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LECTURE 2: DNA, RNA & Proteins Information flow : DNA -> RNA -> Protein 2.1 DNA Structure Eukaryotes: DNA found in nucleus and in certain organelles (eg. Mitochondria, chloroplasts) Prokaryotes: DNA not enclosed in a membrane, found in the nucleoid(specialised cell region) •

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DNA is a polymer of nucleotides consisting of: o Nitrogenous base ▪ Purine base (2 carbon ring) ▪ Pyrimidine base (1 carbon ring) o Sugar ▪ Deoxyribose: 5-carbon sugar ▪ Base is attached to the 1’ carbon ▪ Phosphate attached to 5’ carbon o Phosphate ▪ 1 nucleotide consists 3 phosphate groups ▪ When nucleotide join a DNA/RNA chain, it loses 2 phosphate groups. DNA found in double helix structure o Sugar and phosphate on the outside of the helix, forms sugar-phosphate backbone o Nitrogenous bases extend into interior and bound by hydrogen bonds o Antiparallel orientation- the 2 strands run in opposite directions

Properties • A polynucleotide chain has directionality o 2 ends different from each other o At 5’ end, phosphate group sticks out o At 3’ end, hydroxyl group is exposed o DNA written from 5’ to 3’ direction o When new nucleotides are added to a DNA/RNA strand, 5’ phosphate of incoming nucleotide attach to 3’ end of chain with a bond called phosphodiester linkage •

Complimentary base pairing o Weak hydrogen bonds o Watson-Crick pairing ▪ Adenine and Thymine (A = T) → double bond ▪ Cytosine and Guanine (C = G) → triple bond ▪ C-G stronger than A-T bond o Chargaff’s rule: ▪ A and T in equal amounts ▪ C and G in equal amounts

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DNA complementarity o Base-pair complement + antiparallel → sequence of one strand completely determines sequence of complementary strand

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DNA replicates to pass down genetic information

Replication • Semi-conservative replication o Replication of one parent DNA helix → 2 identical daughter helices o Synthetisation: by addition of nucleotides complementary to parent strand o Semi-conservative: one parent strand always passed on to daughter helix of DNA •

DNA replication o 1. DNA Helicase: ▪ unzips the 2 strands ▪ replication origin forms Y-shaped replication fork o 2. Primase: ▪ adds RNA bases → makes primer (small piece of RNA) o 3. DNA Polymerase: ▪ binds to primer, replicate bases ▪ Leading strand: replication occurs continuously (from 5’ to 3’) ▪ Lagging strand: Makes Okazaki fragments on other end (from 5’ to 3’) o 4. Exonuclease: ▪ removes all RNA fragments o 5. DNA Polymerase: ▪ fill in gaps with DNA o 6. DNA Ligase: ▪ seals up fragments to form 2 continuous double strand

2.2 RNA Structure • Similar to DNA, but ribose instead of deoxyribose • Bases are A, U (not T), G, C • RNA is single stranded & shorter • RNA less stable than DNA→ does not persist in cell for long

Properties • Can form secondary structures Transcription • Transcription occurs in the nucleus • Transcription begins at transcription start site, terminates when terminator region encountered o 1. RNA Polymerase: ▪ binds to DNA helix at promoter region ▪ Open complex formed as DNA unwinds ▪ RNA Nucleotides polymerised from nucleotide triphosphates to make messenger RNA in 5’ to 3’ direction → form mRNA o Spliceosome: Splicing ▪ remove introns and leave exons ▪ provides more diversity in proteins that can be coded

2.3 Genetic code 20 amino acids- building blocks to make all proteins Properties • Each 3 consecutive base on mRNA is a code word/codon that specifies an amino acid o Code is non-overlapping: bases not shared between codons o 64 codons in total: 61 code amino acid, 3 signal terminators (UAA,UAG,UGA) o AUG is the start codon and codes for methionine Translation Synthesizing a protein from amino acids based on sequences of nucleotides on mRNA • Occurs at ribosome o Consists of large subunit and small subunit o mRNA binds to small subunit • Transfer RNA (tRNA) o Carries specific amino acid to ribosome o Folded o Each tRNA recognises correct codon on mRNA molecule o Anticodon on tRNA codon on mRNA

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Steps: 1. mRNA leaves nucleus and migrates to ribosome 2. mRNA binds to small ribosomal subunit 3. tRNA brings amino acid to ribosome, tRNA anticodon binds to mRNA codon 4. Amino acid bonds to adjoining amino acid → forms growing polypeptide 5. Molecule

