🧪Biophysical Chemistry Unit 6 – Nucleic Acid Structure & Stability

Key Concepts

• Nucleic acids DNA and RNA store, transmit, and express genetic information in living organisms
• Nucleotides are the building blocks of nucleic acids consisting of a sugar, phosphate, and nitrogenous base
• Complementary base pairing through hydrogen bonding enables the double helix structure of DNA
• DNA typically exists as a right-handed double helix with major and minor grooves
• Various factors influence the stability of nucleic acid structures including temperature, pH, and ionic strength
• Advanced structures beyond the canonical double helix include triplexes, quadruplexes, and Z-DNA
• Experimental techniques such as X-ray crystallography and NMR spectroscopy elucidate nucleic acid structures
• Understanding nucleic acid structure and stability has profound implications for fields like genetics, biotechnology, and medicine

DNA and RNA Basics

• DNA (deoxyribonucleic acid) is the primary genetic material in most organisms while RNA (ribonucleic acid) plays crucial roles in gene expression
• DNA is composed of deoxyribose sugar, phosphate backbone, and four nitrogenous bases: adenine (A), thymine (T), guanine (G), and cytosine (C)
• RNA contains ribose sugar instead of deoxyribose and uracil (U) in place of thymine
• DNA is typically double-stranded and more stable while RNA is usually single-stranded and more prone to degradation
• The directionality of nucleic acid strands is denoted as 5' to 3' based on the carbon positions in the sugar
• DNA primarily serves as the long-term storage of genetic information while RNA acts as a messenger and plays functional roles (mRNA, tRNA, rRNA)
• The central dogma of molecular biology describes the flow of genetic information from DNA to RNA to proteins

Nucleotide Structure

• Nucleotides are the monomeric units of nucleic acids composed of three main components: a pentose sugar, a phosphate group, and a nitrogenous base
• The pentose sugar is either deoxyribose (in DNA) or ribose (in RNA) with the difference being the presence or absence of a hydroxyl group at the 2' position
• The phosphate group is attached to the 5' carbon of the sugar and enables the formation of the sugar-phosphate backbone through phosphodiester bonds
• Nitrogenous bases are heterocyclic aromatic compounds classified as purines (adenine and guanine) or pyrimidines (cytosine, thymine, and uracil)
  • Purines have a double-ring structure derived from adenine and guanine
  • Pyrimidines have a single-ring structure and include cytosine, thymine (in DNA), and uracil (in RNA)
• The nitrogenous base is attached to the 1' carbon of the sugar via a glycosidic bond
• Nucleosides are nucleotides without the phosphate group and consist of just the sugar and base

Base Pairing and Hydrogen Bonding

• Complementary base pairing between purines and pyrimidines is a fundamental aspect of nucleic acid structure and function
• In DNA, adenine (A) pairs with thymine (T) through two hydrogen bonds while guanine (G) pairs with cytosine (C) through three hydrogen bonds
• In RNA, uracil (U) replaces thymine and pairs with adenine (A) through two hydrogen bonds
• Hydrogen bonds are electrostatic attractions between a hydrogen atom bonded to an electronegative atom (like nitrogen or oxygen) and another electronegative atom
• The specificity and directionality of hydrogen bonding between complementary bases enable the formation of stable double-stranded structures
• Proper base pairing is crucial for processes like DNA replication, transcription, and translation
  • Errors in base pairing can lead to mutations and potentially deleterious effects on cellular function
• The difference in hydrogen bonding patterns between A-T (two bonds) and G-C (three bonds) affects the stability and melting properties of DNA

Double Helix Structure

• The iconic double helix structure of DNA was elucidated by James Watson and Francis Crick in 1953 based on X-ray crystallography data from Rosalind Franklin and Maurice Wilkins
• DNA typically exists as a right-handed double helix with two antiparallel polynucleotide strands wound around a central axis
• The sugar-phosphate backbones are on the outside of the helix while the nitrogenous bases face inward and engage in complementary base pairing
• The double helix has a diameter of approximately 20 Å and makes a complete turn every 34 Å, or about every 10 base pairs
• The spacing between adjacent base pairs is about 3.4 Å, resulting in a compact and efficient packaging of genetic information
• The double helix structure gives rise to major and minor grooves that differ in size and accessibility
  • The major groove is wider and more accessible to proteins like transcription factors
  • The minor groove is narrower and can be a target for small molecule binding
• The double helix structure provides stability, protection, and a mechanism for the faithful replication and transmission of genetic information

