The site of the nitrogenous base attached to the sugar residue (glycosidic bond) is shown in red.

DNA and RNA Hydrogen-bonded structures

Figure \(\PageIndex{3}\) below shows a "flattened" structure of double-stranded B-DNA that best shows the backbone and hydrogen-bonded base pairs between two antiparallel strands of the DNA. Unlike the protein α-helix, where the R-groups of the amino acids are positioned to the outside of the helix, in the DNA double-stranded helix, the nitrogenous bases are positioned inward and face each other. The backbone of the DNA is made up of repeating sugar-phosphate-sugar-phosphate residues. Bases fit the double-helical model if a pyrimidine on one strand is always paired with a purine on the other. From Chargaff’s rules, the two strands will pair A with T and G with C. This pairs a keto base with an amino base and a purine with a pyrimidine. Two H‑bonds can form between A and T, and three can form between G and C. This third H-bond in the G:C base pair is between the additional exocyclic amino group on G and the C2 keto group on C. The pyrimidine C2 keto group is not involved in hydrogen bonding in the A:T base pair.

Furthermore, the orientation of the sugar molecule within the strand determines the directionality of the strands. The phosphate group that makes up part of the nucleotide monomer is always attached to the 5′ position of the deoxyribose sugar residue. The free end that can accept a new incoming nucleotide is the 3′ hydroxyl position of the deoxyribose sugar. Thus, DNA is directional and is always synthesized in the 5′ to 3′ direction. Interestingly, the two strands of the DNA double helix lie in opposite directions or have a head-to-tail orientation.

Figure \(\PageIndex{3}\): "Flattened" Structure of DNA Madeleine Price Ball. https://en.Wikipedia.org/wiki/File:D..._structure.svg. Wikimedia Commons

By analogy with proteins, DNA and RNA can be loosely thought of as having primary and secondary structures. For a single strand, the primary sequence is just the base sequence read from the 5' to 3' end of the strand, with the bases thought of as "side chains" as illustrated in Figure \(\PageIndex{4}\) for an RNA strand which contains U instead of T.

Figure \(\PageIndex{4}\): "Primary" sequence of a single RNA strand. https://en.Wikipedia.org/wiki/Nucleic_acid_sequence. Creative Commons Attribution-ShareAlike License

Since it is found paired with another DNA molecule (strand), the double-stranded DNA, which consists of two strands held together by hydrogen bonds, might be considered to have secondary structure (analogous to alpha- and beta-structures in proteins). Of course, the hydrogen bonds are not between backbone atoms but between side chain bases in double-stranded DNA.

Figure \(\PageIndex{5}\) shows an interactive iCn3D model of the iconic structure of a short oligomer of double-stranded DNA (1BNA).

Figure \(\PageIndex{5}\): Iconic structure of a short oligomer of double-stranded DNA (1BNA). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...b5HUbmuQrCobg8

The backbones of the antiparallel strands are magenta (chain A) and cyan (chain B). Each chain's 5' sugar-phosphate end is shown in spacefill and colored magenta (chain A) and cyan (chain B). The hydrogen-bonded interstrand base pairs are shown in two ways: as spacefill and sticks to illustrate how the bases stack over each other.

Figure \(\PageIndex{6}\) shows types of "secondary (flat representations) and their 3D or tertiary representations found in nucleic acids.

Figure \(\PageIndex{6}\): https://en.Wikipedia.org/wiki/Nucleic_acid_sequence. Creative Commons Attribution-ShareAlike License

Figure \(\PageIndex{7}\) shows an interactive iCn3D model of the tertiary structure of the T4 hairpin loop on a Z-DNA stem (1D16).

Figure \(\PageIndex{7}\): T4 hairpin loop on a Z-DNA stem (1D16). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...8C7qBqgh8ZTJH9

The hairpin shown is from a synthetic DNA oligomer C-G-C-G-C-G-T-T-T-T-C-G-C-G-C-G, which adopts an alternative Z-DNA conformation (which we will explore below) with a loop at one end. The thymine bases 7, 8, and 9 are generally perpendicular to one another and stack together, along with the ribose of T7.

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of pseudoknot in RNA (437D).

Figure \(\PageIndex{8}\): Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot (437D). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ZtdeJqQXvjCKfA

The pseudoknot has two stems that form a "helix" and two loops. The knot consists of a hairpin in the nucleic acid structure, with the loop between the helices paired to another part of the nucleic acid. Pseudoknots can be found in mRNA and ribosomal RNA and affect translation (the decoding process that instructs the synthesis of a protein sequence from RNA). RNA viruses have pseudoknots, which likewise affect protein synthesis and RNA replication. Pseudoknots also occur in DNA.

Synthesis and structure of DNA

The nucleotide required as the monomer for the synthesis of both DNA and RNA is a nucleoside triphosphate. During the incorporation of the nucleotide into the polymeric structure, two phosphate groups (PP_i, called pyrophosphate) from each triphosphate are cleaved from the incoming nucleotide and further hydrolyzed during the reaction, leaving a nucleoside monophosphate that is incorporated into the growing RNA or DNA chain, as shown in Figure \(\PageIndex{9}\) below. The nucleophilic attack of the 3′-OH of the growing DNA polymer mediates the attack on the incoming nucleoside triphosphate. Thus, DNA synthesis is directional, only occurring at the 3′-end of the molecule.