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5. tRNA without amino acid released from ribosome 6. Other tRNA bring amino acids to ribosome (repeat 3 to 6) 7. Protein is completed Protein synthesis o Occurs in cytoplasm o Many ribosomes can occupy same mRNA strand for simultaneous protein synthesis

Summary 1. Cell nucleus: RNA produced by transcription 2. RNA single stranded; substitute ribose for deoxyribose; substitute U for T 3. Cytoplasm: mRNA convey DNA recipe for protein synthesis 4. mRNA binds to ribosome→ 3 base codon of mRNA = 3-base anti-codon of tRNA 5. tRNA transfer 1 amino acid to growing protein chain 6. Each codon directs addition of 1 amino acid to protein

LECTURE 3: Genetic engineering + DNA fingerprinting 3.1 Genetic engineering – Recombinant DNA cloning DNA cloning • •

DNA cloning → process of making multiple identical copies of DNA Process: 1. Gene of interest is inserted into a plasmid (small circular DNA molecules, duplicate separately from chromosome) → CUT AND PASTE a. Restriction enzyme cuts DNA into fragments b. Gene of interest cut with same restriction enzyme c. Fragments stick together by base pairing d. DNA ligase joins fragment into strands 2. Recombinant plasmid introduced into bacteria through heat shock 3. Bacteria carrying plasmid is selected and grown to produces copies (eg. insulin) a. Plasmid contains antibiotic resistant gene b. Bacteria placed in antibiotic, those with plasmid survives c. Bacteria with plasmid survives and produce desired protein

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Restriction enzyme o Each restriction enzyme only cuts a specific sequence (recognition site) of DNA o “sticky ends” → restriction enzyme cut leaves overhanging chains → overhanging chains will glue the two pieces together even though back bone cut o “blunt ends” → restriction enzyme cut leaves no overhang

3.2 DNA fingerprinting 1. Extract DNA 2. Amplify DNA amount with Polymerase Chain Reaction (PCR) Polymerase Chain Reaction (Kary Mullis- 1983)→ replicate DNA in test tube •

Process o Denaturation to open up template DNA o Primer (short synthetic DNA) bind to template → initialise DNA polymerisation o Taq polymerase extends primers o Taq polymerase used as it is heat resistant

Variable Number tandem Repeat (VNTR) • • •

VNTR is a location in a genome where a short nucleotide sequence is organised as a repeat Show variations in length(no. of repeats) among individuals Variable lengths of VNTR → different fragment length → use Restriction Fragment Length Polymorphism (RFLP) to differentiate VNTR alleles

Gel electrophoresis • • •

Seperates DNA of different lengths A current is run through a gel containing DNA fragments DNA fragments travels at different speed (due to diff in size and charge)

LECTURE 4: Mutation and Diseases 4.1 Mutation Chromosomal Mutation • • • • •

Deletion → loss of whole/part chromosome (part/whole) Duplication → produce extra copy of whole/part chromosome Inversion → reverse direction of some part of chromosomes Translocation → part of chromosome breaks and attach to another chromosome Nondisjunction → both chromosome go one side during mitosis

Point Mutation (change in nucleotides) •

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Substitution of base o Silent → no effect on amino acid sequence o Missense → produce malfunctioning protein (eg. Sickle cell anemia) o Nonsense → shorten protein due to reading STOP codon Insertion/Deletion o Insertion/Deletion of base changes order of triplet codes (DNA reads in 3s) o Frameshift mutation (eg. Tay-Sachs disease)

Cancer •

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Mutation in cell checkpoint regulators → uncontrolled cell division o proto-oncogenes: increase cell division o Tumor suppressor genes: inhibit cell division Factors affecting cancer o Chemical mutagens (eg. tar in cigarettes) o Ultraviolet/Ionising radiation o Viruses o Genetic predisposition

4.2 DNA sequencing - Next Generation sequencing •

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First Generation: Sanger Sequencing (serial sequencing) o Electrophoretic separation of chain-termination products produced in individual sequencing reactions o DNA polymerisation with dideoxy (ddNTPs) chain terminators create fragments of different lengths and colour o DNA fragments arranged by size and length Second Generation: Massive parallel sequencing via spatially separated, clonally amplified DNA template in a flow cell Third Generation: Nanopore sequencer: single molecule sequencing Uses o Sequencing bacteria to control disease outbreak o Genetic information can guide cancer treatment and prevention o Detection of chromosomal anomalies in fetus

LECTURE 6: Amino Acids and Proteins

6.1 Amino acids pH and pKa •

Amino acid structure: amino group, carboxyl group, H, R group

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Lower pKa → stronger acid → easily release protons If pH > pKa: o plenty of protons in the environment (pH), protonated form predominates If pH > pKa: o a lack of protons in the environment (pH), de-protonated form predominates pKa of carboxyl group < pKa of amino group

