Lecture 1: Structure, Synthesis & Functions of Nucleic Acids

Functions of nucleotides (7)

1. Energy currency in metabolic transactions

2. Chemical link response of cells to hormones and other extracellular stimuli

3. Structural components of enzyme cofactors and metabolic intermediates

4. Constituents of nucleic acids

5. Some coenzymes contain adenine

6. Function as second messenger in the cells + work as regulatory molecules (cAMP, cGMP)

7. Can function as neurotransmitters and ligands

Give examples of nucleotides functioning as neurotransmitters and ligands

• ATP binds to P2x receptors in post-synapsis (taste, inflammation, smooth muscle contraction)

• ADP binds to P2γ receptors in platelets, promoting clotting (clopidogrel interferes)

Which bonds are hydrolysed to release energy from nucleotides, and how much energy is released?

Ester (α-P) = 14 kJ/mol

Anhydride (β-P, γ-P) = 30.5 kJ/mol

Nucleotide components

nitrogenous base, pentose (sugar with 5 C atoms), phosphate group

A nucleotide without a phosphate group is called…?

A nucleoside

Phosphate group structure

sp3, regular tetrahedron

Nitrogenous bases are derivates of…?

Pyrimidine + purine (heterocyclic rings)

Properties of pyrimidine and purine

aromatic (planar, hydrophobic), basic

Conformation of ribose in nucleic acids

Only β-D-furanose ring form (no equilibrium with aldehydic form)

How many different ribose conformations?

4 different puckered conformations.

4 out of 5 atoms are nearly in a single plane. C-2’ or C-3’ is either on the same (endo) or opposite (exo) side of the plane relative to the C-5’ atom.

The bond between the nitrogenous base and the pentose is called…?

N-glycosidic

The phosphate group and nucleoside are linked by ______________. Therefore, the nucleotide contains a ____________ bond.

An ester bond, phosphodiester

How are successive nucleotides linked in nucleic acids?

Through the formation of phosphodiester bonds between C-3’ and C-5’ of the two nucleotides

Phosphodiester linkages are the ________ of DNA and RNA

covalent backbone

Levels of nucleic acid structure

Primary: The covalent structure and nucleotide sequence

Secondary: Any regular, stable structure taken up by some or all of the nucleotides (e.g. DNA)

Tertiary: The complex folding of large chromosomes or the elaborate folding of large tRNA or rRNA

Chargaff’s rules (4)

For DNA base composition:

1. [A] = [T] and [G] = [C], therefore [A] + [G] = [T] + [C]

2. Base composition varies from one species to another

3. Same composition from different tissues of the same species

4. No variations with age, nutritional state or changing environment

Watson & Crick model (1953)

• Hydrophilic groups exposed to H₂O, inner hydrophobic groups and H bonds (fulfils the thermodynamic requirements)

• Base complementarity (fulfils [A] + [G] = [T] + [C])

  • Atoms of complementary nitrogenous bases lie on the same plane (almost perpendicular to the helix axis)

• Antiparallel strands

• Vertically stacked bases inside the double helix, 3.4 Å apart

  • Secondary repeat distance of ~ 34 Å (10 bases per helical turn)

DNA structure in aqueous solution vs fibres

Secondary repeat distance altered in aqueous: 10.5 base pairs (36 Å or 3.6 nm) per helical turn

“Space filling” model

Atoms are at the centre of spheres with van der Waals radius

R_C = 1.7 Å, R_N =1.5 Å

DNA structural variation

Free rotation about C-1’-N-glycosidic bond.

Purines: syn, anti

Pyrimidines: anti (steric interference between sugar and C=O)

Compare the different DNA forms:

	A-form	B-form	Z-form
Right-/left-handed?
Helical rise per base pair
Base pairs per turn
Width/diameter
Base tilt normal to helix axis

	A-form	B-form	Z-form
Right-/left-handed?	Right-handed	Right-handed	Left-handed
Helical rise per base pair	2.6 Å	3.4 Å	3.7 Å
Base pairs per turn	11	10.5	12
Width/diameter	26 Å	20 Å	18 Å
Base tilt normal to helix axis (not perpendicular to helix axis, more tilt)	20°	6°	7°

Describe Z-form DNA

• Structure appears more slender and elongated

• Backbone takes on a zigzag appearance

• Major groove is barely apparent and minor groove is narrow and deep

• May play a role (unknown) in gene expression regulation and in genetic recombination

Conditions for A and B-form DNA

A: solutions devoid of water, DNA crystallisation

B: most stable form under physiological conditions (aqueous solution)

Certain nucleotide sequences fold into __-form DNA more readily than others. Give examples.