The further hydrolysis of the pyrophosphate (P_i-P_i) releases a large amount of energy, ensuring that the overall reaction has a negative ΔG. Hydrolysis of P_i-P_i ↔ 2P_i has a ΔG = -7 kcal/mol (-29 kJ/mol) and is essential to provide the overall negative ΔG  (-6.5 kcal/mol, -27 kJ/mol) of the DNA synthesis reaction. Hydrolysis of the pyrophosphate also prevents the reverse reaction, pyrophosphorolysis, which would remove the newly incorporated nucleotide from the growing DNA chain.

This reaction is mediated in DNA by a family of enzymes known as DNA polymerases. Similarly, RNA polymerases are required for RNA synthesis. A more detailed description of polymerase reaction mechanisms will be covered in Chapter 24, which deals with DNA replication and repair, and in Chapter 25, which covers DNA transcription.

Figure \(\PageIndex{9}\): Nucleic Acid Synthesis: In nucleic acid synthesis, the 3’ OH of a growing chain of nucleotides attacks the α-phosphate on the next NTP to be incorporated (blue), resulting in a phosphodiester linkage and the release of pyrophosphate (PPi). The DNA polymerase also catalyzes the hydrolysis of pyrophosphate, preventing the reverse reaction and releasing energy to drive the reaction forward. The synthesis of DNA is shown in this diagram. Image modified from Michal Sobkowski

DNA was first isolated by Friedrich Miescher in 1869. The double-helix model of DNA structure was first published in the journal Nature by James Watson and Francis Crick in 1953 based upon the crucial X-ray diffraction image of DNA from Rosalind Franklin in 1952, followed by her more clarified DNA image with Raymond Gosling, Maurice Wilkins, Alexander Stokes, and Herbert Wilson, and base-pairing chemical and biochemical information by Erwin Chargaff. The prior model was triple-stranded DNA.

The realization that DNA is a double helix elucidated the mechanism of base pairing by which genetic information is stored and copied in living organisms, and it is widely considered one of the most important scientific discoveries of the 20th century. Crick, Wilkins, and Watson each received one-third of the 1962 Nobel Prize in Physiology or Medicine for their contributions to the discovery. Rosalind Franklin, whose breakthrough X-ray diffraction data were used to determine the structure of DNA, died in 1958 and was unfortunately ineligible for a Nobel Prize.

Watson and Crick proposed that DNA consists of two strands, each in a right-handed helix, wound around the same axis. The two strands are held together by H-bonding between the complementary base pairs (A pairs with T and G pairs with C) as shown in Figure \(\PageIndex{10}\) below. Note that, in a top-down view, the attachment point of a DNA base pair to the DNA backbone is not equidistant. This creates unequal gaps or spaces in the DNA: the major groove for the larger gap and the minor groove for the smaller gap (Figure 4.5). Based on the DNA sequence within the region, the hydrogen-bond potential between the nitrogen and oxygen atoms in the nitrogenous base pairs creates unique recognition features in the major and minor grooves, allowing specific protein recognition sites to form.

Figure \(\PageIndex{10}\): The Major and Minor Grooves of DNA. Top view of an (A) A-T base pair and a (B) G-C base pair showing the formation of the major and minor groove sides of the DNA. (C) Side view of the DNA double helix with the major and minor grooves indicated. The DNA backbone is green, potential nitrogen hydrogen-bonding locations are indicated in blue, and oxygen hydrogen-bonding locations are red. Figure C modified from dullhunk

Figure \(\PageIndex{1}\) shows a schematic representation of available hydrogen bond donors and acceptors in the major and minor grooves for TA and CG base pairs.

Figure \(\PageIndex{11}\): Available hydrogen bond donors and acceptors in the major and minor groove for TA and CG base pairs

Figure \(\PageIndex{12}\) shows an interactive iCn3D model of DNA showing the major and minor grooves.

Figure \(\PageIndex{12}\): Major and minor grooves of ds-DNA (1D66). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?3xicqSv9ERCBPHvd6

The two sugar-phosphate backbones are shown in green and yellow. Some of the red (oxygen) and blue (nitrogen) atoms in the major groove (and to a much lesser extent in the minor groove) are not involved in inter-strand G-C and A-T base pairing and so would be available to hydrogen bond donors with specific binding proteins that would display complementary shape and hydrogen bond acceptors and donors. (The white spheres are Cd ions.)

Figure \(\PageIndex{13}\) shows an interactive iCn3D model of the N-terminal fragment of the yeast transcriptional activator GAL4 bound to DNA (1D66).

Figure \(\PageIndex{13}\): N-terminal fragment of the yeast transcriptional activator GAL4 bound to DNA (1D66). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...5kLYSSfG7rsmS9

The N-terminal fragment binds to conserved CCG triplets found at both ends of the DNA in the major groove. The protein shown is a dimer held together by a short coiled-coil interaction domain, so the site has 2-fold symmetry. A small Zn²⁺-containing secondary structure motif in each member of the dimer interacts with the major groove. An extended chain connects the DNA-binding and interaction domains of each protein.

