CONFORMATION OF NUCLEIC ACIDS Nucleic acids can adopt different conformations.

B-DNA is found at low salt concentrations. It is believed to be the native conformation occurring in chromatin. In the cell nucleus DNA is complexed with about an equivalent mass of protein to form a structure known as chromatin. Chromatin is a periodic structure made up of repeating, regularly spaced subunits, the subunit being the nucleosome. Within the nucleosomes the major part of DNA is wrapped around histones. The remaning DNA joining each nucleosome is known as linker DNA. Recently, the X-ray crystal structure of the nucleosome core particle of chromatin has been determined. It shows in atomic detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it (Luger et al., Nature 1997, 389, 251260). The PDB code of this structure is 1aoi.

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In solutions with higher salt concentrations or with alcohol added A-DNA is found. Z-DNA occurs for alternating poly(dG-dC) sequences in solutions with high salt concentrations or alcohol. RNA occurs (contrary to DNA) almost exclusively in the A-conformation (or in a related A'-form). There are further nucleic acid conformations like C-DNA, H-DNA or others which are not discussed here.

Geometrical features: The distance between two subsequent base pairs along the helical axis is called helical rise (h).The pitch (p) is the length of the helix axis for one complete helix turn. The turn angle per nucleotide or twist angle (t) is given by 360 / number of nucleotides per turn. C2'-endo and C3'-endo are descriptions of sugar conformations. The most frequently occurring nucleic acid model conformations are characterized by the following geometrical parameters :
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A-DNA o right-handed helix; sugar pucker: C3'-endo; number of nucleotides per pitch: 11; h: 2.56 ; t: +32.7. B-DNA o right-handed helix; sugar pucker: C2'-endo; number of nucleotides per pitch: 10; h: 3.38 ; t: +36 . Z-DNA o left-handed helix; G: syn conformation; sugar pucker: C3'-endo; C: anti conformation, sugar pucker: C2' endo; number of nucleotides per pitch: 6x2; h: 3.7x2 ; t= -30x2 (for Z-DNA the repeat unit is the dimer (G-C).

These geometrical features lead to different widths and depths of the minor and major grooves of the nucleic acid double helix (data from Jeffrey, Saenger, Hydrogen Bonding in Biological Structures, Springer-Verlag, 1991, Table 20.1, p. 401). Groove Width Groove Depth Major Minor Major Minor A-DNA 2.7 11.0 13.5 2.8 Z-DNA 11.7 5.7 8.5 7.5

Nucleic Acid Stability

Nucleic acid structures are stabilized by non-covalent intramolecular interactions between the bases. All biological processes involving DNA and RNA require these structures to be in the stable and in the appropriate conformation. It is important to know how nucleic acids form their biologically active states and how these active states are stabilized. There have been rapid advances in structural biology and relating structure to biochemical function and mechanism. However, knowledge of nucleic acid structure alone does not ensure accurate prediction of stability, function and biological activity. The complete characterization of any biomolecule requires stability determination and the forces which lead to stability and correct folding. Differential Scanning Calorimetry (DSC) is a powerful analytical tool which directly measures the stability and unfolding of biomolecules. In DSC, the sample is heated at a constant rate, and there is a detectable heat change associated with thermal denaturation. A single DSC experiment can determine:
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Transition midpoint (T m) Enthalpy (H) and heat capacity change (Cp) associated with uncoiling Presence of multiple melting site domains

A nucleic acid in aqueous solution is in equilibrium between the native conformation and its uncoiled conformation. The stability of the native state is based on the magnitude of the Gibbs free energy (G) of the system and the thermodynamic relationships between enthalpy (H) and entropy (S) changes. A positive G indicates the native state is more stable than the denatured state the more positive the G, the greater the stability. For a DNA molecule to melt, stabilizing forces need to be broken. The transition midpoint (T m) is the temperature where 50% of the DNA is in its native confirmation and the other 50% is melted. In general, the higher the Tm, the more stable the DNA. DSC measures H due to heat denaturation. Nucleic acid unfolding is typically endothermic. During the same experiment, DSC also measures the change in heat capacity (Cp) for denaturation. Many factors are responsible for the stability of nucleic acids, including hydrogen bonding, conformational entropy, and the physical environment (pH, buffer, ionic strength, excipients, etc.). DSC data, either used alone or in conjunction with stability and structural data, can provide information on:
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Effects of DNA and RNA sequence Effects of buffer, pH, salt, additives Duplex, triplex and quadruplex structures Formation of RNA and DNA complexes Formation of nucleic acid-protein complexes Effects of small molecule drugs on nucleic acid stability

Protein Folding & Stability

Protein structures are stabilized by non-covalent intramolecular interactions between amino acid side chains. Protein complexes are also formed by specific non-covalent intermolecular interactions. All biological processes depend on proteins being stable and in the appropriate folded conformation. It is important to know how proteins fold into their biologically active states, and how these active states are stabilized. A primary goal of protein engineering, rational drug design and biopharmaceutical production is the development, production, and storage of stable proteins with full functionality. There have been rapid advances in structural biology and relating structure to biochemical function and mechanism. However, knowledge of protein structure alone does not ensure accurate prediction of stability, function and biological activity. The complete characterization of any protein requires stability determination and the forces which lead to stability and correct folding. Differential Scanning Calorimetry (DSC) is a powerful analytical tool which directly measures the stability and unfolding of a protein. In DSC, the protein is heated at a constant rate, and there is a detectable heat change associated with thermal denaturation A single DSC experiment can determine:
y y y

Transition midpoint (T m) Enthalpy ( H) and heat capacity change ( Cp) associated with unfolding Presence of multiple unfolding domains

A protein in aqueous solution is in equilibrium between the native (folded) conformation and its denatured (unfolded) conformation. The stability of the native state is based on the magnitude of the Gibbs free energy ( G) of the system and the thermodynamic relationships between enthalpy ( H) and entropy ( S) changes. A positive G indicates the native state is more stable than the denatured state the more positive the G, the greater the stability. For a protein to unfold, stabilizing forces need to be broken. Conformational entropy overcomes stabilizing forces allowing the protein to unfold at temperatures where entropy becomes dominant. The transition midpoint T m is the temperature where 50% of the protein is in its native confirmation, and the other 50% is denatured. In general, the higher the Tm, the more stable the protein. Proteins which are more stable are less susceptible to unfolding and precipitation. DSC measures H of unfolding due to heat denaturation. Protein unfolding is typically endothermic. During the same experiment, DSC also measures the change in heat capacity ( Cp) for denaturation. Heat capacity changes associated with protein unfolding are primarily due to changes in hydration of side chains that were buried in the native state, but become solvent exposed in the denatured state. Many factors are responsible for the folding and stability of native proteins, including hydrophobic interactions, hydrogen bonding, conformational entropy, and the physical environment (pH, buffer, ionic strength, excipients, etc.).DSC data, either used alone or in conjunction with sequence, stability and structural data, can provide information on:
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Effects of protein mutagenesis and engineering Effects of buffer, pH, salt, additives Effects of post-translational modification Reversibility of unfolding, by changes in H Presence of multiple unfolding domains Stability contributions of individual domains Formation of protein complexes Effects of lipid, nucleic acid, or other biopolymer on protein stability

Protein stability characterization is a critical element of Biopharmaceutical and Vaccines Development. Using Tm data from DSC, one can design and select the most stable engineered protein/variant, optimize process development, and screen for the most stable liquid formulations.