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R group- 20 naturally occuring

Zwitterions • • • •

amino acid exists as zwitterions (net charge = 0) isoelectric point → pH when net charge = 0 zwitterion → least soluble state of amino acid increasing or decreasing pH improve solubility

6.2 Proteins Amino acids linked by covalent peptide bond→ through condensation between NH3+ and COOPrimary Structure • • •

Directionality: N to C terminus R groups: identities of i=maino acids Peptide bonds (stronger than disulfide bonds)

Secondary Structure • • • •

Peptide bond rigid and planar Hydrogen bond of peptide bond → polypeptide backbone form α-helix and β-pleated sheet α-helix: R groups pointing out β-pleated sheet: R groups point above and below the plane of the sheet

Tertiary Structure • •

3-D structure of 1 polypeptide Disulfide bond > Electrostatic interaction > Hydrogen bonds > Hydrophobic interactions

Quartenery Structure • • •

Spatial arrangement of multiple polypeptides in the protein Protein size measured in daltons (Da) 1 amino acid ~ 110 Da o α-chain: 141 amino acids = 16kDa o β chain: 147 amino acides = 16kDa

LECTURE 7: Protein functions, enzyme, inhibitors 7.1 Functions of protein structures

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Ligand o molecule that a protein can bind Binding site o 3D cavity made by specific arrangement of amino acid o interact with specific ligand due to structural specificity(shape) and binding specificity(non-covalent bonds) Correction 1: Post translational modification o Covalent addition to polypeptide by adding R side chains ot C or N terminus Correction 2: Protein folding o Crowding occurs at high concentration of macromolecule (eg. protein) o Crowding can reduce yield of correctly folded proteins o Molecular chaperons: 1. assist protein folding 2. stabilise unfolded/partially folding proteins and 3. facilitate correct order of folding

Native structure and denaturation • •

Native structure – normal physiological structure of protein Protein denaturation o Process where protein loses its native structure (secondary, tertiary, quartenery) o Primary structure not changed o Solubility reduced → due to exposure of hydrophobic groups o Loss of binding site o Causes:

7.2 Enzymes and inhibitors Enzymes •

catalytic proteins that bind to ligands(substrates) to speed up reaction o Highly specialised proteins o Can be reused o Regulated different metabolic activities for life o Some enzymes require additional non-protein components ▪ Inorganic metal ions (eg. Zn2+, Mg2+, Fe2+, Mn2+) ▪ Organic molecules (coenzymes) (eg. vitamins, biotin) o Enzyme specificity ▪ Absolute specificity (1 to 1 substrate) ▪ Group specificity (1 to group of substrates) ▪ Linkage specificity (1 to specific bond)

Enzyme mechanism

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Free energy: higher the energy, less stable the complex Reactants → transition state → products

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Enzyme catalysed reaction → transition state energy level lowered Activation energy reduced Net energy change same Steps: o Enzyme binds to substrate through non-covalent bonds in active site o Enzyme can change shape when substrate binds to fit better o Formation of enzyme-substrate complex o Substrate bonds weakened

Enzyme Kinetics

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Slope changes over time (at equilibrium V =0)

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As substrate increases: o Low substrate concentration: rate of reaction V increases, more enzymes work o High substrate concentration: Vin at max, all enzymes already working (saturation) o Km = concentration of substrate to achieve ½Vmax o Km is an intrinsic property of the active enzyme (active site)

Enzyme inhibitors Enzyme inhibitors: molecules that prevents enzyme from working • •

Non-specific → affects all enzyme via same mechanism o Physical/chemical changes that denature (eg. temperature, pH) Specific → affects a type of enzyme o Irreversible o Reversible ▪ Competitive ▪ Non-competitive

7.3 Applications of enzymes Cell signalling •

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Protein activity regulated through the addition of a phosphate group o Phosphate group negatively charged → attract positively charged R groups o Switch due to electrostatic interaction and structural changes Reversible addition and removal of phosphate group turns on and off the protein activity

• Bio-stoning • •

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Enzymes selectively modify the fabric surface Cellulase o Binds to exposed cellulose and break molecular bonds o Indigo dye particles loosnened o Back-staining: released dye redeposited on the garment Protease More controllable, less waste and pollution compared to conventional stone washing

LECTURE 8: Diseases, Protein quantitation and immunoblotting 8.1 EGFR- Epidermal Growth Factor Receptor

Mechanism • • • •

Dimerization of EGFR→ Kinase active site of one EGFR close to substrate on another EGFR Phosphorylate each other (at tyrosine residue) Turns on protein activities and enhance growth EGFR activated (phosphorylated only when there is growth factors