In addition to the major and minor grooves providing variation within the double helix structure, the axis alignment of the helix, along with other influencing factors such as the degree of solvation, can give rise to three forms of the double helix, the A-form (A-DNA), the B-form (B-DNA), and the Z-form (Z-DNA), as shown in Figure \(\PageIndex{14}\).

Figure \(\PageIndex{14}\): Structures of A-DNA (left), B-DNA (middle), and Z-DNA (right). https://en.Wikipedia.org/wiki/File:A..._and_Z-DNA.png. Creative Commons Attribution-Share Alike 3.0 Unported

Both the A- and B-forms of the double helix are right-handed spirals, with the B-form being the predominant form found in vivo. The A-form helix arises when conditions of dehydration below 75% of normal occur and has mainly been observed in vitro during X-ray crystallography experiments when the DNA helix has become desiccated. However, the A-form of the double helix can occur in vivo when RNA adopts a double-stranded conformation or when RNA-DNA complexes form. The 2′-OH group of the ribose sugar backbone in the RNA molecule prevents the RNA-DNA hybrid from adopting the B-conformation due to steric hindrance.

The third double-helical structure formed is a left-handed helix known as Z-DNA, or Z-form. Within this structural motif, the backbone phosphates appear to zigzag, giving rise to the name Z-DNA. In vitro, the Z-form of DNA is adopted in short sequences that alternate pyrimidine and purines when high salinity is present. However, the Z-form has been identified in vivo in short regions of DNA, indicating that DNA is quite flexible and can adopt a variety of conformations. A comparison of features between A-, B-, and Z-form DNA is shown in Table 4.1.

Table 4.1 Comparisons of B-DNA, A-DNA, and Z‑DNA

	B-DNA	A-DNA	Z-DNA
helix sense	Right Handed	Right Handed	Left Handed
base pairs per turn	10	11	12
vertical rise per bp	3.4 Å	2.56 Å	19 Å
rotation per bp	+36°	+33°	-30°
helical diameter	19 Å	19 Å	19 Å

The double-stranded helix of DNA is not always stable. This is because the attractions between the strands are noncovalent, reversible interactions. Depending on the DNA sequence, denaturation (melting) can be local or widespread, enabling crucial cellular processes such as DNA replication, transcription, and repair.

Both sequence specificity and interaction (whether covalent or not) with a small compound or a protein can induce tilt, roll, and twist effects that rotate the base pairs in the x, y, or z axis, respectively, as seen in Figure \(\PageIndex{15}\), and can therefore change the helix’s overall organization. Furthermore, slide or flip effects can alter the helix's geometric orientation. Hence, the flip effects and (to a lesser extent) the other movements described above modulate the double-strand stability within the helix or at its ends. Indeed, under physiological conditions, local DNA ‘breathing’ has been observed at both ends of the DNA helix, and B-DNA-to-Z-DNA structural transitions have been observed in internal DNA regions. These locally open DNA structures are good substrates for specific proteins, which can also induce the opening of a ‘closed’ helix. The DNA replication and repair processes will be discussed in more detail in Chapter 24.

Figure \(\PageIndex{15}\): Localized Structural Modification of the DNA Double Helix. (a) Base pair orientations with the x, y, and z axes result in different kinds of rotation (tilt, roll, or twist) or slipping of the bases (slide, flip) regarding the central helix axis. (b) Mature B-DNA has nearly 11 base pairs within one helical turn. (c) Mono- or bis-intercalation of a small molecule (shown in blue) between adjacent base pairs, resulting in an unwinding of the DNA helix (orange arrow on the top) and a lengthening of the DNA helix (ΔLength) depending on the X and y Å values that are specific for a defined DNA intercalating compound. (d) Representation of the DNA bending, base flipping, or double-strand opening induced by some DNA destabilizing alkylating agents (adducts shown in blue). Adapted from Calladine and Drew’s schematic box representation. Lenglet and David-Cordonnier (2010) Journal of Nucleic Acids, http://dx.doi.org/10.4061/2010/290935. Creative Commons Attribution License,

Figure \(\PageIndex{16}\) shows interactive iCn3D models of A-DNA (top), B-DNA (center), and Z-DNA (bottom). (Copyright; author via source). Click the image for a popup or use the external links in column 1.

A-DNA (440D)
B-DNA (1BNA)
Z-DNA (4OCB)

Figure \(\PageIndex{16}\): A, B, and Z-DNA. Click the image for a popup or use the links in column 1

We studied the structure of proteins in depth, discussing resonance in the peptide backbone, allowed backbone angles φ, ψ, and ω, side-chain rotamers, Ramachandran plots, and various structural motifs. We explored them dynamically using molecular dynamics simulations. We also discussed the thermodynamics of protein stability and how stability can be altered by environmental factors, nucleic acid structure, and solution composition and temperature.

In contrast, our understanding of nucleic acid structure and dynamics is less advanced. This may seem paradoxical, especially given the apparent simplicity of the iconic DNA structure presented in textbooks. Yet, we should first look at the types of secondary structures of nucleic acids, then at the more complex tertiary and quaternary structures of RNA.

