Nucleic Acids
Nucleic acids, originally discovered in 1868 by Friedrich Miescher from leukocyte nuclei and termed “nuclein,” are the fundamental informational macromolecules of all living systems. These polymers, primarily deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are composed of monomeric units called nucleotides.
A nucleotide is built from three distinct components: a nitrogenous base, a five-carbon pentose sugar, and a phosphate group. The nitrogenous bases are planar, heterocyclic aromatic molecules divided into two classes: purines and pyrimidines. Purines, which include adenine (A) and guanine (G), feature a double-ring structure and are found in both DNA and RNA. Pyrimidines feature a single ring; cytosine (C) is present in both nucleic acids, whereas thymine (T) is exclusive to DNA and uracil (U), which lacks a methyl group at the C5 position compared to thymine is exclusive to RNA.
The sugar component is a pentose: ribose in RNA, possessing hydroxyl groups on both the 2′ and 3′ carbons, and deoxyribose in DNA, which lacks the 2′-hydroxyl group. These furanose rings are non-planar, adopting a “puckered” conformation to minimise steric strain. The ring can exist in an envelope form (four atoms coplanar, one out of plane) or a twisted form (three atoms coplanar, two out of plane). The most structurally significant variations are C2′-endo and C3′-endo puckers, which profoundly dictate the overall nucleic acid conformation; B-DNA predominantly features C2′-endo puckering, while A-DNA and RNA generally adopt C3′-endo puckering.
When a base covalently bonds to the C1′ carbon of the sugar via a beta-N-glycosidic linkage, a nucleoside is formed. This bond allows for rotation, yielding two primary conformations: syn (where the base rests above the sugar) and anti (where the base points away). Pyrimidines almost exclusively adopt the anti conformation due to severe steric interference between their O2 atom and the sugar’s C5′, whereas purines can freely exist in both syn and anti states. The addition of one to three phosphate groups via esterification at the C5′ position converts a nucleoside into a nucleotide. Beyond synthesising nucleic acids, nucleotides act as critical energy currencies (like ATP, which links catabolism and anabolism through high-energy phosphoanhydride bonds), precursors to coenzymes (NAD+, FAD), and secondary messengers (cAMP, cGMP).
Polynucleotide chains are formed when nucleotides condense, establishing a phosphodiester bond between the 5′-phosphate of one unit and the 3′-hydroxyl of the next. This gives the polymer intrinsic directionality (5′ rightarrow 3′) and makes it a highly acidic polyanion at physiological pH. The foundational understanding of how these chains interact came from Erwin Chargaff, whose parity rules revealed that in double-stranded DNA (dsDNA), the molar ratio of adenine equals thymine, and guanine equals cytosine, meaning the total sum of purines equals the total sum of pyrimidines.
Building on Chargaff’s rules and Rosalind Franklin’s X-ray diffraction data, Watson and Crick deduced the structure of B-DNA, the most stable and predominant form under physiological conditions. B-DNA is a right-handed double helix consisting of two antiparallel strands intertwined around a central axis. The hydrophobic bases stack internally, while the hydrophilic sugar-phosphate backbones face outward, minimising charge repulsion. The strands are held together by complementary hydrogen bonding: A pairs with T (two H-bonds) and G pairs with C (three H-bonds). The geometry of the intertwined strands creates uneven spaces known as the major groove (wide and deep) and the minor groove (narrow and deep). A standard B-DNA helix has 10.4 base pairs per helical turn, a pitch of 33.2 Ã…, and a diameter of approximately 20 Ã….
DNA exhibits structural polymorphism depending on hydration and sequence. A-DNA is a wider, right-handed helix that forms under dehydrated conditions; it accommodates 11 base pairs per turn, features a C3′-endo sugar pucker, and has a very deep, narrow major groove. Z-DNA is a radically different left-handed helix characterized by a zig-zag sugar-phosphate backbone. It forms primarily in sequences with alternating purines and pyrimidines (like GCGC) and relies on an alternating sequence of glycosidic bond conformations (purines adopt syn, pyrimidines adopt anti). Z-DNA is favored by high salt concentrations and negative supercoiling.
