Lecture 6 - DNA

Nucleic Acid Structure

• Primary structure: The linear sequence of nucleotides in a DNA or RNA molecule.

• Nucleobases: The nitrogenous bases (Adenine, Guanine, Cytosine, Thymine, and Uracil) that form the core of nucleotides.

• Sugars: Ribose in RNA and deoxyribose in DNA; these pentose sugars provide the backbone for nucleic acids.

• Phosphates: Phosphate groups link the sugars in nucleic acids, forming the phosphodiester bonds.

• Nucleic acids: Polymers of nucleotides; DNA and RNA are the two main types.

• Secondary structure: The three-dimensional arrangement of the nucleic acid chain (e.g., the double helix in DNA).

• B-form: The most common form of DNA under physiological conditions; a right-handed helix.

• A-form and Z-form: Alternative DNA structures; A-form is favored in dehydrated conditions, while Z-form is a left-handed helix.

• Triplex and quadruplex DNA: More complex DNA structures involving three or four strands, respectively.

• RNA structures: RNA can form a variety of secondary structures like hairpins, loops, and bulges due to its single-stranded nature.

• Tertiary structure: The overall three-dimensional arrangement of all atoms in the nucleic acid, including interactions beyond secondary structure elements.

• Nucleosomes: Basic structural units of chromatin, where DNA is wrapped around histone proteins.

• DNA nanotechnology: Using DNA as a building material to create nanoscale structures and devices.

Components of Nucleic Acids

• Includes base pairs and phosphodiester/phosphate linkages: These components are essential for the structure and function of nucleic acids.

• Deoxyribose structure: The pentose sugar lacking an oxygen atom at the 2' position in DNA.

Nucleobases

• Pyrimidine: Thymine (T), Uracil (U), Cytosine (C): Single-ring structures.

• Purine: Adenine (A), Guanine (G): Double-ring structures.

• Nucleobases are based on pyrimidine and purine structures.

Nucleobases: H-bond donors and acceptors

• Thymine, Uracil, Cytosine are represented with donors and acceptors indicated: These bases have specific sites that can donate or accept hydrogen bonds.

• Adenine, Guanine are represented with donors and acceptors indicated: These bases also have specific sites for hydrogen bonding, essential for base pairing.

Nucleobases: Structures and Hydrogen Bonding

• Remember the structures of the nucleobases: Critical for understanding their interactions.

• Adenine (A) pairs with Thymine (T) via 2 hydrogen bonds (DA/AD).

• Guanine (G) pairs with Cytosine (C) via 3 hydrogen bonds (ADD/DAA).

• Nucleobases form strong dimeric pairs with each other.

Ribose and Deoxyribose

• Ribose and 2-deoxyribose structures are shown (Fisher projection).

• Ribose has a hydroxyl group at the 2' position, while deoxyribose does not.

• Nucleobase attaches to the sugar: Forms a nucleoside.

• Phosphate group attaches to the 5' position: Creates a nucleotide.

Phosphates/Phosphodiesters

• pKa ~ 0: Indicates they are negatively charged at physiological pH.

• Phosphodiester links connect the 5' to 3' positions: Forming the backbone of nucleic acids.

• Asymmetric linkage gives DNA strands directionality.

Nucleosides and Nucleotides

• Nucleoside: Nucleobase + Sugar: A building block of nucleic acids without the phosphate group.

• Nucleotide: Nucleobase + Sugar + Phosphate: The basic unit of nucleic acids.

• Structures of nucleosides and nucleotides are shown with Adenine as the base.

Nucleic Acids

• DNA strands run antiparallel (5' to 3' and 3' to 5').

• Always give sequences 5' to 3'.

Primary Structure and Hybridisation

• Primary structure = sequence of nucleotides (e.g., ATGCGAATCGA).

• Duplex formed with strand of complementary sequence (complementary bases in opposite order).

• A complements T, G complements C.

• Longer duplex with higher GC content is more stable: Due to the three hydrogen bonds in G-C pairs compared to two in A-T pairs.

Hybridisation

• Given the sequence ATGTCTTGAACA:1. Split strand into three-base units: ATG TCT TGA ACA

  2. Write down the complementary bases: TAC AGA ACT TGT

  3. Reverse the order: TGT TCA AGA CAT

• Hybridization is the process in which two complementary single-stranded DNA and/or RNA molecules bond together to form a double-stranded molecule. Important in polymerase chain reaction.

Problem: Hybridisation

• Process of finding the complementary sequence to a given strand.

• Important in polymerase chain reaction.

Hybridisation

• Hybridisation = ‘melting temperature’ (Tm).

• Longer duplex, higher GC content = more stable = higher Tm.

• The ‘melting temperature’ of DNA is when half of the strands are in a random coil single strand state (i.e., duplex dissociates).