The nucleic acid backbone has a 5-membered sugar ring, which adds rigidity, linked to another sugar ring by CH₂O(PO₃)O- connectors, providing some additional conformational freedom. We'll explore the effects of the pentose ring geometry in RNA and DNA in Chapter 8.3. To illustrate a yet unexplored complexity of nucleic acid structure, consider just the orientation of rings in double-stranded DNA and in regions of RNA where double-stranded structures form. The variants in the orientation of the hydrogen-bonded base pairs and the corresponding parameters that define them are shown in Figure \(\PageIndex{17}\).

Figure \(\PageIndex{17}\): Base pair orientation and corresponding parameters in nucleic acids. http://x3dna.org/highlights/schemati...air-parameters (with permission). 2008 3DNA Nature Protocols paper (NP08), the initial 3DNA Nucleic Acids Research paper .

Consider just two of these: the propeller and twist angles. If you examine the iCn3D models of nucleic acids presented above, you will see that the base pairs are not perfectly flat but are twisted. Larger propeller angles are associated with increased rigidity. The propeller angles for A, B, and Z DNA are + 18 ^o, + 16 +/-7 ^o, and about 0 ^o, respectively. The twist angles of A, B, and Z DNA are +33 ^o, +36 ^o, and -30 ^o, respectively. The lower the twist angle, the higher the number of base pairs per turn. This, of course, affects the pitch of the helix (the length of one complete turn). These terms should be minimized to computationally determine the lowest-energy state of a given double-stranded nucleic acid.

Alternative Base Pairing in DNA and RNA

A first glance at a DNA or RNA structure reveals a myriad of possible hydrogen bond donors and acceptors in the nucleic acid bases. Hence, it should come as no surprise that a variety of alternative or noncanonical (not in the canon or dogma) intermolecular hydrogen bonds can form between and among bases, leading to alternatives to the classical Watson-Crick base pairing. There are 28 possible base pairs, each with two hydrogen bonds. As structure determines function and activity, these alternative structures influence DNA/RNA function. We will consider four types of noncanonical base pairing: reverse Watson-Crick, wobble, Hoogsteen, and reverse Hoogsteen base pairs.

These noncanonical base pairs can form in DNA when bases mismatch in double-stranded regions. In RNA, which we will explore more fully in Chapter 8.2, double-stranded molecules formed by separate RNA molecules aren't common. Instead, the molecule folds into a complex tertiary structure, with regions of helical secondary structure. RNAs also form quaternary structures when bound to other nucleic acids and proteins. Larger RNAs have loops with complex secondary and tertiary structures that often require noncanonical base pairing, thereby stabilizing alternative structures. Noncanonical structures are also important for RNA-protein interactions in the region of the RNA that binds proteins. As with protein-ligand interactions, protein binding to RNA might also induce conformational changes, specifically the formation of noncanonical base pairs. For example, the HIV Rev peptide binds to a target site in the envelope gene of HIV (which has an RNA genome), forming an RNA loop with hydrogen bonding between two purines.

Figure \(\PageIndex{18}\) shows an interactive iCn3D model of the REV Response element RNA complexed with REV peptide (1ETF).

Figure \(\PageIndex{18}\): REV Response element RNA complexed with REV peptide (1ETF). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...T8CJ3pCe986Vx9

The peptide is shown in cyan, and its arginine side chains are shown as cyan lines. An extraordinary number of arginines form ion-ion interactions with the negatively charged phosphates in the major groove of this double-stranded A-RNA. The noncanonical base pairs are shown in CPK-colored sticks. A wobble base, U43-G77 (see below), can be seen, as well as three homopurine base pairs, G47-A73, G55-A58, and G48-G71. The solitary A68 base is shown projecting away from the RNA.

Figure \(\PageIndex{19}\) shows the Watson-Crick model and the first set of alternative non-canonical base pairs.

Figure \(\PageIndex{19}\): Some noncanonical base nucleic acid base pairs

Let's look at them in more detail.

Reverse Watson Crick: The reverse Watson-Crick AT (AU) and GC pairs can sometimes be found at the end of DNA strands and RNA. In forming the reverse base pairs, the pyrimidine can rotate 180 ^o along the axis shown, then rotate in the plane to align the hydrogen bond donors and acceptors, as shown in the top part of the figure. The glycosidic bond between the N in the base and the sugar (the circled R group) is now in an "antiparallel" arrangement in the reverse base pair.

Wobble Base Pairs

The bases in nucleic acids can undergo tautomerization to produce forms that can base pair noncanonically. They are termed wobble base pairs and include G-T(U) base pairs from keto-enol tautomerism and A-C base pairs from amino–imino tautomerism, as illustrated in Figure 18 above.

Figure \(\PageIndex{20}\) shows an interactive iCn3D model of the GT Wobble Base-Pairing in Z-DNA form of d(CGCGTG) (1VTT). Two such GT pairs are found in the structure.

Figure \(\PageIndex{20}\): GT Wobble Base-Pairing in Z-DNA form of d(CGCGTG) (1VTT). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...LtwfzyeqDCaPEA

Water molecules around the wobble base pairs can form hydrogen bonds, stabilizing the pair if a hydrogen bond is missing.

Figure \(\PageIndex{21}\) shows an interactive iCn3D model of dsRNA with G-U wobble base pairs (6L0Y).

Figure \(\PageIndex{21}\): dsRNA with G-U wobble base pairs (6L0Y). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...reyeD6JQM1djq6

The structure contains many GU wobble base pairs and two CU base pairs between two pyrimidine bases.

