Glossary

6.2 Thermodynamics of nucleic acid interactions

7 min read•august 1, 2024

Nucleic acid stability is a crucial aspect of DNA and RNA function. It's all about the delicate balance between forces that hold these molecules together and those that pull them apart. Understanding this balance helps us predict how nucleic acids behave in different conditions.

Hydrogen bonds and are the main players in nucleic acid stability. These interactions work together to keep DNA and RNA in their proper shapes, but they can be affected by things like and pH. Knowing how these factors influence stability is key for many biological processes and lab techniques.

Nucleic acid stability

Thermodynamic principles governing nucleic acid stability

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  • The stability of nucleic acid structures (DNA and RNA duplexes) can be understood through the application of thermodynamic principles (, , and )
  • The formation of nucleic acid structures is driven by the balance between enthalpic and entropic contributions to the Gibbs free energy of the system
    • Enthalpic contributions arise from the formation of hydrogen bonds and base stacking interactions, which stabilize the nucleic acid structure
    • Entropic contributions are related to the loss of conformational freedom and the release of bound water molecules upon nucleic acid structure formation, which tend to destabilize the structure
  • The stability of nucleic acid structures can be quantified by the change in Gibbs free energy (ΔG) associated with their formation, which is determined by the equation: ΔG=ΔH−TΔSΔG = ΔH - TΔS
    • ΔH is the change in enthalpy
    • ΔS is the change in entropy
    • T is the absolute temperature
  • The (Tm) of a nucleic acid structure serves as a measure of the structure's stability
    • Tm is the temperature at which half of the molecules are in the folded state and half are in the unfolded state

Nearest-neighbor model for predicting nucleic acid stability

  • The nearest-neighbor model is a widely used approach to predict the stability of nucleic acid structures based on the sequence-dependent contributions of base pair stacking interactions and loop formations
  • The model takes into account the specific interactions between adjacent base pairs in a nucleic acid sequence
  • The stability contributions of each nearest-neighbor base pair stack are derived from empirically determined thermodynamic parameters
  • The total stability of a nucleic acid structure is calculated by summing the contributions of all nearest-neighbor interactions in the sequence
  • The nearest-neighbor model allows for the prediction of the stability of various nucleic acid structures, such as duplexes, hairpins, and loops

Melting temperature calculations

Calculating the melting temperature (Tm) of DNA and RNA duplexes

  • The melting temperature (Tm) of a DNA or RNA duplex is the temperature at which half of the molecules are in the double-stranded state and half are in the single-stranded state
  • The Tm of a nucleic acid duplex can be calculated using the nearest-neighbor model, which takes into account the sequence-dependent contributions of base pair stacking interactions
  • The basic equation for calculating the Tm of a DNA duplex is: Tm=(ΔH/(ΔS+Rln⁡(C/4)))−273.15Tm = (ΔH / (ΔS + R \ln(C/4))) - 273.15
    • ΔH is the change in enthalpy
    • ΔS is the change in entropy
    • R is the gas constant
    • C is the total strand concentration
  • The ΔH and ΔS values for each nearest-neighbor base pair stack can be obtained from empirically determined tables, and the total ΔH and ΔS for the duplex are calculated by summing the contributions of each base pair stack

Factors affecting the melting temperature of nucleic acid duplexes

  • The Tm of an RNA duplex can be calculated using a similar approach, but with different nearest-neighbor parameters to account for the presence of uracil instead of thymine and the different stabilities of RNA base pairs compared to DNA
  • The Tm of a nucleic acid duplex is affected by various factors:
    • GC content: Higher GC content leads to higher Tm due to the stronger base pairing interactions between G and C
    • Length of the duplex: Longer duplexes generally have higher Tm due to increased base stacking interactions
    • Concentration of salts and other solutes in the solution: Higher salt concentrations (NaCl, KCl) tend to stabilize the duplex and increase Tm by screening the negative charges on the phosphate backbone
  • Accurate determination of Tm is crucial for designing primers, probes, and other oligonucleotides used in PCR, hybridization assays, and other molecular biology techniques

