Week 10 - Nucleic Acid Chem + Techniques

Ribonucleotides

• structural units of RNA = ribonucleoside 5′-monophosphates.

• Ribonucleotide nomenclature, symbols, and corresponding nucleosides:

  • Adenylate (adenosine 5′-monophosphate) | Symbols: A, AMP | Nucleoside: Adenosine.

  • Guanylate (guanosine 5′-monophosphate) | Symbols: G, GMP | Nucleoside: Guanosine.

  • Uridylate (uridine 5′-monophosphate) | Symbols: U, UMP | Nucleoside: Uridine.

  • Cytidylate (cytidine 5′-monophosphate) | Symbols: C, CMP | Nucleoside: Cytidine.

RNAs Types + Functions

• Major cellular RNA classes:

  • Ribosomal RNAs (rRNAs): Components of ribosomes required for protein synthesis.

  • Messenger RNAs (mRNAs): Intermediaries carrying genetic information from one or a few genes to a ribosome for protein synthesis.

  • Transfer RNAs (tRNAs): Adapter molecules that translate information in mRNA into a specific sequence of amino acids.

• Additional specialized types of RNA produced in cells:

  • snRNA: Functions in various nuclear processes (e.g., splicing).

  • snoRNA: Facilitates chemical modification of RNAs.

  • miRNA: Regulates gene expression.

  • siRNA: Silences gene expression.

  • lncRNA: Regulates gene expression.

RNA Primary Structure

• mRNA is always single-stranded + right-handed helical conformation.

• Base-stacking interactions are strongest between two purines.

• Can base pair with complementary regions of DNA or RNA.

• Paired strands run antiparallel.

RNA Secondary Structure

• Complementary RNA strands is an A-form right-handed double helix.

• Breaks caused by mismatched or unmatched bases —> in bulges or internal loops.

• Internal loops form between palindromic sequences.

• Extensive base-paired helical segments form in RNAs.

• Hairpins are the most common type of secondary structure.

RNA (3D) Tertiary Structure

• Multiple secondary structures and unusual base-pairing forms create the 3D structure of rRNA, mRNA, and tRNA.

Messenger RNA (mRNA)

• synthesized by DNA transcription.

• mRNA sequence is complementary and antiparallel to the template strand of DNA.

• Functions as the blueprint for protein synthesis (translation) by encoding amino acid sequences.

Ribosomal RNA (rRNA)

• Integral components of ribosomes —> serve as cellular machinery for protein synthesis.

• In translation, the genetic codes of mRNA are converted into functional proteins.

• Highly conserved across species, underscoring their fundamental role in translation.

• Many antibiotics target bacterial ribosomes, disrupting protein synthesis (tetracyclin)

Transfer RNA (tRNA)

• tRNA has a distinctive cloverleaf-like structure containing 4 critical regions:

  1. Amino acid arm.

  2. D arm.

  3. Anticodon arm.

  4. T or TψC arm.

• Amino acid arm: Carries a specific amino acid esterified by its carboxyl group to the 2′-OH or 3′-OH group of the A residue at the 3′ end of the tRNA.

• Anticodon arm: Contains the anticodon sequence.

• D arm: Helps in the proper folding and stabilization of the tRNA structure.

• T or TψC arm: Contains thymine (T) or pseudouridine (Ψ), involved in binding tRNA to the ribosome during protein synthesis.

The Wobble Hypothesis

• Proposes that the first base of the anticodon (5′ → 3′) pairs loosely with the corresponding codon base.

• Loose pairing permits rapid dissociation of the tRNA from its codon during protein synthesis.

• Rules of the Wobble Hypothesis:

  • First base U can recognize A or G.

  • First base G can recognize U or C.

  • First base I (inosine) can recognize U, C, or A.

Genetic Information Flow

DNA Denaturation + Heat

• Denaturation: Defined as the unwinding of the double helix into two single strands.

• Induced by pH extremes or high temperatures.

• Process disrupts hydrogen bonds and base-stacking interactions between strands.

• Annealing: Rewinding of separated complementary strands when temperature or pH is returned to physiological ranges (occurs in 2 steps).

• Denaturation temperature (tm): The temperature at which 50% of double-stranded DNA is converted to single strands.

• (tm) increases proportionally with higher(G triple bond C) base pair content.

DNA Partial Denaturation + UV Absorbance Effects

• Partially denatured regions within DNA form bubbles, which are typically rich in A=T base pairs.

• Hypochromic effect: Observed decrease in UV light absorption when complementary strands pair into a double helix.

• Hyperchromic effect: Observed increase in UV light absorption when double-stranded nucleic acid undergoes denaturation.