Inosine, a variant of the base adenine, can be found in RNA. It is formed by the deamination of adenosine by the enzyme adenosine deaminase. A nucleotide having inosine is named hypoxanthine. Hypoxanthine can form the wobble base pairs I-U, I-A, and I-C when incorporated into RNA, as illustrated in Figure \(\PageIndex{22}\).

Figure \(\PageIndex{22}\): Wobble bases pairs using hypoxanthine with the base inosine

Wobble base pair interactions are especially important in translation when a protein sequence is made from a messenger RNA template (which will be discussed in Unit III). For that decoding process, two RNA molecules, messenger RNA (mRNA) and transfer RNA (tRNA), which is covalently attached to a specific amino acid, such as glutamic acid, must bind to each other through a 3-base-pair interaction. The three bases on the mRNA are called the codon, and the three complementary bases on the tRNA are called the anticodon. The triplet base pairs are antiparallel to each other. The interaction between mRNA and tRNA is illustrated in Figure \(\PageIndex{23}\).

Figure \(\PageIndex{23}\): The wobble uridine (U34) of tRNA molecules that recognize both AA and AG-ending codons for Lys, Gln, and Glu, is modified by the addition of both a thiol (s2) and a methoxy-carbonyl-methyl (mcm5). This double modification enhances the translational efficiency of AA-ending codons. Goffena, J et al. Nat Commun 9, 889 (2018). https://doi.org/10.1038/s41467-018-03221-z. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

The third 3' base in the mRNA is less restricted and can form noncanonical, specifically wobble, base pairs with the 5' base in the anticodon triplet of tRNA. The term wobble arises from subtle conformational changes used to optimize triplet pairing. Wobble bases occur much more in tRNA than in other nucleic acids.

Hoogsteen base pairing

Flexibility in DNA allows rotation around the C1'-N glycosidic bond connecting the deoxyribose and base, allowing different orientations of AT and GC base pairs. The normal "anti" orientation allows "Watson-Crick" (WC) base pairing between AT and GC base pairs, while the altered rotation allows "Hoogsteen" base pairs. Figure \(\PageIndex{24}\) shows the different orientations for an AT base pair.

Figure \(\PageIndex{24}\): Xu, Y., McSally, J., Andricioaei, I. et al. Modulation of Figure \(\PageIndex{xx}\)Hoogsteen dynamics on DNA recognition. Nat Commun 9, 1473 (2018). https://doi.org/10.1038/s41467-018-03516-1Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

Hoogsteen base pairing is usually observed when DNA is distorted by interactions with bound proteins or by intercalating drugs. Figure \(\PageIndex{25}\) shows an interactive iCn3D model of a Hoogsteen base pair embedded in undistorted B-DNA - MATAlpha2 homeodomain bound to DNA (1K61).

Figure \(\PageIndex{25}\): A Hoogsteen base pair embedded in undistorted B-DNA - MATAlpha2 Homeodomain bound to DNA (1K61). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...SLLRv1m8HQXKcA

The same DNA without bound protein has no Hoogsteen base pairs. To form Hoogsteen base pairs, a rotation around the glycosidic-base bond must occur. Hoogsteen base pairs between G and C can also occur on rotation, but in addition, the N3 of cytosine is protonated, as shown in Figure 14 above.

Evidence suggests that Hoogsteen base pairing may be important in DNA replication, binding, damage, or repair. They can induce kinking of the DNA near the major groove.

There are also reverse Hoogsteen base pairing examples, as shown in Figure \(\PageIndex{26}\).

Figure \(\PageIndex{26}\): The reverse Hoogsteen AT base pair

Additional Alternative Structures: Quadruplexes,  Triple Helices, and 4-Way Junctions

Quadruplexes

These can form in DNA and RNA from G-rich sequences, involving tetrads of hydrogen-bonded guanine bases. They are a bit hard to describe in words, so let's first examine one particular structure.

In human cells, telomeres (the ends of chromosomes) contain 300-8000 repeats of a simple TTAGGG sequence. The repetitive TTAGGG sequences in telomeric DNA can form quadruplexes. Figure \(\PageIndex{27}\) shows an interactive iCn3D model of parallel quadruplexes from human telomeric DNA (1KF1). The structure contains a single DNA strand (5'-AGGGTTAGGGTTAGGGTTAGGG-3') which contains four TTAGGG repeats.

Figure \(\PageIndex{27}\): parallel quadruplexes from human telomeric DNA (1KF1). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...y5joFHDgWJQsQ6

Rotate the model to see three parallel layers of quadruplexes. In each layer, 4 noncontiguous guanine bases interact with a K⁺ ion. Hover over the guanine bases in one layer, and you will find that one layer consists of guanines 4, 10, 16, and 22, which derive from the last G in each of the repeats in the sequence of the oligomer used (5'-AGGGTTAGGGTTAGGGTTAGGG-3'). These quadruplexes certainly serve in recognition and as binding sites for telomerase proteins. The guanine-rich telomere sequences, which can form quadruplexes, may also function to stabilize chromosome ends.

