Chapter 8: Nucleosides, Nucleotides, Nucleobases

Functions of Nucleotides and Nucleic Acids

• Nucleotide Functions:

  • Source of energy for metabolism (e.g., ATP)

  • Serve as enzyme cofactors (e.g., NAD+)

  • Involved in signal transduction (e.g., cAMP)

• Nucleic Acid Functions:

  • Storage of genetic information (DNA)

  • Transmission of genetic information (mRNA)

  • Processing of genetic information (ribozymes)

  • Protein synthesis (tRNA and rRNA)

Nucleotides and Nucleosides

• Nucleotide:

  • Composed of:

  • Nitrogenous base

  • Pentose sugar

  • Phosphate group

• Nucleoside:

  • Composed of:

  • Nitrogenous base

  • Pentose sugar

• Nucleobase:

  • This is the nitrogen-containing molecule itself, such as adenine (A), guanine (G), cytosine (C), thymine (T), or uracil (U).

Phosphate Group

• Characteristic:

  • Contains a negative charge at neutral pH.

• Attachment:

  • Typically bonded at the 5’ position of the pentose ring.

  • Nucleic acids are polymerized from 5’-triphosphates such as ATP, GTP, TTP, CTP known as NTPs (nucleotide triphosphates).

• In nucleic acid polymers (DNA/RNA):

  • Each nucleotide has one phosphate moiety.

• Alternative attachment:

  • Phosphate may be attached at 2’ or 3’ position in specific cases.

Sugar Group

• Structure:

  • A 5-carbon sugar known as D-ribose is fundamental to RNA.

  • 2’-deoxyribose is found in DNA.

• Conformational variation:

  • Different puckered conformations of the sugar ring are possible, crucial for biological function.

Nucleobases

• Classification:

  • Divided into pyrimidines (1 ring) and purines (2 rings).

• Structure:

  • Comprises nitrogen-containing aromatic rings that exhibit planar or near-planar structures.

• Spectroscopy:

  • Capable of absorbing UV light around 250–270 nm, which is relevant for understanding their detection and quantification.

b-N-Glycosidic Bond

• Function:

  • Links the pentose ring to the nucleobase via the b-N-glycosidic bond.

• Formation specifics:

  • The bond forms at the C1’ (anomeric) carbon of the sugar in b configuration.

  • Attachment occurs to position N1 in pyrimidines and position N9 in purines.

• Stability:

  • This bond exhibits significant resistance to hydrolysis, especially in pyrimidines; bond cleavage necessitates acid catalysis.

Conformation around N-Glycosidic Bond

• Flexibility:

  • Free nucleotides exhibit relatively free rotation around the N-glycosidic bond, possessing dihedral angles near 0° (syn conformation) or 180° (anti conformation).

  • Conformational Types:

  • Syn: Angle near 0° (nucleobase over the sugar)

  • Anti: Angle near 180° (nucleobase away from sugar)

  • Anti conformation is typically found in B-DNA.

Tautomerism of Nucleobases

• Definition:

  • Prototropic tautomers are structural isomers differing by proton location.

• Key types:

  • Keto-enol tautomerism is prevalent in ketone compounds.

  • Lactam-lactim tautomerism occurs in select heterocycles.

• Stability:

  • Both tautomers are present in solution, but the lactam forms predominate at neutral pH.

• Historical Note:

  • Watson & Crick's model of DNA was initially impeded by their modeling of incorrect tautomers, which significantly influenced their understanding (e.g., keto forms are favored over enol forms).

UV Absorption of Nucleobases

Mechanism:

• UV light absorption in nucleobases, at wavelengths of $250\text{–}270$ nm, is caused by $p \to p^*$ electronic transitions, where electrons jump from a lower to a higher energy state.

• Photoprotection:

  • When nucleobases absorb UV light and become excited, they quickly release this energy as heat. This process protects the genetic material from damage, though thymine (TT) is an exception and can still be harmed.

• Fluorescence:

  • Nucleic acids do not exhibit fluorescence, which is significant for molecular biology applications.

Minor Nucleosides in DNA

• Post-Synthesis Modifications:

  • Modifications of nucleosides commonly occur after DNA synthesis.

