🔬Biological Chemistry I Unit 11 – Nucleotides and Nucleic Acids

What Are Nucleotides?

• Nucleotides are the fundamental building blocks of nucleic acids (DNA and RNA)
• Consist of three components: a nitrogenous base, a pentose sugar, and a phosphate group
• Nitrogenous bases include purines (adenine and guanine) and pyrimidines (cytosine, thymine, and uracil)
• Pentose sugar is either deoxyribose (in DNA) or ribose (in RNA)
• Phosphate group is attached to the 5' carbon of the pentose sugar
• Nucleotides are joined together by phosphodiester bonds to form nucleic acid polymers
• Play crucial roles in storing and transmitting genetic information, as well as in various cellular processes (ATP, cAMP, and coenzymes)

Building Blocks: Nucleotide Structure

• Nitrogenous bases are heterocyclic aromatic compounds that form hydrogen bonds with complementary bases
  • Purines have a double-ring structure and include adenine (A) and guanine (G)
  • Pyrimidines have a single-ring structure and include cytosine (C), thymine (T), and uracil (U)
• Pentose sugar is a five-carbon monosaccharide that forms the backbone of nucleic acids
  • Deoxyribose lacks a hydroxyl group at the 2' position compared to ribose
• Phosphate group is a negatively charged moiety that contributes to the acidic nature of nucleic acids
• Nucleosides are formed when a nitrogenous base is attached to a pentose sugar (e.g., adenosine, guanosine)
• Nucleotides are nucleosides with one, two, or three phosphate groups attached to the 5' carbon of the pentose sugar
• The 5' and 3' carbons of the pentose sugar are important for forming the phosphodiester bonds that link nucleotides together

Types of Nucleic Acids: DNA and RNA

• DNA (deoxyribonucleic acid) is a double-stranded nucleic acid that stores genetic information
  • Contains deoxyribose sugar and the nitrogenous bases adenine, guanine, cytosine, and thymine
  • Follows base pairing rules: A pairs with T, and G pairs with C
• RNA (ribonucleic acid) is a single-stranded nucleic acid that plays various roles in gene expression and regulation
  • Contains ribose sugar and the nitrogenous bases adenine, guanine, cytosine, and uracil (instead of thymine)
  • Follows base pairing rules: A pairs with U, and G pairs with C
• Both DNA and RNA are composed of nucleotides joined by 3'-5' phosphodiester bonds
• DNA is more stable than RNA due to the absence of the 2' hydroxyl group in deoxyribose
• RNA is more versatile and can form complex secondary structures (hairpins, loops, and pseudoknots)

DNA Structure: The Double Helix

• DNA is a double-stranded helix with two antiparallel polynucleotide chains
• The sugar-phosphate backbones are on the outside, while the nitrogenous bases face inward
• Nitrogenous bases form hydrogen bonds with their complementary bases on the opposite strand (A-T and G-C)
• The double helix has a right-handed twist and makes a complete turn every 10.5 base pairs
• The diameter of the double helix is approximately 2 nm, and the distance between adjacent base pairs is 0.34 nm
• Major and minor grooves are formed due to the asymmetric spacing of the sugar-phosphate backbones
  • Major groove is wider and deeper, allowing proteins to interact with specific DNA sequences
  • Minor groove is narrower and shallower, providing a binding site for some small molecules (antibiotics and regulatory proteins)
• The double helix structure provides stability and protection for the genetic information stored in DNA

RNA: Single-Stranded and Versatile

• RNA is a single-stranded nucleic acid that can fold into complex secondary structures
• The presence of the 2' hydroxyl group in ribose makes RNA more susceptible to hydrolysis compared to DNA
• RNA can form intramolecular base pairs, leading to the formation of hairpins, loops, and pseudoknots
• These secondary structures are important for RNA function and interaction with other molecules
• There are several types of RNA, each with specific roles in the cell:
  • Messenger RNA (mRNA) carries genetic information from DNA to ribosomes for protein synthesis
  • Transfer RNA (tRNA) delivers amino acids to ribosomes during translation
  • Ribosomal RNA (rRNA) is a structural and catalytic component of ribosomes
  • Small nuclear RNA (snRNA) is involved in splicing pre-mRNA to form mature mRNA
  • MicroRNA (miRNA) and small interfering RNA (siRNA) regulate gene expression through RNA interference
• RNA can also function as enzymes (ribozymes) that catalyze chemical reactions, such as self-splicing and peptide bond formation