Environmental effects on nucleic acids

Effects of salt concentration on nucleic acid stability

  • The stability of nucleic acid structures is influenced by the ionic strength of the solution, as salt ions affect the electrostatic interactions between the negatively charged phosphate groups on the nucleic acid backbone
  • Increasing the salt concentration (Na+ or K+) in the solution generally stabilizes nucleic acid structures by screening the negative charges on the phosphate backbone, reducing the electrostatic repulsion between the strands
  • The relationship between salt concentration and Tm can be described by the equation: ΔTm=16.6log⁡[Na+]−16.6log⁡[Na+]refΔTm = 16.6 \log[Na+] - 16.6 \log[Na+]_{ref}
    • [Na+] is the salt concentration
    • [Na+]ref is a reference salt concentration
  • The presence of divalent cations (Mg2+) can further stabilize nucleic acid structures by forming salt bridges between the phosphate groups and by promoting the formation of higher-order structures (RNA tertiary structures)

Impact of pH on nucleic acid stability and structure

  • The pH of the solution affects the protonation state of the nucleobases, particularly cytosine and guanine, which can alter the and base stacking interactions in nucleic acid structures
  • Lowering the pH can lead to the protonation of cytosine and the formation of non-canonical base pairs (C•C+ and C•A+), which can stabilize or destabilize nucleic acid structures depending on the sequence context
  • Changes in pH can also influence the formation of alternative nucleic acid structures, such as triplex DNA, i-motifs, and G-quadruplexes, which have distinct stability and functional properties compared to canonical double-stranded structures
  • The stability of nucleic acid structures at different salt concentrations and pH values can be experimentally determined using techniques such as:
    • UV melting curves: Measuring the absorbance of nucleic acids at 260 nm as a function of temperature to determine the Tm and thermodynamic parameters
    • Circular dichroism spectroscopy: Analyzing the secondary structure and conformational changes of nucleic acids based on their differential absorption of left- and right-circularly polarized light
    • Calorimetry: Directly measuring the heat absorbed or released during the formation or melting of nucleic acid structures to determine the thermodynamic parameters

Forces driving nucleic acid stability

Hydrogen bonding in nucleic acid base pairing and stability

  • Hydrogen bonding occurs between the complementary nucleobases (A•T/U and G•C) and plays a crucial role in the formation and stability of base pairs in nucleic acid duplexes
  • The strength of hydrogen bonding depends on the number and arrangement of hydrogen bonds formed between the base pairs
    • G•C base pairs are more stable than A•T/U base pairs due to the formation of three hydrogen bonds compared to two
    • The formation of non-canonical base pairs (G•U wobble pairs in RNA) can also contribute to the stability of nucleic acid structures through hydrogen bonding
  • Hydrogen bonding is a directional and specific interaction that ensures the fidelity of base pairing and the transmission of genetic information during replication and transcription

Role of hydrophobic interactions and base stacking in nucleic acid stability

  • Hydrophobic interactions, also known as base stacking interactions, arise from the tendency of the hydrophobic nucleobases to minimize their exposure to the aqueous environment by stacking on top of each other
  • Base stacking interactions are sequence-dependent and contribute significantly to the stability of nucleic acid duplexes
    • Purine-purine stacks (A•A, G•G) are more stable than pyrimidine-pyrimidine stacks (T•T, C•C) due to their larger surface area and greater polarizability
    • The strength of base stacking interactions is influenced by factors such as the polarizability and dipole moment of the nucleobases, as well as the presence of intercalating agents (ethidium bromide) that can enhance base stacking
  • Base stacking interactions are non-directional and provide a significant to the stability of nucleic acid structures by reducing the exposure of the hydrophobic nucleobases to water

Experimental and computational methods to evaluate the contributions of hydrogen bonding and hydrophobic interactions