• Common Modifications:

  • 5-Methylcytosine is prevalent in eukaryotes and some bacteria.

  • N6-Methyladenosine is noted in prokaryotes but not in eukaryotes; serves as an epigenetic marker.

  • Purpose of modifications:

  • Marks own DNA for degradation of foreign DNA in prokaryotes.

  • Indicative of gene activation statuses in eukaryotes.

  • Questions raised if environmental factors could toggle gene expression in an inheritable manner.

Minor Nucleosides in RNA

• Inosine:

  • Found in tRNA's wobble position.

  • Made by changing adenosine.

  • Helps expand how genes are read.

• Pseudouridine (Ψ):

  • Found in tRNA and rRNA.

  • Made by changing uridine after RNA is built.

  • Makes tRNA more stable and helps rRNA fold correctly.

Polynucleotides

• Structure:

  • Covalent bonds through phosphodiester linkages form a negatively charged backbone.

• Stability:

  • DNA backbone demonstrates stability, with notable finds like ancient mammoth DNA enduring for eons.

  • Hydrolysis accelerated by enzymes (DNAse).

  • RNA backbone is less stable; survives few years in water, while cellular mRNA degrades in hours.

• Characteristics:

  • Linear polymers: no branching or cross-links.

  • Directionality: distinctions between 5’ and 3’ ends; sequences read from 5’ to 3’.

  • Nomenclature includes different lengths: dinucleotide, trinucleotide, oligonucleotide, polynucleotide.

Hydrolysis of RNA

• Instability:

  • RNA is reasonably unstable, particularly under alkaline conditions.

• Hydrolysis:

  • Enzymes such as RNase catalyze hydrolysis.

• RNase Examples:

  • RNase A: A digestive enzyme produced by the pancreas.

  • RNase P: A ribozyme that processes tRNA precursors.

  • Dicer: Cleaves double-stranded RNA into oligonucleotides for protection against viral genomes and facilitates RNA interference technology (RNAi).

Hydrogen-Bonding Interactions

• Base Pairing:

  • Hydrogen bonds between nucleobases result in specific base pairing.

  • In monomers, there are numerous possible base pairs, but the polynucleotide structure limits actual pairing possibilities.

• Watson-Crick Base Pairs:

  • A pairs with T (or U in RNA)

  • C pairs with G

  • Each purine pairs with a pyrimidine in nucleic acid structures.

Discovery of DNA Structure

• The discovery of DNA's structure was a major biological breakthrough.

• Watson and Crick (1953) highlighted its "novel features" and biological significance.

• This work emphasized the role of collaboration and addressing challenges in research.

Early Discoveries of DNA's Covalent Structure (1868–1935)

• Friedrich Miescher first isolated "nuclein" from cell nuclei.

• Further analysis showed it contained phosphate, a pentose sugar, and nucleobases.

• Levene & London (1929) and Levene & Tipson (1935) advanced understanding of DNA's structure, including phosphodiester linkages.

The Path to the Double Helix

• Rosalind Franklin and Maurice Wilkins provided crucial calculations and insights suggesting a helical structure and the phosphate-sugar backbone.

• Watson and Crick explained the double helix's alternating major/minor grooves and specified the A-T and G-C hydrogen bonding patterns.

• Watson, Crick, and Wilkins received the Nobel Prize in 1962, though Franklin passed away before her contributions could be formally acknowledged.

Complementarity of DNA Strands

• Strand Differences:

  • Two chains differ in nucleotide sequence (sequence read from 5’ to 3’ direction).

  • Complementary nature allows pairing between the chains.

• Orientation:

  • Each of the two strands runs in an antiparallel configuration.

Replication of Genetic Code

• Process Outline:

  • Initial separation of strands is required.

  • Each strand serves as a template for synthesizing a new complementary strand.

  • Enzyme catalysis occurs through DNA polymerases during synthesis.

  • Results in one newly synthesized strand (daughter) and one original strand (parent).

Messenger RNA: Code Carrier for the Sequence of Proteins

• Synthesis:

  • mRNA is transcribed from a DNA template.

• Composition:

  • Contains ribose as opposed to deoxyribose and uracil instead of thymine.

• Multiplicity:

  • Individual mRNA molecules can code for multiple proteins, linking genetic information to protein synthesis.