A Quadruplex can be formed in 1 strand of nucleic acid (as in the above model) or from 2 or 4 separate strands. They also must have at least two stacked triads. As in the example above, single-stranded sections can form an intramolecular G-quadruplex from a G_mX_nG_mX_oG_mX_pG_m sequence, where m is the number of Gs in each short segment (3 in the structure above). A G might be in a loop if a segment is longer than the others.

Triple Helices

These structures can occur in DNA (and RNA) and contain homopurine and homopyrimidine sequences with mirror-repeat symmetry. Hence, they can occur naturally. A mirror repeat contains a center of symmetry on a single strand. Here is an example: 5'-GCATGGTACG-3'.

They can also occur when a third single-stranded DNA (a triplex-forming oligonucleotide or TFO) binds to a double-stranded DNA. The TFOs bind through Hoogsteen base pairing in the major groove of the ds-DNA. They can bind tightly and specifically, in parallel or antiparallel orientations. Specific, locally higher concentrations of divalent cations or positively charged polyamines, such as spermine, stabilize the excess negative charge density induced by the binding of a third polyanionic DNA strand.

An example of a triple helix system that has been studied in vitro is shown in Figure \(\PageIndex{28}\).

Figure \(\PageIndex{28}\): Intermolecular triplex formation and their oligonucleotide sequences (where “•” and “-” indicate Hoogsteen and Watson–Crick base pairings, respectively). Inset: chemical structure of a parallel T•AT triplet. Guerrini, L. and Alvarez-Puebla, R.A. Nanomaterials 2021, 11, 326. https://doi.org/10.3390/nano11020326. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

The double-stranded canonical helix (D1D2) consists of 31 base pairs, in which strand D1 is pyrimidine-rich, and D2 is purine-rich (D2). A 22-nucleotide Triple helix-forming oligonucleotide (TFO) that is rich in pyrimidines binds the 19 AT and 2 C-GC base triplets. The TFO binds along the major groove of the D2 strand, which is purine-rich.

If the binding of the third strand in the major groove occurs at the site where RNA polymerase binds to a gene, then the third strand can inhibit gene transcription. Binding can also lead to a mutation or recombination at the site.

Figure \(\PageIndex{29}\) shows the base pairing of purine and pyrimidines of the third strand to the canonical AT dn GC base pairs of the original double-stranded DNA.

Figure \(\PageIndex{29}\): Base pairing in triple helix motifs. (after Jain et al. Biochimie. 2008. doi: 10.1016/j.biochi.2008.02.011

Figure \(\PageIndex{30}\) shows an interactive iCn3D model of a solution conformation of a parallel DNA triple helix (1BWG).

Figure \(\PageIndex{30}\): Solution conformation of a parallel DNA triple helix (1BWG). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...5JU813eNjND8E7

Triple helix formation can also occur within a single strand of DNA. The resulting structure is called H-DNA. An example is shown below. Note that the central blue, black, and red sequences are all mirror-image repeats (around a central nucleotide). During processes that unravel DNA (replication, transcription, repair), the self-association of individual mirror repeats can form a locally stable triple helix, as shown in Figure \(\PageIndex{31}\).

Figure \(\PageIndex{31}\): Schematic illustrations of (A) the H-DNA or intramolecular triplex structure used in this study; del Mundo et al. (2019) Nucleic acids research. 47. e73. 10.1093/nar/gkz237. Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/)

The * between in the G*G and A*A denote Hoogsteen hydrogen bonding (purine motifs) in this intramolecular triple helix. Reverse Hoogsteen hydrogen bonds can also occur.

Triple helices can form when single-stranded DNA, formed during replication, transcription, or DNA repair, with only half of the required mirror symmetry, folds back into the adjacent major groove and base pairs, using Hoogsteen/reverse Hoogsteen bonding, which Mg²⁺ can stabilize.

Recent Updates: Four-Way Junctions

As we will see in the next section on RNA, nonhelical sections of DNA can bind small target molecules through noncovalent interactions.  (RNA examples that we will see in the next chapter section include aptamers, ribozymes, and riboswitches.)  One example is "lettuce" single-stranded DNA that can bind small fluorophores modeled after the intrinsic fluorophore of the green fluorescent protein.  The fluorophore's fluorescence is dramatically enhanced upon binding to lettuce DNA.  Figure \(\PageIndex{32}\) below shows the structure of extrinsic DNA fluorophores based on GFP that bind to the single-stranded "lettuce" DNA.

Figure \(\PageIndex{32}\): Structure of extrinsic DNA fluorophores based on GFP that bind to DNA.  The font color of the names indicates the fluorescence color emitted.

Figure \(\PageIndex{33}\) shows an interactive iCn3D model of a solution conformation of a ssDNA:DFAME fluorophore complex (8FI0). The blue dotted lines show π-π stacking interactions, and the green dotted line a hydrogen bond.

Figure \(\PageIndex{33}\): Solution conformation of a ss-53 mer DNA:DFAME fluorophore complex (8FI0). (Copyright; author via source). Click the image for a popup or use this external link:  https://structure.ncbi.nlm.nih.gov/i...97eLqwNWmaTNC9

The DFAME ligand is shown as sticks.

Figure \(\PageIndex{34}\) shows a close-up of DFAME (colored spacefill) bound to the lettuce DNA.

Figure \(\PageIndex{34}\): Pi stacking interactions (blue dotted lines) between the extrinsic fluorophore DFAME (spacefill) and ssDNA.

