Nucleic Acids Structure and Function

Introduction to Nucleic Acids

Nucleic acids are fundamental macromolecules essential for all known forms of life. They are biological polymers that store and transmit genetic information, and they play crucial roles in gene expression. There are two primary types of nucleic acids: Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA). These long molecules are formed by the repetition of building blocks called nucleotides. From a chemical perspective, nucleic acids are weak acids.

Key Locations and Functions

• DNA: Primarily located in the nucleus of cells, DNA serves as the main carrier of genetic information, which is passed down to offspring, thus acting as the principal vehicle of heredity. Mitochondrial DNA (mtDNA) is an exception, found in the cytoplasm and responsible for mitochondrial (maternal) inheritance.
• RNA: Essentially found in the cytoplasm, though also present in the nucleus, RNA plays diverse roles in expressing the genetic information encoded in DNA.

The Structure of DNA and RNA

Both DNA and RNA are polymers of nucleotides. Each nucleotide is composed of three main parts:

1. An nitrogenous base
2. A five-carbon sugar (pentose) — either ribose (in RNA) or deoxyribose (in DNA)
3. A phosphate group

The phosphate groups and sugar molecules provide the structural backbone of nucleic acids, while the nitrogenous bases carry the genetic information.

Nitrogenous Bases

Nitrogenous bases are essential components of nucleic acids. They are aromatic molecules derived from either a purine or a pyrimidine ring structure.

Purine Bases

Purine bases are derived from a nine-atom aromatic ring containing five carbon atoms and four nitrogen atoms. They are formed by substituting hydrogen atoms of the heterocyclic ring with hydroxyl, amine, or methyl radicals.

• Adenine (Ade): 6-amino-purine. Found in DNA, RNA, ATP, and various coenzymes.
• Guanine (Gua): 2-amino-dihydroxy-purine. Found in DNA, RNA, and GTP.

Pyrimidine Bases

Pyrimidine bases are derived from a six-atom aromatic ring containing four carbon atoms and two nitrogen atoms. They are formed by substituting hydrogen atoms of the heterocyclic ring with hydroxyl, amine, or methyl radicals.

• Cytosine (Cyt): 2-hydroxy-4-amino-pyrimidine. Found in DNA and RNA.
• Uracil (Ura): 2,4-dihydroxy-pyrimidine. Found exclusively in RNA.
• Thymine (Thy): 5-methyl-uracil. Found exclusively in DNA.

Physicochemical Properties of Nitrogenous Bases

The aromatic nature of purine and pyrimidine bases confers several important properties:

• Resistance to oxidation.
• Characteristic UV absorption, which is used for their identification and quantification. The maximum absorption for DNA is at $260\,\mathrm{nm}$.
• Tautomerism: The presence of hydroxyl and amino substituents allows purine and pyrimidine bases to exist in several tautomeric forms:
  • Keto-enol tautomerism: The keto (lactam) form predominates at physiological pH over the enol (lactim) form.
  • Amino-imino tautomerism: For amino groups.
  Tautomerism involves the simultaneous displacement of a hydrogen atom and a pair of electrons from an adjacent double bond, transforming one functional group into another.

Pentose Sugars (Oses)

The sugars found in nucleic acids are pentoses (five-carbon sugars) in their furanose (five-membered ring) form. These are β-D-(-)-ribose (in RNA) and β-D-(-)-2-deoxyribose (in DNA).
The carbon atoms of the pentose sugars are numbered with a prime symbol (e.g., C1', C2') to differentiate them from the atoms in the nitrogenous bases.

Phosphoric Acid

The acidic character of nucleic acids is primarily due to the presence of phosphoric acid residues, which bear negative charges at physiological pH.

Nucleosides

A nucleoside is formed by the covalent combination of a pentose sugar and a nitrogenous base through a β-N-glycosidic bond. This bond links the C1' atom of the sugar to the N9 atom of a purine base or the N1 atom of a pyrimidine base.

Nomenclature of Nucleosides

The names of nucleosides are formed by adding suffixes to the base names:

• For purine bases, "-osine" is added (e.g., Adenine → Adenosine, Guanine → Guanosine).
• For pyrimidine bases, "-idine" is added (e.g., Cytosine → Cytidine, Uracil → Uridine, Thymine → Thymidine).

Bases	Ribonucleosides	Deoxyribonucleosides
Adenine	Adenosine	Deoxyadenosine
Guanine	Guanosine	Deoxyguanosine
Cytosine	Cytidine	Deoxycytidine
Thymine	-	Deoxythymidine (Thymidine)
Uracil	Uridine	-

Nucleotides

Nucleotides are phosphoric esters of nucleosides, meaning a phosphate group is attached to a hydroxyl group of the sugar.