• 260 nm = transition from double-stranded to single-stranded DNA.

RNA Denaturation

• RNA duplexes exhibit higher heat denaturation stability than DNA duplexes.

• RNA-DNA hybrid duplexes generally possess intermediate stability.

DNA Non-Enzymatic Transformations

• Definition: Spontaneous chemical modifications occurring in DNA without enzyme catalysis.

• Common Types of Transformations:

  1. Mutation

  2. Deamination (removal of an exocyclic amino group)

  3. Depurination (loss of a purine base)

  4. Pyrimidine dimers (cross-linking caused by UV light)

  5. Oxidation (damage from reactive oxygen species)

  6. Methylation (\(-\text{CH}_3\))

• Causes: Exposure to chemical reagents, radiation, or reactive oxygen species (ROS) produced during cellular metabolism.

Mutations + Alkylating Agents

• Mutations: Structural alterations in DNA producing permanent changes in encoded genetic information.

• Strongly linked to aging processes and carcinogenesis.

• Alkylating agents: Modified nucleotides and chemical agents that add alkyl groups

Deamination + Depurination

Deamination: Spontaneous loss of exocyclic amino groups from bases.

Depurination: Hydrolysis of the N-beta-glycosyl bond connecting the base to the pentose sugar.

Pyramidine Dimers

• Pyrimidine dimers: Covalent bond formation between adjacent pyrimidine bases (T or C) caused by UV radiation.

• Forms Thymine-Thymine, Cytosine-Cytosine, or Thymine-Cytosine dimers.

• Structural types include Cyclobutane dimers or 6-4 photoproducts.

• Abnormal bonds introduce structural kinks that distort the DNA backbone, causing replication errors.

Oxidation

• Reactive Oxygen Species (ROS): Metabolic byproducts including hydrogen peroxide, hydroxyl radicals, and superoxide radicals.

• Guanine most susceptible to oxidative damage, lowest one-electron reduction potential among DNA bases

• Cellular organisms maintain elaborate defence systems against oxidative stress.

Enzymatic Transformations: Methylation

• Adenine (A) and Cytosine (C) residues undergo enzymatic methylation (CH₃) more frequently than Guanine (G) or Thymine (T).

• DNA methylases transfer methyl groups using S-adenosylmethionine as a methyl donor.

• In eukaryotic genomes, 5% of cytidine residues are methylated.

• Biological functions: DNA replication, gene expression regulation, development, cell differentiation, and epigenetic control.

Chemical Synthesis of DNA (Phosphonamidite Method)

• Phosphonamidite method: Automated chemical approach for oligonucleotide synthesis.

• Nucleotides are added sequentially in the 3' —> 5' direction.

• Solid-Phase Synthesis: Performed on a solid support matrix, allowing rapid separation of the growing DNA chain from reaction byproducts.

• Four-Step Reaction Cycle:

  1. Detritylation

  2. Activation and coupling

  3. Capping

  4. Oxidation

• Features high yield, precision, and efficiency for producing custom DNA sequences.

Polymerase Chain Reaction (PCR)

• PCR: In vitro method used to exponentially amplify targeted DNA segments.

• Relies on DNA polymerases to synthesize new strands from dNTPs using a DNA template.

• DNA polymerases attach new nucleotides to the 3' ends of preexisting oligonucleotides called primers.

• Three Core Thermal Steps (1 Cycle):

  1. Denaturation (95°C): Separates template strands.

  2. Annealing (55°C): Synthetic primers bind to template strands.

  3. Extension (72°C): Thermostable Taq DNA polymerase synthesizes new strands in the 5' —> 3' direction.

• Capable of detecting and amplifying a single DNA molecule.

• Key Applications:

  • Amplification of rare DNA from ancient samples (40,000+ years old).

  • Tracing evolutionary relationships.

  • Forensic investigations.

  • Asymptomatic detection of viral infections and oncogenic mutations.

  • Prenatal genetic disease diagnosis.

DNA Sequencing

• Sanger Sequencing:

  • Known as dideoxy chain-termination sequencing.

  • Uses modified dideoxynucleoside triphosphates (ddNTPs) lacking a 3'-OH group to terminate strand extension.

  • Fragments are separated by size using agarose gel electrophoresis.

• Automated DNA Sequencing:

  • Each of the four ddNTPs is tagged with a distinct fluorescent dye.

  • Uses capillary gel electrophoresis paired with laser detection to record sequence chromatograms.

• Next-Generation Sequencing (NGS):

  • Illumina NGS: Delivers 300 bp read lengths, 300 Gb per run, with high accuracy.

  • Nanopore Sequencing: Reads up to 4 Mb, 48 Gb per run, with reasonable accuracy.