  • The relative contributions of hydrogen bonding and hydrophobic interactions to nucleic acid stability can be evaluated using experimental techniques, such as:
    • UV melting curves: Analyzing the temperature-dependent changes in the absorbance of nucleic acids to determine the thermodynamic parameters and the relative contributions of hydrogen bonding and base stacking
    • Calorimetry: Measuring the heat absorbed or released during the formation or melting of nucleic acid structures to determine the enthalpic and entropic contributions of hydrogen bonding and base stacking
    • NMR spectroscopy: Investigating the local structural and dynamic properties of nucleic acids at the atomic level to identify the specific hydrogen bonding and base stacking interactions
  • Computational methods, such as molecular dynamics simulations, can provide insights into the dynamic behavior and energetic contributions of hydrogen bonding and hydrophobic interactions in nucleic acid structures
    • Force fields (AMBER, CHARMM) are used to model the interactions between atoms in nucleic acids and to calculate the energy and stability of different conformations
    • Free energy calculations (MM-PBSA, MM-GBSA) can be used to estimate the relative contributions of hydrogen bonding and hydrophobic interactions to the stability of nucleic acid structures
  • The interplay between hydrogen bonding and hydrophobic interactions determines the overall stability and conformational dynamics of nucleic acid structures, and understanding these forces is crucial for predicting the behavior of nucleic acids in biological systems and for designing nucleic acid-based technologies (antisense oligonucleotides, aptamers)

Key Terms to Review (19)