Z

• Pyrimidines alternating with purines

  • Purines flip to syn alternating with pyrimidines in anti conformation

• C-G alternating with G-C

• 5-methyl-C and G residues

What kind of nucleotides for hairpins and cruciforms?

hairpin: ssDNA, ssRNA

cruciform: dsDNA

Hoogsteen pairing

• Triple helix + quadruplex

• Stabilisation occurs between one purine and two pyrimidine bases (in triple helix)

Hoogsteen pairing may originate from…?

• virus

• if bases are methylated (epigenetic effect)

• fragment generated during DNA replication

• free nucleotides in nucleus (excision)

DNA denaturation

Reversible disruption of hydrogen bonds between paired bases and of base-stacking interactions causing unwinding of the double helix to form two single strands, completely separated from each other along the entire length or part of the length (partial denaturation) of the molecule

Are covalent bonds broken during denaturation?

No (hydrogen bonds aren’t covalent)

Factors promoting DNA denaturation

high temperature, pH

Melting temperature (t_m)

Temperature at which half of the DNA is present as separate single strands

Properties of t_m

• Each species of DNA has a characteristic t_m

• Increases with %GC content

• dsDNA < DNA:RNA hybrid < dsRNA

  • (More H-bonds in RNA, due to 2’-OH)

Effect of denaturation of UV light absorption

Hypochromic effect: Close interaction between stacked bases decreases UV light absorption compared to a solution with the same concentration of free nucleotides. Absorption is further decreased when 2 DNA strands are paired.

Hyperchromic effect: Increased UV absorption when a double stranded nucleic acid is denatured.

The transition from dsDNA to denatured ssDNA can be detected by monitoring UV absorption at 260 nm.

Appearance of partially denatured DNA in electron microscopy

Bubbles

What makes denaturation reversible? What is the process called?

• Nitrogen base complementarity

  • Creates a strong specificity in the interaction between polynucleotides: two complementary DNA strands can “search” for each other in solution and pair, leading to stable double helix

• Re-annealing

Which bases are methylated more often?

A and C (more often than G and T)

Methylation is confined to _______?

Specific DNA regions (CpG islands)

All known DNA methyltransferases use __________ as a methyl group donor

S-adenosylmethionine

What % of cytidine residues in DNA are methylated to 5-methylcytidine

5%

DNA methylation plays a role in ______?

Gene expression regulation

PCR (polymerase chain reaction)

1. Heat to separate strands

2. Add synthetic DNA oligonucleotide primers; cool

3. Add thermostable Taq DNA polymerase to catalyse 5’ → 3’ DNA synthesis

4. Repeat steps 1 - 3

DNA sequencing

Sanger method (dideoxy chain-termination sequencing)

1. Target DNA denatured

2. Oligonucleotide primers annealed to template strand (starting point for DNA synthesis)

3. Mix of dNTPs and ddNTPs + DNA polymerase used to make complementary chains

4. Chain-terminating ddNTPs result in DNA fragments of different lengths

5. Fragments separated using gel electrophoresis (shorter fragments migrate faster to other end)

Automated DNA sequencing

1. Each ddNTP linked to a different fluorescent dye

2. All 4 ddNTPs added together

3. Resulting coloured DNA fragments are separated by size in an electrophoretic gel in a capillary tube

4. All fragments of a given length migrate in a single band

5. The colour associated with each band is detected with a laser beam

6. DNA sequence is read by identifying the colour sequences in the bands

7. Amount of fluorescence in each band is represented as a peak in the computer output

Next-generation pyrosequencing

Requires initial DNA fragmentation; ~ 400-500 nucleotides

1. 4 nucleotides added as pulses one at a time, in repeating sequence

2. Excess is destroyed by apyrase before next pulse

3. When the nucleotide binds, pyrophosphate (PP) is released

4. Sulfurylase uses PP to generate ATP

5. ATP activates luciferase that reacts with luciferin and releases a detectable signal (peak)

Reversible Terminator Sequencing

Illumina; ~100-200 nucleotides

1. Primer and template are fixed to solid support

2. Add blocked, fluorescently (different colours) labelled dNTPs

3. Fluorescent colour is observed and recorded

4. Remove labels and blocking groups; wash

5. Repeat steps 2-4

SMRT Sequencing

Single-Molecule Real Time Sequencing; ~30-40 kb fragments; 10-15% error, mitigated by multiple readings

1. Zero Mode Wavelength (ZMW) pores: smaller diameter than visible light wavelength (70 nm).

2. DNA anchored by adapter sequences

3. Diameter hosts one DNA polymerase

4. Nucleotide-specific fluorescence emitted and detected as the correct nucleotide is inserted

What are contigs?

SMRT can translate sequences of millions of short DNA fragments into complex and contiguous sequences called contigs, with the help of computerised alignment of overlapping segments