• Ribonucleosides (containing ribose) can be phosphorylated at the 2', 3', and 5' positions. Cyclic nucleotides, such as adenosine 3':5'-cyclic monophosphate (cAMP), can form when the phosphate group esterifies two hydroxyl functions of the sugar (e.g., at 2' and 3', or 3' and 5').
• Deoxyribonucleosides (containing deoxyribose) can only be phosphorylated at the 3' and 5' positions, as the 2' carbon atom lacks a hydroxyl group. Consequently, only one type of cyclic ester (at 3' and 5') can exist.

Polynucleotide Structure

A polynucleotide is a polymer formed by numerous nucleotides (or deoxyribonucleotides) linked by phosphodiester bonds. Specifically, the 3'-hydroxyl group of the sugar of one nucleotide is joined to the 5'-hydroxyl group of the sugar of an adjacent nucleotide via a phosphate group.

Convention for Reading Polynucleotides

Polynucleotides are always read in the 5'p to 3'OH free direction. This means starting from the phosphate group attached to the 5' carbon of the first sugar and proceeding towards the free hydroxyl group at the 3' carbon of the last sugar.
Example: pApGpCpApT (AGCAT) implies a sequence starting with Adenine at the 5'-end and ending with Thymine at the 3'-end.

DNA Structure

DNA is typically found as a double-stranded molecule.

Characteristics of the DNA Molecule

• Sugar: Deoxyribose
• Bases: Adenine (A), Guanine (G), Cytosine (C), and Thymine (T)
• Structure: Consists of two polynucleotide chains, or strands.

The two DNA strands are said to be antiparallel. This means that while both strands run parallel to each other, their orientations are opposite. If one strand runs in the 5'→3' direction from top to bottom, the complementary strand runs in the 5'→3' direction from bottom to top.

Secondary Structure: Watson and Crick Model

The two DNA chains form a helical configuration, coiling around a central imaginary axis to create a double helix.

• Diameter: Approximately 2 nm.
• Pitch (one complete turn): 3.4 nm, containing 10 base pairs.
• Distance between two adjacent nucleotides: 0.34 nm.

The two strands are connected by hydrogen bonds between complementary bases. This complementarity rule is crucial:

• Adenine (A) always pairs with Thymine (T) via two hydrogen bonds.
• Guanine (G) always pairs with Cytosine (C) via three hydrogen bonds.

This pairing is dictated by steric reasons (a purine pairs with a pyrimidine) and the optimal formation of hydrogen bonds.

Chargaff's Rules

The complementarity principle leads to Chargaff's rules: $$\mathrm{A} = \mathrm{T} \quad \text{and} \quad \mathrm{G} = \mathrm{C}$$ Therefore: $$\mathrm{A} + \mathrm{G} = \mathrm{C} + \mathrm{T}$$ However, the ratio of (A+T) to (G+C) is not necessarily equal: $$\mathrm{A} + \mathrm{T} \neq \mathrm{G} + \mathrm{C}$$

Conformations of the DNA Molecule

DNA is a flexible double helix, and its conformation can vary depending on environmental and physicochemical conditions. Three main forms are defined: A, B, and Z.

	DNA A-form (Dehydrated form)	DNA B-form (Hydrated form)	DNA Z-form (Zig-zag)
Helix handedness	Right-handed	Right-handed	Left-handed
Grooves	Major groove: large; Minor groove: compressed, inaccessible	Major groove: large; Minor groove: small	Both grooves are equivalent
Base pair inclination	19° tilt relative to the plane perpendicular to the helix axis	Perpendicular to the helix axis	Perpendicular to the helix axis
Glycosidic bond	Anti	Anti	Purines: syn; Pyrimidines: anti
Prevalence	Relatively frequent (in vitro)	Very frequent (in vivo)	Rare
Helix pitch	2.8 nm	3.4 nm	4.5 nm
Base pairs per turn	11 bp	10 bp	12 bp
Base pairs per nm	3.9 bp/nm	2.9 bp/nm	2.7 bp/nm
Sugar puckering	C3'-endo	C2'-endo	C2'-endo (pyrimidines), C3'-endo (purines)

Physicochemical Properties of DNA

• Molecular Weight: DNA has a very high molecular weight, which can reach up to $3 \times 10^{12}$ Daltons. For double-stranded DNA, a length of 1 micron corresponds to a molecular weight of $2 \times 10^{6}$ Daltons (average molecular weight of a base pair is 600).
• Fibrous Nature and Solubility: The double helix structure gives DNA a fibrous nature. It is insoluble in organic solvents but precipitates in ethanol as long fibers, a property used for purification.
• Density: DNA molecules have a density that allows for separation by ultracentrifugation in density gradients (e.g., cesium chloride). RNA is denser than DNA, and proteins are less dense than both.
• Electrical Charge: At physiological pH, nucleic acids carry a negative charge solely due to the phosphate groups. This property allows for separation by electrophoresis, where negatively charged DNA migrates towards the anode (+) in an electric field.
• Solubility and Viscosity: Nucleic acids are soluble in water, forming highly viscous solutions. Double-stranded DNA (dsDNA) exhibits higher viscosity than single-stranded DNA (ssDNA).
• UV Absorption: Nucleic acids absorb UV radiation due to the conjugated aromatic nature of their bases. The maximum absorption wavelength for DNA is $260\,\mathrm{nm}$. This property is used for:
  • Detection
  • Quantification
  • Assessment of purity
• Denaturation and Renaturation: The two strands of the DNA helix are held together by weak hydrogen bonds. These bonds can be broken by increasing the temperature (typically 90-95°C), causing the two strands to separate in a process called denaturation. This process does not destroy the DNA and is reversible: cooling the solution allows the complementary strands to re-anneal, a process called renaturation. DNA can also be denatured in mildly alkaline conditions.