Palindromic Sequences and Structural Forms

• Palindromes:

  • Defined as sequences that read the same forwards and backwards, both in language and nucleic acids.

• Structural formations:

  • Palindromic sequences may lead to hairpin and cruciform structures in nucleic acids.

RNA Molecules and Their Complex Structures

• Secondary Structure:

  • RNA exhibits complex tertiary and secondary structures given its potential for non-canonical base pairs.

• Example:

  • G-U pairs and various other interactions contribute to the overall stability and functionality of RNA.

DNA Denaturation

• Characteristics:

  • Retains covalent bonds while hydrogen bonds and base stacking are disrupted, causing strand separation.

• Induction Factors:

  • Denaturation can be induced by exposure to elevated temperatures or pH changes, potentially reversible through annealing.

• Monitoring:

  • UV absorbance increases can measure denaturation, typically monitored at 260 nm.

Thermal DNA Denaturation (Melting)

• Heating:

  • At normal temperatures, DNA exists as a double helix; heating causes strand dissociation.

  • Re-annealing occurs upon reduced temperatures.

• Relevance:

  • This thermal property underpins polymerase chain reaction (PCR) methodologies.

Denaturation of Large DNA Molecules

• Variability:

  • Denaturation is non-uniform; AT-rich regions have lower melting temperatures compared to GC-rich regions.

Hybridization of Complementary DNA Strands

• Applications:

  • Detection of specific DNA molecules can be done through hybridization techniques (radioactive detection, fluorescent chips).

  • Amplification techniques (e.g., PCR) allow for targeted amplification of genes.

• Insights:

  • Provides perspectives on evolutionary relationships and therapies such as antisense techniques.

Molecular Mechanisms of Spontaneous Mutagenesis

• Deamination:

  • Defined as the slow conversion and significant daily occurrence of C to U mutations (100 events/cell/day).

• Depurination:

  • Hydrolysis of the N-glycosidic bond primarily affects purines (daily loss of 10,000 purines per cell).

• Cellular Responses:

  • Mechanisms exist to correct most spontaneous modifications, maintaining genomic stability.

Molecular Mechanisms of Oxidative and Chemical Mutagenesis

• Oxidative Damage:

  • Hydroxylation of guanine is notable, particularly in mitochondrial DNA.

• Chemical Alkylation:

  • Methylation of guanine can lead to mutagenesis with corrective cellular systems in place.

Molecular Mechanisms of Radiation-Induced Mutagenesis

• UV Light:

  • Causes pyrimidine dimerization, a mechanism strongly linked to skin cancers.

• Ionizing Radiation:

  • X-rays and gamma rays can induce ring opening and strand breaks, often challenging to repair.

• Mutation Links:

  • Accumulation of mutations correlates to aging and increased cancer risk.

Mechanism of DNA Polymerases

• DNA Synthesis Overview:

  • DNA polymerases play a crucial role in elongating the DNA strand based on the template.

• Key requirements:

  • Presence of magnesium ions (Mg2+) and dNTPs fueling the synthesis process.

Importance of a Primer in DNA Synthesis

• Role of Primers:

  • Serves as necessary initiators for DNA polymerases.

  • A diagram illustrates primer-bound and template-bound base pairing during synthesis.

Base-Pair Geometry in DNA

• Importance of Correct Pairing:

  • The geometric fit of base pairs in the active site is vital for accurate DNA synthesis.

  • Mispairing leads to structural instability, impacting replication fidelity.

Polymerase Chain Reaction (PCR)

• Overview:

  • Technique designed to amplify specific DNA fragments, useful in numerous applications such as gene cloning and diagnostic testing.

• Protocol:

  • Comprises cycles of heating, cooling, and extension phases using a thermocycler to achieve target amplification.

General Steps of PCR

• Detailed Sequence:

  • Step 1: Heat to separate DNA strands.

  • Step 2: Add synthetic primers and cool for annealing.

  • Step 3: Addition of Taq polymerase to synthesize new strands in 5’ to 3’ direction.

  • Repeat cycles significantly enhance target sequence amplification (up to 106-fold after 20 cycles).

Expression of Cloned Genes

• Purpose:

  • Focused on studying protein product derived from cloned genes.