Factors Affecting Stability

• The stability of nucleic acid structures is influenced by various physical and chemical factors
• Temperature plays a significant role, with higher temperatures leading to denaturation or melting of the double helix
  • The melting temperature (Tm) is the point at which half of the DNA is denatured and depends on factors like GC content and ionic strength
• pH affects the protonation of nitrogenous bases and can alter their hydrogen bonding and stacking interactions
  • Extremes of pH (highly acidic or alkaline conditions) can lead to depurination or depyrimidination and strand breakage
• Ionic strength, particularly the concentration of cations like Na+ and Mg2+, influences the electrostatic repulsion between the negatively charged phosphate groups
  • Higher ionic strength tends to stabilize the double helix by shielding the negative charges
• Hydrophobic interactions between the planar aromatic bases contribute to the stability of the double helix through base stacking
• Modifications to the nucleotide structure, such as methylation or oxidative damage, can affect the stability and function of nucleic acids
• Interactions with proteins (like histones or single-stranded binding proteins) and small molecules (like intercalators or groove binders) can modulate nucleic acid stability

Advanced Structures and Conformations

• Beyond the canonical B-form DNA double helix, nucleic acids can adopt various advanced structures and conformations
• A-form DNA is a wider, more compact helix with a shorter rise per base pair and is favored under dehydrating conditions or in RNA-DNA hybrids
• Z-DNA is a left-handed helix with a zig-zag phosphate backbone and alternating purine-pyrimidine sequence that can form under certain conditions (high salt, methylation, negative supercoiling)
• Triple helix (triplex) structures can form when a third strand binds to the major groove of a DNA duplex through Hoogsteen hydrogen bonding
  • Triplex-forming oligonucleotides (TFOs) have potential applications in gene regulation and targeted mutagenesis
• Quadruplex structures (G-quadruplexes) can form in guanine-rich sequences through the stacking of planar G-tetrads stabilized by monovalent cations (like K+)
  • G-quadruplexes are found in telomeric regions and promoters and may play regulatory roles in transcription and replication
• i-Motifs are four-stranded structures that can form in cytosine-rich sequences under acidic conditions through intercalated C-C+ base pairs
• Hairpins, cruciforms, and pseudoknots are examples of secondary structures that can form within single-stranded regions of nucleic acids

Experimental Techniques

• Various experimental techniques are used to study the structure, stability, and interactions of nucleic acids
• X-ray crystallography provides high-resolution three-dimensional structures of nucleic acids and their complexes with proteins or small molecules
  • Requires the growth of high-quality crystals and the generation of diffraction patterns to solve the structure
• Nuclear magnetic resonance (NMR) spectroscopy allows the determination of nucleic acid structures in solution and provides dynamic information
  • Relies on the magnetic properties of certain nuclei (like 1H, 13C, and 31P) and their chemical environment
• Circular dichroism (CD) spectroscopy measures the differential absorption of left- and right-circularly polarized light and is sensitive to the secondary structure and conformation of nucleic acids
• Thermal denaturation (melting curve) analysis monitors the absorbance at 260 nm as a function of temperature to determine the melting temperature and thermodynamic parameters of nucleic acid structures
• Footprinting techniques (like DNase I or hydroxyl radical footprinting) probe the accessibility and protein binding sites on nucleic acids
• Single-molecule techniques (like atomic force microscopy or fluorescence resonance energy transfer) enable the manipulation and visualization of individual nucleic acid molecules
• Computational methods (like molecular dynamics simulations and structure prediction algorithms) complement experimental approaches in understanding nucleic acid structure and dynamics

Real-World Applications

• Understanding the structure and stability of nucleic acids has profound implications for various fields and real-world applications
• In genetics and genomics, knowledge of DNA structure and function is crucial for understanding inheritance, genetic variation, and disease
  • Techniques like DNA sequencing, genotyping, and genetic engineering rely on the principles of base pairing and stability
• In biotechnology and synthetic biology, the ability to design and manipulate nucleic acid structures enables the development of new tools and products
  • Examples include DNA origami, aptamers, and CRISPR-based gene editing
• In medicine, nucleic acid structure and stability are relevant for diagnosing and treating genetic disorders, infections, and cancer
  • Antisense oligonucleotides, siRNAs, and mRNA vaccines are examples of therapeutic approaches that target or exploit nucleic acid structures
• In forensic science, DNA profiling based on short tandem repeats (STRs) and other markers relies on the stability and reproducibility of DNA structures
• In nanotechnology, the programmability and self-assembly properties of nucleic acids are harnessed for creating nanoscale structures and devices
  • DNA-based nanomachines, sensors, and computing elements are active areas of research
• In origin of life studies and astrobiology, understanding the structure and stability of nucleic acids is crucial for elucidating the emergence and evolution of genetic systems
  • The RNA world hypothesis posits that RNA played a central role as both genetic material and catalyst in early life forms