Association Constant: The association constant is a measure of the strength of the interaction between two or more molecules in a binding reaction, indicating how likely they are to form a complex. A higher association constant signifies a stronger binding affinity, which is crucial for understanding interactions such as nucleic acid hybridization and protein-ligand binding. This constant plays a vital role in thermodynamic calculations and helps predict the behavior of biomolecules in various conditions.
Base stacking: Base stacking refers to the non-covalent interactions between the aromatic bases in nucleic acids, such as DNA and RNA, which contribute to the stability of their helical structures. This phenomenon occurs due to hydrophobic interactions and van der Waals forces that arise from the close proximity of the bases when they are stacked on top of one another. Base stacking plays a critical role in maintaining the structural integrity and overall thermodynamic stability of nucleic acids.
Binding Constant: The binding constant is a quantitative measure of the affinity between a ligand and a biomolecule, representing how tightly the two interact in a reversible reaction. This constant is crucial for understanding the stability and dynamics of molecular interactions, as it provides insights into how likely a ligand will bind to its target and how effectively the interaction occurs. The binding constant is influenced by factors such as temperature, concentration, and the specific chemical properties of both the ligand and the biomolecule.
Duplex stability: Duplex stability refers to the strength and durability of the double-stranded structure formed by nucleic acids, primarily DNA and RNA. This stability is crucial as it determines how well the strands can maintain their base pairing under various conditions, which is vital for processes like replication and transcription. Factors such as temperature, ionic strength, and the presence of stabilizing agents can significantly impact duplex stability, making it a key concept in understanding nucleic acid interactions.
Enthalpic contribution: Enthalpic contribution refers to the part of the total free energy change in a system that is related to the heat absorbed or released during a reaction or interaction at constant pressure. This concept is crucial for understanding how nucleic acids, such as DNA and RNA, interact through processes like base pairing and hybridization, where changes in heat indicate the strength of these interactions.
Enthalpy: Enthalpy is a thermodynamic quantity that represents the total heat content of a system, typically denoted as H. It accounts for the internal energy of the system as well as the energy required to make room for it by displacing its environment, which makes it crucial for understanding energy changes in biological reactions and processes.
Entropic contribution: Entropic contribution refers to the change in entropy associated with a reaction or interaction, which influences the spontaneity and stability of molecular complexes. In the context of molecular interactions, particularly nucleic acids, the entropic contribution arises from the degrees of freedom and configurational states available to the system. This aspect is crucial in understanding how changes in entropy can affect binding affinity and the overall thermodynamics of nucleic acid interactions.
Entropy: Entropy is a measure of the disorder or randomness in a system, reflecting the number of ways in which the system can be arranged. In biological contexts, entropy is crucial for understanding energy transformations, molecular interactions, and the behavior of biological macromolecules as they strive for equilibrium. The concept also helps explain phenomena such as the spontaneous nature of processes and the stability of complex structures like proteins and nucleic acids.
First Law of Thermodynamics: The First Law of Thermodynamics states that energy cannot be created or destroyed, only transformed from one form to another, meaning the total energy of an isolated system is constant. This principle is crucial in understanding how energy transformations occur in biological processes, as it helps explain how organisms convert food into usable energy and how this impacts their biochemical reactions.
Gibbs Free Energy: Gibbs Free Energy (G) is a thermodynamic potential that measures the maximum reversible work obtainable from a thermodynamic system at constant temperature and pressure. It is crucial in determining the spontaneity of reactions, as reactions with a negative change in Gibbs Free Energy (ΔG < 0) occur spontaneously, while those with a positive change do not.
Hydrogen bonding: Hydrogen bonding is a type of attractive interaction that occurs between a hydrogen atom covalently bonded to an electronegative atom and another electronegative atom. This relatively weak bond plays a crucial role in stabilizing the structures of biological molecules, influencing their interactions and properties. The specific geometry and strength of hydrogen bonds can greatly affect molecular behavior, impacting processes such as DNA base pairing, protein folding, and the formation of lipid bilayers.
Isothermal Titration Calorimetry: Isothermal titration calorimetry (ITC) is a technique used to measure the heat change that occurs during a chemical reaction, particularly in binding interactions. This method is widely used to determine thermodynamic parameters such as binding affinity, enthalpy, and entropy by monitoring the heat released or absorbed during the titration process. ITC is invaluable across various fields, providing insights into molecular interactions and contributing to our understanding of biological systems and drug development.
Mcghee-von hippel model: The McGhee-von Hippel model is a theoretical framework that describes how nucleic acids interact with proteins, specifically focusing on the binding of proteins to specific DNA sites. This model emphasizes the importance of the intrinsic and extrinsic factors that influence the affinity and specificity of these interactions, ultimately impacting processes like gene regulation and DNA replication.
Melting temperature: Melting temperature, often denoted as Tm, is the specific temperature at which a solid becomes a liquid, marking the transition from a crystalline state to a disordered liquid state. In the context of nucleic acids, the melting temperature is crucial as it indicates the stability of double-stranded structures, such as DNA, under varying thermal conditions. Understanding Tm allows researchers to predict how nucleic acid strands will behave in different environments, particularly during processes like hybridization and PCR.
Nearest-neighbor thermodynamic model: The nearest-neighbor thermodynamic model is a statistical approach used to understand the stability and interactions of nucleic acids based on the influence of neighboring base pairs on the free energy of formation. This model is essential for predicting how different sequences of nucleotides will behave when forming structures like DNA or RNA, as it takes into account the local context provided by adjacent bases. By evaluating the contributions of each base pair's interactions, this model helps explain the thermodynamics behind nucleic acid hybridization and stability.
Salt concentration: Salt concentration refers to the amount of salt present in a solution, typically measured in moles per liter (M). In the context of nucleic acid interactions, salt concentration plays a critical role in stabilizing the structure of nucleic acids such as DNA and RNA, influencing their binding affinities and thermodynamic properties. Higher salt concentrations can shield negative charges on the phosphate backbone of nucleic acids, reducing electrostatic repulsion and promoting more stable interactions between nucleic acid strands.
Screening effect: The screening effect refers to the phenomenon where the effective nuclear charge experienced by an electron is reduced due to the presence of other electrons in an atom. This concept is particularly relevant in understanding how nucleic acids interact, as the screening effect influences the electrostatic interactions between charged groups in nucleotides and their surroundings, affecting stability and binding affinities.
Second Law of Thermodynamics: The Second Law of Thermodynamics states that the total entropy of an isolated system can never decrease over time and is often understood in the context of energy transformations. This principle is crucial in understanding how biological systems use energy, highlighting that processes occur in a direction that increases overall disorder, thus impacting metabolic pathways, free energy changes, and interactions between biomolecules.
Uv-vis spectroscopy: UV-Vis spectroscopy is a technique that measures the absorption of ultraviolet and visible light by a substance, allowing for the analysis of its electronic transitions. This method is crucial for studying the properties of biomolecules, as it provides insight into their electronic structure and interactions, particularly in nucleic acids and other key biological compounds.
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