- Denaturation of protein describes its conversion from the physiological conformation to some other (inactive) conformation.
- Renaturation describes the reassociation of denatured complementary single strands of a DNA double helix.
- Annealing of DNA describes the renaturation of a duplex structure from single strands that were obtained by denaturing duplex DNA.
- Hybridization describes the pairing of complementary RNA and DNA strands to give an RNA-DNA hybrid.
- Heating causes the two strands of a DNA duplex to separate.
- The Tm is the midpoint of the temperature range for denaturation.
- Complementary single strands can renature when the temperature is reduced.
- Denaturation and renaturation/hybridization can occur with DNA-DNA, DNA-RNA, or RNA-RNA combinations, and can be intermolecular or intramolecular.
- The ability of two single-stranded nucleic acid preparations to hybridize is a measure of their complementarity.
The concept of base pairing is central to all processes involving nucleic acids. Disruption of the base pairs is a crucial aspect of the function of a double-stranded molecule, while the ability to form base pairs is essential for the activity of a single-stranded nucleic acid.Figure 1.16 shows that base pairing enables complementary single-stranded nucleic acids to form a duplex structure.
- An intramolecular duplex region can form by base pairing between two complementary sequences that are part of a single-stranded molecule.
- A single-stranded molecule may base pair with an independent, complementary single-stranded molecule to form an intermolecular duplex.
Formation of duplex regions from single-stranded nucleic acids is most important for RNA, but single-stranded DNA also exists (in the form of viral genomes). Base pairing between independent complementary single strands is not restricted to DNA-DNA or RNA-RNA, but can also occur between a DNA molecule and an RNA molecule.
The lack of covalent links between complementary strands makes it possible to manipulate DNA in vitro. The noncovalent forces that stabilize the double helix are disrupted by heating or by exposure to low salt concentration. The two strands of a double helix separate entirely when all the hydrogen bonds between them are broken.
The process of strand separation is called denaturation or (more colloquially) melting. ("Denaturation" is also used to describe loss of authentic protein structure; it is a general term implying that the natural conformation of a macromolecule has been converted to some other form.)
Denaturation of DNA occurs over a narrow temperature range and results in striking changes in many of its physical properties. The midpoint of the temperature range over which the strands of DNA separate is called the melting temperature (Tm). It depends on the proportion of G·C base pairs. Because each G·C base pair has three hydrogen bonds, it is more stable than an A·T base pair, which has only two hydrogen bonds. The more G·C base pairs are contained in a DNA, the greater the energy that is needed to separate the two strands. In solution under physiological conditions, a DNA that is 40% G·C—a value typical of mammalian genomes—denatures with a Tm of about 87°C. So duplex DNA is stable at the temperature prevailing in the cell.
The denaturation of DNA is reversible under appropriate conditions. The ability of the two separated complementary strands to reform into a double helix is called renaturation. Renaturation depends on specific base pairing between the complementary strands. Figure 1.17 shows that the reaction takes place in two stages. First, single strands of DNA in the solution encounter one another by chance; if their sequences are complementary, the two strands base pair to generate a short double-helical region. Then the region of base pairing extends along the molecule by a zipper-like effect to form a lengthy duplex molecule. Renaturation of the double helix restores the original properties that were lost when the DNA was denatured.
Renaturation describes the reaction between two complementary sequences that were separated by denaturation. However, the technique can be extended to allow any two complementary nucleic acid sequences to react with each other to form a duplex structure. This is sometimes called annealing, but the reaction is more generally described as hybridization whenever nucleic acids of different sources are involved, as in the case when one preparation consists of DNA and the other consists of RNA. The ability of two nucleic acid preparations to hybridize constitutes a precise test for their complementarity since only complementary sequences can form a duplex structure.
The principle of the hybridization reaction is to expose two single-stranded nucleic acid preparations to each other and then to measure the amount of double-stranded material that forms. Figure 1.18 illustrates a procedure in which a DNA preparation is denatured and the single strands are adsorbed to a filter. Then a second denatured DNA (or RNA) preparation is added. The filter is treated so that the second preparation can adsorb to it only if it is able to base pair with the DNA that was originally adsorbed. Usually the second preparation is radioactively labeled, so that the reaction can be measured as the amount of radioactive label retained by the filter.
The extent of hybridization between two single-stranded nucleic acids is determined by their complementarity. Two sequences need not be perfectly complementary to hybridize. If they are closely related but not identical, an imperfect duplex is formed in which base pairing is interrupted at positions where the two single strands do not correspond.