The DNA fold is characterized by a four-way junction (also seen in RNA, but it is more L- or H-shaped).  On either end are B-DNA duplexes, and the ssDNA between them forms stem-loops with odd base pairings in the stems.  The overall structure is like a cloverleaf.  Two coaxial stacks of nucleotides form a G-quadruplex where the fluorophore binds.  Pi base stacking between diagonally packed bases and the binding of Mg²⁺ and K⁺ stabilize the structure. 

Stability of nucleic acids

After looking at the myriad of structures showing the nearly parallel hydrogen-bonded base pairs and from ideas from most textbooks and classes you have taken, you probably think that double-stranded DNA is held together and stabilized by hydrogen bonds between the bases. It is well known that the greater the GC content relative to AT, the greater the stability of dsDNA. This translates into a higher "melting temperature (T_M)," the temperature at which the dsDNA is converted to ssDNA. There is a linear relationship between GC content and T_M^. The figures above show that GC base pairs have three inter-base hydrogen bonds compared to 2 in AT base pairs. These observations support the notion that inter-base hydrogen bonds are the source of dsDNA stability.

You would be, in general, correct in this belief. Still, you'd be missing the more important contributor to ds-DNA stability, base (π) stacking, and the noncovalent interactions associated with the stacking. The main contributors to stability are hydrophobic interactions in the anhydrous hydrogen-bonded base pairs in the helix. Given that the hydrogen bond donors and acceptors that contribute to base pairing are present in the absence of competing water, they are free to fully engage in bonding. The hydrogen bond interaction energy is more favorable in the stack. The stacking energy is similar for an AT-AT stack and a GC-GC stack (about -9.8 kcal/mol, 41 kJ/mol). Hence, the AT and GC base pairs contribute equally to stability. The excess stability of dsDNA enriched in GC base pairs can still be explained by the extra stabilization for an additional hydrogen bond per GC base pair

A myriad of interactions stabilizes proteins, but the folded state is marginally more stable than the ensemble of the unfolded state. Marginal stability is important, as protein conformation is often perturbed by binding and subsequent functions. The same must be true of double-stranded DNA, which must "unfold' or separate on replication, transcription, and repair. It is well known that dsDNA structure is sensitive to hydration (see the section on A, B, and Z DNA). As we saw with proteins, small molecules like urea can also denature DNA into single strands.

DNA must be stable enough to carry genetic information but dynamic enough to allow events that require partial unfolding. Other water-soluble molecules like ethylene glycol ethers (polyethylene glycol-400) and diglyme (dimethyl ether of diethylene glycol), which are more hydrophobic than water, appear to reduce base stacking interactions while maintaining them and, at the same time, allow a longitudinal extension or breathing of the helix. This dynamic extension may be required for transitions of B-DNA to Z-DNA, for example. The extensions also allow transient "holes" to form between base pairs, which might assist in binding intercalating agents, such as some transition-metal complexes. The extension caused by these ethers and natural extensions would decrease base stacking but also strengthen the hydrogen bonding between bases.

Longitudinal helical extensions may be important during homologous gene recombination. In that process, the homologous DNA strand is exchanged with a paired homolog. This processing is associated with strand extension and disruption of base pairs at every third base. Recombination must also allow chain extension, as it maintains base-pairing fidelity.

DNA structures get more complicated as they pack into the nucleus of a cell and form chromosomes, as shown in Figure \(\PageIndex{35}\). We will study DNA packing in other sections.

Figure \(\PageIndex{35}\): Packing of DNA into the chromosome. https://commons.wikimedia.org/wiki/F...omosome_en.svg. Creative Commons Attribution 3.0 Unported

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter introduces the structure and stability of nucleic acids — DNA and RNA — from their monomer building blocks through the iconic B-DNA double helix, its alternative forms, noncanonical base pairing, and higher-order structures, establishing the structural foundation for understanding DNA replication, transcription, translation, and repair.

The monomer building blocks of both DNA and RNA are nucleotides: nucleosides (a five-membered deoxyribose or ribose sugar attached at the anomeric C1' position to N1 of a pyrimidine or N9 of a purine base via a glycosidic bond) bearing one to three phosphate groups at C5'. DNA contains the four bases adenine, guanine, cytosine, and thymine (a pyrimidine methylated at C5 relative to uracil); RNA uses uracil instead of thymine and ribose instead of deoxyribose. Nucleoside triphosphates are the substrates for polymerization: the 3'-OH of the growing chain attacks the α-phosphate of an incoming NTP in a nucleophilic substitution reaction, forming a new 3'→5' phosphodiester bond and releasing pyrophosphate (PPᵢ). The subsequent hydrolysis of PPᵢ to 2Pᵢ (ΔG = −7 kcal/mol) provides the thermodynamic driving force for the overall reaction (net ΔG ≈ −6.5 kcal/mol) and prevents the reverse reaction (pyrophosphorolysis). Chain elongation proceeds strictly 5'→3', and synthesis requires a template strand to specify the sequence of incoming nucleotides through Watson-Crick base pairing.