RNA Structure

RNA primarily functions in the expression of genetic information.

Primary Structural Characteristics of RNA

• Sugar: Ribose
• Bases: Adenine (A), Uracil (U), Guanine (G), Cytosine (C)
• Chains: Typically a single polynucleotide chain.

The genetic code in DNA is deciphered through the formation of an RNA molecule via a process called transcription. RNA is a polymer of ribonucleotides (purine or pyrimidine) linked by 3',5'-phosphodiester bonds, similar to DNA. While native RNA exists as a single strand, this strand is capable of folding back on itself to form complex secondary structures, such as hairpins.

Types of RNA

RNA molecules are categorized into two main groups: coding RNA and non-coding RNA.

Coding RNA

Messenger RNA (mRNA):

• Its sequence is complementary to one of the two strands of the DNA molecule.
• It carries the genetic information for protein biosynthesis.
• Contains specific sequences for translation initiation (e.g., AUG or GUG) and termination (e.g., UAA, UAG, UGA).

Non-coding RNA

Non-coding RNAs do not directly code for proteins but play crucial regulatory and structural roles.

• Ribosomal RNA (rRNA):
  • Represent over 80% of total cellular RNA.
  • Associate with proteins to form ribosomes, the cellular machinery for protein synthesis.
  • Ribosomes consist of two subunits (Prokaryotes: 50S and 30S; Eukaryotes: 60S and 40S).
• Transfer RNA (tRNA):
  • Transports specific amino acids to the ribosome for protein biosynthesis.
  • Single chain of 73-93 ribonucleotides, typically folding into a cloverleaf structure.
  • Key structural elements of cloverleaf:
    • Acceptor arm: Formed by base-pairing of the 5' and 3' ends of the tRNA. A conserved CCA sequence at the 3' end serves as the amino acid attachment site.
    • D-arm (or DHU arm): A hairpin structure containing dihydrouracil, an unusual pyrimidine nucleotide.
    • Anticodon arm: Contains the anticodon loop with a three-base anticodon that recognizes and binds to a complementary codon on mRNA.
    • Variable arm: Present in some tRNAs, with variable size.
    • TΨC arm: Contains the sequence TΨC, where Ψ (pseudo-uridine) is a modified nucleotide.
  • Each tRNA is specific for a particular amino acid.

Other Non-coding RNA Types

These are classified by size: * Small Non-coding RNAs (less than 200 nucleotides):

• snRNA (small nuclear RNA): Involved in mRNA splicing and maturation as part of small nuclear ribonucleoprotein particles (snRNPs).
• snoRNA (small nucleolar RNA): Involved in rRNA maturation.
• microRNA (miRNA): Short RNAs (21-23 bp) that regulate gene expression by promoting mRNA degradation or repressing translation.
• Interfering RNA (RNAi): Double-stranded RNA that interferes with mRNA to cleave it or reduce its translation into protein.
• siRNA (small interfering RNA): Small interfering RNAs (20-25 nucleotides) that are 100% complementary to their target mRNAs.

* Long Non-coding RNAs (lncRNA) (greater than 200 nucleotides):

• Can facilitate protein interactions, guide protein complexes to target genes, or sequester proteins or miRNAs.
• Widely involved in post-transcriptional processes related to mRNA biogenesis, including splicing, transport, translation, and degradation of mRNA.

Comparison of RNA and DNA Structural Characteristics

| Feature | DNA | RNA | | :---------------- | :----------------------------------------- | :----------------------------------------------- | | **Sugar** | Deoxyribose | Ribose | | **Bases** | Adenine, Guanine, Cytosine, Thymine | Adenine, Guanine, Cytosine, Uracil | | **Strands** | Double-stranded | Single-stranded (can fold) | | **Primary Function** | Storage and transmission of genetic info | Gene expression (synthesis, regulation) | | **Stability** | More stable (deoxyribose, double helix) | Less stable (ribose 2'-OH, single strand) | | **Location** | Nucleus, mitochondria | Cytoplasm, nucleus, ribosomes | | **Helix Type** | B-form right-handed double helix (most common) | Generally no stable double helix, forms complex secondary structures | | **Length** | Very long | Shorter than DNA |