• Expression Vectors:

  • Specialized plasmids designed to promote transcription, containing essential sequences such as promoter and operator regions, ribosome binding sites, and transcription termination signals.

Purification of Recombinant Proteins

• Challenge:

  • Natural proteins prove difficult to purify, leading to necessity in recombinant methods.

• Strategies:

  • Utilization of affinity tags to enable specific binding to purification columns.

1. Structures of Nucleobases (A, T, G, C, U)

Purines (2 rings)

• Adenine (A) – has an amino group (–NH₂) at C6.

• Guanine (G) – has an amino group at C2 and a carbonyl (=O) at C6.

Pyrimidines (1 ring)

• Cytosine (C) – has an amino group at C4 and a carbonyl at C2.

• Thymine (T) – has two carbonyls (C2 and C4) and a methyl group (–CH₃) at C5.

• Uracil (U) – same as thymine but no methyl group at C5.
  → T = DNA only, U = RNA only

2. Structures of Nucleotides

Nucleotide = phosphate + sugar + base

• Phosphate group → attached to 5′ carbon of sugar (negatively charged).

• Sugar (pentose)

  • Ribose (RNA): has –OH on 2′ carbon.

  • Deoxyribose (DNA): has –H on 2′ carbon (no oxygen).

• Base → attached to 1′ carbon of sugar by β-N-glycosidic bond.

🔗 Connections in a strand:

• Phosphodiester bond between 3′-OH of one sugar and 5′-phosphate of the next → gives 5′ → 3′ directionality.

3. Naming of Nucleic Acids

4. Base-Pairing Rules & % Calculations

• A–T (2 hydrogen bonds) → weaker

• G–C (3 hydrogen bonds) → stronger (↑ melting temp)

• In double-stranded DNA:

  • %A = %T

  • %G = %C

  • %A + %T + %G + %C = 100%

5. Secondary Structures (mostly in RNA)

• Stem-loop (hairpin): base-pairing within the same RNA strand → loop = unpaired region.

• Occurs when sequence has inverted repeats (ex: 5′-GCCGA...UCGGC-3′).

• More G≡C pairs = more stable stem.

Structure of Double-Stranded DNA

• Antiparallel: one strand 5′→3′, the other 3′→5′.

• Double helix: right-handed spiral (B-DNA most common).

• Base stacking: flat bases stack like pancakes → stabilizes via hydrophobic & van der Waals forces.

• Major & minor grooves: allow protein interactions.

• Hydrogen bonding: pairs of bases (A-T and G-C) connect the strands, contributing to overall stability of the structure.

1. Overall Stability Comparison

🧪 2. Chemical Basis of Stability

• DNA (Deoxyribonucleic acid)

  • Deoxyribose lacks a hydroxyl group on the 2′ carbon → makes it chemically inert.

  • Resistant to hydrolysis (breaking down in water).

  • Thymine is more stable than uracil; it allows for easier error detection (uracil can result from cytosine deamination).

  • Double helix protects nitrogenous bases from chemical attack.

• RNA (Ribonucleic acid)

  • Ribose contains a 2′-OH group → can attack the phosphate backbone, causing self-cleavage (hydrolysis) especially under basic conditions.

  • Usually single-stranded, so bases are exposed and prone to degradation.

  • Uracil is less stable and more prone to mutations (cannot distinguish between original uracil and deaminated cytosine).

🌡 3. Structural and Environmental Factors

• DNA:

  • Double helix → base stacking and hydrogen bonding stabilize structure.

  • Stable at a wide range of pH and temperature.

  • Can last for years under proper storage.

• RNA:

  • Sensitive to heat, pH, and RNases (enzymes that degrade RNA).

  • Secondary structures (hairpins, stem-loops) can locally stabilize RNA, but not overall.

DNA Polymerase

• Requires:

  • Template strand

  • Primer (short RNA/DNA with free 3′-OH)

  • dNTPs (A, T, G, C triphosphates)

  • Mg²⁺ (stabilizes negative charges on phosphate)

• Synthesizes: always 5′ → 3′ direction (adds to 3′-OH).

• Proofreading: 3′ → 5′ exonuclease removes mispaired bases.