The B-DNA double helix, elucidated by Watson and Crick in 1953 using Franklin's X-ray diffraction data and Chargaff's base equivalence rules (A = T, G = C), consists of two antiparallel complementary strands wound in a right-handed helix with the bases stacked inward and the sugar-phosphate backbone on the outside. A pairs with T (2 hydrogen bonds) and G pairs with C (3 hydrogen bonds), with each base pair approximately planar and stacked ~3.4 Å apart, giving ~10 base pairs per helical turn and a helical pitch of 34 Å. The asymmetric attachment of the two bases to their respective sugars creates unequal grooves: the major groove (wide, ~12 Å) and minor groove (narrow, ~6 Å). The major groove presents a richer pattern of hydrogen bond donors (NH) and acceptors (C=O, ring N) in a sequence-specific arrangement that allows proteins (transcription factors, restriction enzymes) to read the base sequence without opening the helix. The minor groove presents fewer distinguishing features but is accessed by some proteins and small molecule drugs. B-DNA is the predominant in vivo form under physiological hydration. The A-form arises under dehydrating conditions (~75% relative humidity) and is also found in RNA-DNA hybrids and double-stranded RNA; the 2'-OH of ribose sterically excludes the B-conformation by clashing with adjacent phosphates, forcing the A-form's wider, shallower major groove and deeper, narrower minor groove. The Z-form is a left-handed helix with a characteristic zigzag backbone (giving the name "Z") that forms at alternating purine-pyrimidine sequences (especially CG repeats) under high ionic strength or cytosine methylation; it has been detected in vivo and may play roles in gene regulation and DNA-protein recognition. The helix can undergo localized structural fluctuations (tilt, roll, twist, slide, and base-pair flip relative to the helical axis) and "breathing" — transient local strand separation — that are essential for processes requiring DNA strand access. Long-range chromosome compaction proceeds through histone-mediated nucleosome formation and higher-order folding into chromatin.

DNA stability is dominated by two forces whose relative contributions are less appreciated than their textbook descriptions suggest. While Watson-Crick hydrogen bonds clearly contribute (GC base pairs with 3 H-bonds are more stable than AT pairs with 2, explaining the linear correlation between GC content and Tm), base stacking — the hydrophobic and van der Waals π-π interactions between parallel aromatic base planes in the anhydrous interior of the helix — is the larger stabilizing force, contributing approximately −9.8 kcal/mol per stacked pair for both AT·AT and GC·GC stacks. Because the bases are sequestered from water within the helix, H-bond donors and acceptors operate without competition from solvent, further stabilizing the structure. Like proteins, dsDNA is only marginally stable (the melting transition is relatively cooperative), which is biologically essential — the helix must be easily unwound during replication, transcription, and repair without being spontaneously unstable.

Noncanonical base pairing significantly expands the structural versatility of nucleic acids beyond Watson-Crick pairs. Reverse Watson-Crick pairs form when a pyrimidine rotates 180° around its long axis, placing the glycosidic bonds in antiparallel arrangement; these occur at strand ends and in RNA tertiary structure. Wobble base pairs arise from tautomeric shifts: keto-enol tautomerism produces G·T (DNA) and G·U (RNA) pairs with a shifted hydrogen bond geometry, while amino-imino tautomerism produces A·C pairs. Wobble is biologically critical in tRNA-mRNA codon-anticodon pairing, where the 5' anticodon base (position 34, the wobble position) can pair with multiple third-codon-position bases — inosine (from adenosine deamination), which forms I·U, I·A, and I·C wobble pairs, is particularly important in expanding the decoding capacity of tRNAs. Hoogsteen base pairs form when a purine rotates ~180° around its glycosidic bond (the "syn" conformation), reorienting the Hoogsteen face (N7 and C6 substituent) of the purine toward the pyrimidine; G·C⁺ Hoogsteen pairs additionally require protonation of cytosine N3. Hoogsteen pairs occur in protein-bound DNA, drug-intercalated DNA, and DNA damage responses, and can induce local kinking. Reverse Hoogsteen pairs use the same purine rotation but in an antiparallel arrangement.

Higher-order DNA structures beyond the double helix include G-quadruplexes, triple helices, and four-way junctions. G-quadruplexes form from G-rich sequences (motif GmXnGmXoGmXpGm) in which four non-contiguous guanine bases form a planar tetrad through reciprocal Hoogsteen hydrogen bonds, with a monovalent cation (K⁺ or Na⁺) coordinated at the center of each tetrad; multiple stacked tetrads produce a stable four-stranded structure. These are well-documented at human telomeres (TTAGGG repeats), where they may regulate telomerase access, and at gene promoters, where they may modulate transcription. Triple helices form when a third strand binds in the major groove of dsDNA through Hoogsteen or reverse Hoogsteen base pairing with the purine-rich strand of the duplex; they require homopurine-homopyrimidine sequences with mirror symmetry and are stabilized by Mg²⁺ and polyamines. Intramolecular triple helices (H-DNA) form within single strands during replication or transcription when the required symmetry is present; external triplex-forming oligonucleotides (TFOs) can inhibit transcription and are explored as gene-silencing tools. Four-way junctions are complex folds — exemplified by "lettuce" DNA — that combine B-DNA duplexes, stem-loops, and G-quadruplex elements stabilized by K⁺, Mg²⁺, and π-stacking interactions, and can selectively bind small fluorophores through stacking interactions, with applications in biosensing and molecular imaging.

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