The results of hybridization reactions of nucleic acids depend on the solution conditions under which they are performed. Some conditions will favor hybridization of the probe nucleic acid to the target nucleic acid. Low stringency conditions favor duplex formation. High stringency conditions destabilize the duplex, thereby selecting only the most stable duplexes. Under high stringency solution conditions, probes will bind only to regions of high sequence homology. Under low stringency conditions, the requirement for sequence specificity is relaxed, and significant numbers of mismatches, bulges, or hairpins may be tolerated. Regions of the target nucleic acid can be discriminated as a function of sequence homology by adjusting the stringency of the hybridization reaction.
Typically temperature, salt concentration, and chemical denaturant concentration are adjusted to optimize the stability of probe-target nucleic acid duplexes. Duplex stability decreases with decreasing salt concentration, with increasing temperature, and with increasing denaturant concentration. Selection of the appropriate level of stringency requires consideration of the influence of the various adjustable conditions on duplex stability.
Several factors, in addition to the base composition, sequence, and length, affect the stability of nucleic acid complexes. These factors include environmental variables such as temperature, the nature and concentration of salts, concentration of added denaturant, and in some cases pH. The concentration of nucleic acid strands is a significant determinant of thermal stability when oligonucleotides are involved in the equilibrium.
The influence of cations on nucleic acid complex stability may be due to specific interactions or to nonspecific binding. Nonspecific, or "territorial", binding is characterized by an elevated cation concentration over that in the bulk solution in the vicinity of the nucleic acid. This type of binding, which is also called "counterion condensation," occurs when the linear charge density of a polymer exceeds a critical value.
Both single-stranded and higher order complexes of nucleic acids possess condensed counterions. The linear charge density of the isolated strands is less than 1/n (where n is the molecularity) of the linear charge density of the complex, due to elongation of the single strands. Therefore, the complex binds more cations than do the isolated strands. When the strands separate, the excess cations are released into solution. As the bulk cation concentration increases, this cation release is increasingly unfavorable. Thus, more energy is required to effect the transition, and the complex is stabilized. This effect depends on the counterion valence, with higher valence counterions promoting greater stabilization than monovalent ions at the same concentration.
Because of the large negative charge of nucleic acid duplexes, anions seldom bind strongly to these molecules. The influence of anions is exerted via effects on cations and on water structure. Anion effects are usually small; at very high concentration, however, chaotropic anions can reduce the thermal stability of DNA.
Chemical denaturants are used in hybridization reactions to reduce the Tm into an experimentally convenient range. This is particularly important for experiments using RNA, which is degraded readily at high temperature. Formamide is used frequently as a chemical denaturant because it does not react with the DNA to make or break chemical bonds, it reduces the Tm of duplexes significantly at reasonable concentrations, it is readily miscible in water, and it has desirable optical properties (low absorbance in the UV region relative to alternatives).
Because neither the nucleic bases nor the sugar-phosphate backbone have titratable groups near neutral pH, the stabilities of nucleic acid complexes are typically insensitive to pH over a wide range. The primary exception is the pyrimidine-purine-pyrimidine triple helix . Formation of this complex is accompanied by protonation of the third-strand cytosine residues. This protonation equilibrium is coupled to triplex formation and therefore influences the stability of the complex; it can increase the apparent pKa of the cytosine by more than 2 pH units.
Optimization of the hybridization conditions requires consideration of the thermal stability and the rate of formation of the hybrid duplex. The rate depends on temperature and usually reaches a maximum at about Tm-25°C (1). Selection of the solution conditions typically is made with the aid of empirical equations that predict the Tm of the fully formed duplexes (p. 31 of Ref. 1). The estimates of Tm are rather crude, but high precision is usually not required. Different equations are used for DNA-DNA, RNA-RNA, and DNA-RNA duplexes. These equations contain terms for sodium ion concentration, %GC, length (L), and % formamide, but are valid only for duplexes of length L > 50 base pairs. DNA-DNA duplexes:
If additional components are included in the solutions, the equations may fail to provide useful estimates. A pH value near neutrality is normally used, so a pH term is not included in the empirical equations, but significant deviation from neutral pH will compromise use of these empirical equations. Examination of the equations shows that the %GC and L-dependent terms are determined by the construction of the molecule. The Tm can be adjusted by manipulating the concentrations of Na+ and formamide. A given Tm can be obtained by infinitely many different combinations of Na+ and formamide concentration. This gives the experimenter flexibility in the design of hybridization conditions.
The Tm of nucleic acid polymers depends linearly on the GC content, a property that is exploited in the empirical equations shown above. This linearity is due to the sequence effects averaging out over a polymer. For oligonucleotides, the sequence effects become important in determining the thermal stability. Therefore, explicit account of sequence must be made when estimating the Tm of oligonucleotides. Another feature that differs from the polymer case is the dependence of Tm on [Na+], which depends on the length for oligonucleotides. Reliable estimates for the enthapy, DH°, and free-energy, DG°, changes associated with formation of DNA (2) and RNA (3) duplexes and for T m can be computed with knowledge of the base sequence and solution conditions. Thermodynamic data for RNA-DNA duplexes and for higher molecular structures, such as triple helices, are sparse relative to the databases for DNA-DNA and RNA-RNA duplexes. Data for these systems are being accumulated, and reliable predictive models should be forthcoming.
Adjustments in stringency can be used to discriminate among nucleic acids with different backbones, thereby favoring binding of a desired hybridization probe. Use of high formamide concentration (80%) selects for RNA-DNA hybrids over DNA-DNA duplexes, so it is useful when RNA probes are employed. The physiochemical properties of the various backbone-modified nonnatural nucleic acid analogues can be exploited by adjustment of the conditions of the binding reaction. Discrimination also can be made between nucleic acid complexes of different numbers of strands. When specific base composition requirements are met, three-stranded nucleic acid complexes can form. Cytosine protonation is required for the formation of the pyrimidine -purine-pyrimidine triple helix, thereby making its formation strongly dependent on pH. Triple helices also exhibit different salt concentration dependencies than do duplexes. Thus, solution conditions can be adjusted to favor or disfavor formation of three-stranded complexes relative to duplexes.