Nucleic acids can also form higher-order secondary structures. Triplex DNA occurs when a homopyrimidine or homopurine third strand binds to the major groove of a standard dsDNA duplex. This requires non-standard Hoogsteen hydrogen bonding, where the purine ring adopts a syn conformation to form alternative hydrogen bonds, often requiring protonation of cytosine at lower pH levels. Furthermore, guanine-rich sequences can spontaneously fold into G-quadruplexes. These four-stranded structures are built on planar G-quartets, cyclic arrangements of four guanines held together by Hoogsteen bonding and deeply stabilized by the central coordination of monovalent cations, specifically potassium or sodium.
The structural stability of the DNA double helix is driven primarily by base stacking interactions, which rely on hydrophobic effects and van der Waals forces between adjacent planar bases, effectively shielding them from water. Hydrogen bonding between complementary bases is considered a minor contributor to overall thermodynamic stability, though it is essential for specificity. When exposed to extremes of temperature or pH, or chaotropic agents like urea, DNA undergoes denaturation (melting), separating into single strands. Because stacked aromatic bases share delocalized pi-electrons, their unstacking during denaturation leads to a measurable increase in UV light absorbance at 260 nm, a phenomenon known as the hyperchromic shift. The melting temperature (Tm) at which half the DNA is denatured is intrinsically dependent on its GC-content (since the G-C pair’s stacking and three H-bonds confer higher stability) and extrinsic factors like ionic strength; high salt neutralizes phosphate repulsion, increasing Tm, while extreme pH disrupts hydrogen-bonding potentials. This UV absorbance is standardised for quantification, where an A260 reading of 1.0 corresponds to 50 µg/mL of dsDNA.
In living organisms, particularly within confined cellular spaces, DNA is subjected to supercoiling. Topologically constrained DNA, such as covalently closed circular DNA (cccDNA) in bacteria or DNA anchored to proteins, can become overwound (positive supercoiling) or underwound (negative supercoiling). This is defined mathematically by the linking number (Lk), an integer representing the number of times one strand crosses the other, which is the sum of the twist (helical turns) and writhe (supercoils of the helix axis in space). Relaxed B-DNA has about 10.4 bp per turn; negatively supercoiled DNA (found in most organisms) has fewer turns, storing torsional energy that aids in unwinding the helix for replication and transcription. Intercalating agents like ethidium bromide force the stacked base pairs apart, decreasing the twist; to maintain a constant linking number in cccDNA, this forces a compensatory increase in writhe, relaxing negatively supercoiled DNA before eventually inducing positive supercoiling. Enzymes called topoisomerases tightly regulate this topology by transiently breaking and resealing the DNA backbone.
The identification of DNA as the genetic material was established through landmark historical experiments. In 1928, Frederick Griffith discovered the “transforming principle” when he observed that heat-killed virulent Streptococcus pneumoniae could physically transform live, avirulent strains into lethal pathogens. In 1944, Avery, MacLeod, and McCarty purified this principle and definitively proved it was DNA, as only treatment with DNase (and not RNase or proteases) abolished the transforming capability. Finally, in 1952, the Hershey-Chase experiment utilized T2 bacteriophages radiolabeled with either 35S (for proteins) or 32P (for DNA) to demonstrate that only the viral DNA enters E. coli cells to direct the production of new phages.
While DNA is the permanent storage medium, RNA acts as the crucial intermediate, transmitting genetic information (mRNA) and participating in protein synthesis (tRNA, rRNA). Some RNA molecules, termed ribozymes, also exhibit catalytic activity, a discovery by Cech and Altman that forms the foundation of the RNA World hypothesis. This hypothesis suggests RNA was the first life-form, uniquely capable of both encoding information and catalyzing metabolic reactions before the evolution of protein enzymes and DNA. RNA also serves as the primary genetic material for certain viruses; Fraenkel-Conrat and Singer demonstrated this via the reconstitution of the Tobacco Mosaic Virus (TMV), showing that hybrid viruses always produce progeny matching their RNA component, not their protein capsid.
Chemically, RNA is distinct from DNA due to its 2′-hydroxyl group, which makes it highly susceptible to alkaline hydrolysis. In basic conditions, this 2′-OH is deprotonated and nucleophilically attacks the adjacent phosphodiester bond, creating a 2′,3′-cyclic monophosphate intermediate and severing the RNA backbone. DNA, lacking this hydroxyl, remains stable in basic solutions. Despite this chemical fragility, the secondary structure of double-stranded helical RNA (which adopts the A-conformation) is thermodynamically more stable than dsDNA of a comparable sequence, requiring higher temperatures for denaturation.