Nucleotide Functions Beyond DNA/RNA

• Nucleotides play essential roles in various cellular processes beyond their function as building blocks of nucleic acids
• Adenosine triphosphate (ATP) is the primary energy currency of the cell
  • Hydrolysis of ATP to ADP + Pi releases energy that drives many cellular reactions
• Cyclic AMP (cAMP) and cyclic GMP (cGMP) are important second messengers in signal transduction pathways
  • Regulate the activity of protein kinases and ion channels
• Nucleotide derivatives serve as coenzymes in metabolic reactions
  • Nicotinamide adenine dinucleotide (NAD+) and its phosphorylated form (NADP+) are involved in redox reactions
  • Flavin adenine dinucleotide (FAD) is a cofactor for many oxidoreductases
• Nucleotides are precursors for the synthesis of other important biomolecules
  • Uridine diphosphate glucose (UDP-glucose) is a precursor for glycogen synthesis
  • Cytidine diphosphate diacylglycerol (CDP-DAG) is a precursor for phospholipid synthesis
• Nucleotides are also involved in the regulation of enzyme activity and gene expression
  • Allosteric regulation of enzymes by ATP, ADP, and AMP
  • Riboswitches are RNA elements that bind specific nucleotides and regulate gene expression

Lab Techniques: Working with Nucleic Acids

• Extraction and purification of DNA and RNA from biological samples
  • Phenol-chloroform extraction and ethanol precipitation
  • Column-based purification kits
• Quantification of nucleic acids using spectrophotometry (absorbance at 260 nm)
• Agarose gel electrophoresis to separate DNA fragments based on size
  • Visualization of DNA bands using ethidium bromide or other intercalating dyes
• Polymerase chain reaction (PCR) to amplify specific DNA sequences
  • Uses a heat-stable DNA polymerase (Taq) and specific primers
• Reverse transcription PCR (RT-PCR) to convert RNA to cDNA for analysis
• DNA sequencing methods to determine the nucleotide sequence of DNA
  • Sanger sequencing using dideoxy chain termination
  • Next-generation sequencing (NGS) technologies for high-throughput sequencing
• Cloning and recombinant DNA technology to manipulate and express genes
  • Restriction enzymes to cut DNA at specific sites
  • DNA ligase to join DNA fragments
  • Plasmid vectors for cloning and expression in bacteria
• RNA interference (RNAi) to knock down gene expression using siRNA or shRNA
• CRISPR-Cas9 genome editing to make precise changes to DNA sequences

Real-World Applications and Research

• Forensic science and DNA fingerprinting for criminal investigations and paternity testing
• Personalized medicine and pharmacogenomics to tailor treatments based on an individual's genetic profile
• Genetic testing for inherited disorders and predisposition to certain diseases (BRCA1/2 for breast cancer)
• Genetically modified organisms (GMOs) for agriculture and biotechnology
  • Crops with improved yield, resistance to pests, and enhanced nutritional value
  • Production of recombinant proteins and drugs in genetically engineered bacteria or mammalian cells
• Gene therapy to treat genetic disorders by introducing functional copies of genes into cells
• Molecular diagnostics and pathogen detection using PCR and DNA sequencing
  • Identification of infectious agents (viruses, bacteria, and fungi)
  • Monitoring of viral load in HIV and hepatitis patients
• Ancient DNA analysis to study the evolution and migration of species, including humans
• Metagenomics to study the genetic diversity of microbial communities in various environments (gut microbiome, soil, and oceans)
• Synthetic biology and the design of artificial genetic circuits for novel applications (biosensors, biofuels, and biomaterials)