Mutation • •

Change EGFR structure and function Mutated EGFR constantly active (no ligand binding site for growth factors)

Therapy 1. Chemotherapy (kills dividing cells) 2. Targeted therapy with tyrosine kinase inhibitors • • •

Tyrosine kinase inhibitors irreversible and competitive with ATP Major competitive inhibitors Selectively kill cells with abnormal EGFR activation

8.2 Protein changes Post-translational Protein modifications • • • • •

Genetic changes may modify primary structure → change higher order structures Problems in protein folding → disrupts protein structure and function Misfolded protein → form insoluble aggregates → cause toxic deposition → kill cells Major cause of neurodegenerative diseases Prions: misfolded infections proteins o Cause of neurogenerative diseases (eg. Mad Cow’s disease o Infect native structures of good protein into abnormal protein

8.3 Protein quantitation Bicinchoninic acid assay (BCA) • • • •

Proteins reduce Cu2+ to Cu+ BCA compounds bind with Cu+ to form purple complex More purple = more proteins Method: 1. Standard curve from known protein concentration prepared 2. Unknown sample protein concentration interpolated from the curve 3. Limitations: do not specific protein type, size, abundance etc.

Specific protein recognition • • • •

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Antibodies- family of large, Y shaped proteins that bind to any molecule Each antibody recognises a unique target(antigen) → binds at the antigen-binding site(tip of the Y-structure) Variable region confers specific recognition Constant region same for all antibodies in a group

Antibodies can be engineered/modified with enzymes o Antibodies provide the specificity o Enzymes catalyse detection signal (eg. generate light signals) Signal amplification with antibodies o Primary antibody → binds directly to target protein o Secondary antibody → binds to primary antibody’s constant region o Advantages: ▪ multiple secondary antibodies can bind to primary = more signal ▪ even with n primary antibodies, only need modify one type of secondary antibody with enzyme

Protein immunoblotting •

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Widely used analytical technique to: o Determine protein size o Identify specific proteins o Quantify specific proteins Electrophoresis principle o + molecules move to – electrode o – molecules move to + electrode o In gel matrix, rate of movement determined by charge and size of molecule

Steps:

1. Sample preparation • Mercaptoethanol (reducing agent) destroy disulphide bond • Sodium Dedocyl Sulfate (SDS, -ve charge detergent) destroy hydrogen bonds, electrostatic interactions etc. • Denatures protein → quaternary, tertiary and secondary structure disrupted • Coat protein uniformly with large -ve charge 2. Electrophoresis • Gel separation of polypeptides(from sample) by size • SDS-PAGE (Polyacrylamide gel electrophoresis) • Polypeptides put at negatively charged terminal • Gel staining of all polypeptides o Protein ladder: consist of mixture of polypeptides with specific molecular weight to serve as reference o Cell sample : consists of mixture of polypeptides with different molecular weight → multiple bands of proteins 3. Transfer to membrane • Gel is porous → only small chemicals can enter the gel to bind to polypeptides • For antibody binding, polypeptides transferred from gel to a thin membrane • Via perpendicular electric field 4. Stain for Protein • Proteins on membrane react with antibodies • Primary antibody binds to specific polypeptide (detection) • Secondary antibody bind to primary antibody to generate insoluble coloured products (signal)

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8.4

Loading control o Different samples prepared individually o Need intrinsic normalisation to account for these variations o Loading controls are high abundance, ubiquitous proteins o Ensure equal amounts of protein are loaded

LECTURE 9: ELISA • • •

Enzyme-linked immunosorbent assay Rapidly detects specific antigen 3 components: o Enzymatic amplification → allow reaction to be amplified into optical signal o Antibody → specific detection of antigens o Solid phase → allow capture and easy washing away of unbound materials

Direct ELISA

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Antigens direcly adsorbed onto a solid surface Primary antibodies added o If antigen present → antibody will bind o If antigen absent → antibody remain in solution Wash away unbound antibodies Bound enzyme used to generate detection signal (signal intensity proportional to antibody c)

Indirect ELISA

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Similar to direct ELISA, but use secondary antibody to generate signal instead

Problems of complex sample • •

Direct and indirect ELISA require antigen immobilisation onto a solid surface Non specific adsoption

Sandwich ELISA

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Uses a pair of antibodies to enrich antigens from complex samples Captured antibody is immobilised on a solid support Captured antibody binds and retains specific antigen from sample Detection antibody binds to another part of antigen → signal generation Sandwich ELISA needs antibodies that do not cross-talk(overlapping signal)

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Uses ...