Double Helix

• Why does duplex DNA have a helical structure?- Negatively charged phosphate groups repel each other.

  • Stacking of nucleobases through hydrophobic / Van der Waals interactions compacts duplex vertically.

  • Base pairs hydrogen bond.

Double Helix Parameters

• 10.5 nucleotides per turn

• 1 turn = 3.3 nm = 33 Å

• Right-handed double helix

• 2.0 nm = 20 Å wide

• 3.3 Å base stacking distance

• Major groove and Minor groove: These grooves are important for protein binding and regulatory functions.

DNA Forms

• B-DNA: right-handed: The most common form under physiological conditions.

• A-DNA: right-handed: Favored in dehydrated conditions and RNA-DNA hybrids.

• Z-DNA: left-handed: Occurs in regions of DNA with alternating purine-pyrimidine sequences.

Triplex and Quadruplex DNA

• Watson-Crick face, Hoogsteen face: Different faces of nucleobases involved in hydrogen bonding.

• Third strand binds in major groove: Forming a triplex structure.

• Found in telomeres – ends of chromosomes: Quadruplex DNA is common in telomeres, stabilizing chromosome ends.

RNA structures

• DNA vs RNA:- Additional OH group in RNA: Makes RNA less stable than DNA.

  • Further possible hydrogen bonding patterns: Allows RNA to form complex secondary and tertiary structures.

  • Phosphodiesters more easily hydrolysed: RNA is more susceptible to degradation.

  • U replaces T in RNA.

  • Usually single-stranded (RNA), usually double-stranded (DNA).

RNA structures differences between Thymine and Uracil

• Thymine and Uracil's structural formulas are compared.

• Explains the difference between the two bases: Uracil lacks a methyl group compared to Thymine.

RNA structures

• RNA exists in many complex structures.

• Structures include Bulge, Bubble/interior loop, Hairpin, Stem-loop.

• Sequence conservation is shown in the structures.

Nucleosomes

• DNA is coiled around proteins called histones to produce nucleosomes, which are further packed to produce chromosomes.

• Eight protein units are required for each nucleosome: Two copies each of histones H2A, H2B, H3, and H4.

‘The Central Dogma’ of Biology

• “The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid.” – Francis Crick

• DNA → RNA → Protein

• Transcription, reverse transcription, translation, replication.

Replication of DNA

• In their 1953 announcement of a double helix structure for DNA, Watson and Crick stated, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."

• DNA is synthesised 5’ to 3’.

Replication of DNA Mechanism

• Use of 5’ nucleotide triphosphates means addition of new base occurs at 3’ end.

• Microscopic reversibility gives proofreading.

Polymerase Chain Reaction (PCR)

• Double-stranded template DNA

• Denaturation at 94 °C: Separates the DNA strands.

• Hybridise primers at 68 °C: Primers bind to the single-stranded DNA.

• Elongation at 72 °C: DNA polymerase extends the primers, synthesizing new DNA strands.

• 2 x double-stranded DNA

• Repeat…

• Thirty repeats = 2^{30} copies made = ca. 1 billion copies

• Performed by polymerase

Transcription

• The ‘production’ of proteins by DNA.

• Operates very similarly to DNA Replication.

• Protein cluster termed: “Transcription Factors” signals to RNAP (RNA polymerase) where to begin transcription.

• Resulting strand produced is ‘messenger RNA’ or mRNA…used to code specific peptide sequences.

• RNA is also produced 5’ to 3’.

Translation

• process involving tRNA, mRNA, ribosome, and amino acids to produce a new protein.

• Ribosome subunits (large and small) are involved.

• A site and P site are labeled.

Codons

• 20 amino acids but four nucleotides – need multiple bases to specify each amino acid

• All proteins start with Met

tRNA

• tRNA brings in a codon-specific amino acid as an activated ester

• tRNA molecule structure shown with intramolecular base-pairing, anticodon arm, acceptor stem, CCA tail

The Ribosome

• Huge complex of RNA and protein

• Small ribosomal subunit: reads RNA

• Large ribosomal unit: links amino acids

RNA Interference (RNAi)

• siRNA (Short interfering RNA) designed to correspond to gene target

• Modified siRNAs penetrate the cell membrane and harness the RNAi mechanism for gene silencing

• Gene silencing achieves a therapeutic effect

Summary

• Right-handed anti-parallel double helix

• 2.0 nm = 20 Å wide

• 3.3 Å /base

• 10.5 nucleotides/turn

• Negatively charged phosphate groups repel each other

• Stacking of nucleobases through hydrophobic / Van der Waals interactions compacts duplex vertically

• Base pairs hydrogen bond

• DNA → RNA → Protein

• transcription reverse transcription translation replication replication