8. PCR (Polymerase Chain Reaction)

Goal: Amplify a specific DNA fragment.

Steps:

1. Denaturation (≈95°C) – strands separate.

2. Annealing (≈55–65°C) – primers bind to target sequences.

3. Extension (≈72°C) – Taq polymerase adds dNTPs from primers.

Cycle repeats ~30 times → exponential amplification.

Reagents:

Product: double-stranded DNA of defined length between primers.

PCR Primer Design

• Length: 18–25 bases

• GC content: 40–60%

• End with G/C clamp (stabilizes binding).

• Avoid runs of identical bases or complementarity within/between primers.

• Forward primer: matches 5′ end of target; Reverse primer: complementary to opposite strand.

10. Sanger Sequencing (Chain-Termination Method)

Goal:

To find the exact sequence of a DNA strand (the order of A, T, G, and C).

🧩 Key Idea

DNA synthesis stops whenever a special nucleotide called a dideoxynucleotide (ddNTP) is added.

What’s in the reaction mixture?

🧬 How it works (step-by-step):

1. DNA polymerase extends the primer along the template strand.

2. Occasionally, a ddNTP is added instead of a normal dNTP.

3. When this happens, synthesis stops at that exact base (because no 3′-OH = no next bond).

4. As a result, you get a mixture of DNA fragments of different lengths, each ending with a fluorescently labeled ddNTP.

How it’s read:

• The mixture is run through capillary electrophoresis (a very thin tube).

• Smallest fragments (shortest) come out first → represent the 5′ end.

• A laser detects the fluorescent color at each fragment’s end.

• The computer records the color order → gives you the DNA sequence.

🧠 Quick summary to remember:

DNA Gel Electrophoresis

Goal:

To separate DNA fragments by size and visualize them.

🧩 Basic setup

• A gel made of agarose (like a sponge with pores).

• DNA samples are loaded into wells at the top.

• A buffer solution conducts electricity.

• An electric current is applied.

Key principle:

• DNA has a negative charge (from phosphate groups).

• It moves toward the positive electrode (anode).

• Smaller fragments move faster and farther through the gel’s pores.

• Larger fragments move slower and stay closer to the wells.

Visualization:

• After running, the gel is stained with a DNA-binding dye (e.g., Ethidium Bromide or SYBR Safe).

• When exposed to UV light, the DNA bands glow.

• Each band = DNA fragments of the same length.

Applications:

• To check PCR product size (did amplification work?).

• To verify restriction enzyme digests (did cutting happen?).

• To confirm Sanger sequencing fragments were produced.

🧠 Quick summary to memorize:

Sanger Sequencing

• Purpose: Determines the exact nucleotide sequence of DNA.

• Method type: DNA synthesis–based (chain-termination).

• Key components: Template DNA, primer, DNA polymerase, normal dNTPs, fluorescently labeled ddNTPs.

• Mechanism: Random incorporation of ddNTPs → terminates strand elongation.

• Output: Mixture of fragments ending at every possible nucleotide position.

• Separation step: Uses capillary electrophoresis to separate fragments by size.

• Detection: Laser detects fluorescent colors of ddNTPs to read sequence.

• Result type: Exact sequence (ex: 5′-ATGCCA-3′).

• Scale: Analyzes one DNA region at a time (low throughput).

• Visualization: Output displayed as a colored peak chromatogram.

• Product use: Provides the precise order of bases; used for mutation detection, gene identification, etc.

⚡ DNA Gel Electrophoresis

• Purpose: Separates DNA fragments by size (not to read the sequence).

• Method type: Physical separation in agarose gel.

• Key components: Agarose gel, buffer, DNA samples, DNA ladder (size marker), electric current, DNA-staining dye (Ethidium Bromide or SYBR Safe).

• Mechanism: DNA migrates through gel because it’s negatively charged → moves toward positive electrode.

• Smaller fragments travel faster/farther; larger fragments move slower/shorter distance.

• Detection: DNA bands visualized under UV light.

• Result type: Pattern of bands (relative sizes).

• Scale: Can compare many samples at once.

• Visualization: Bands on gel image, not sequences.

• Product use: Used for checking PCR results, verifying DNA purity, or confirming fragment sizes before sequencing.