Denaturation and Renaturation of DNA

Denaturation of DNA:

       The three-dimensional structures of DNA, RNA, and proteins are determined by weak noncovalent interactions, principally hydrogen bonds and hydrophobic interactions. The free energies of these interactions are not much greater than the energy of thermal motion at room temperature, so that at elevated temperatures the structures of these molecules are disrupted. A macromolecule in a disrupted state is said to be denatured; the ordered state, which is presumably that originally present in nature, is called the native state. A transformation from the native to the denatured state is called denaturation. 

         When double-stranded (native) DNA is heated, the bonding forces between the strands are disrupted and the two DNA strands separate; thus, completely denatured DNA is single stranded. Much information about the structure and stabilizing interactions has been obtained by studying denaturation. Some property of DNA that changes as denaturation proceeds is measured, e.g., the absorption of ultraviolet light. A change in the ultraviolet absorbance (or some other property) as a function of temperature is called a melting curve. Many reagents either break hydrogen bonds or weaken hydrophobic interactions, and are powerful denaturants. Thus, denaturation is also studied by varying the concentration of a denaturant at a constant temperature. For DNA, the simplest way to detect denaturation is to monitor the ability of DNA in a solution to absorb ultraviolet light at a wavelength of 260 nm. 

           The absorbance of DNA at 260 nm, A260, is not only proportional to its concentration (as is the case for most light-absorbing molecules) but also depends on the structure of the molecule; the more ordered the structure, the less light is absorbed. Therefore, free nucleotides absorb more light than a single-stranded polymer of DNA (or RNA), which in turn absorbs more light than a double-stranded DNA molecule. For example, solutions of double-stranded DNA, single-stranded DNA, or free bases, each at 50 μg/mL, have the following A260 values:

Double-stranded DNA: A260 = 1.00
Single-stranded DNA: A260 = 1.37
Freebases: A260 = 1.60
If a DNA solution is heated slowly and the A260 is measured at various temperatures, a melting curve is obtained. The following features of this curve should be noted:

1. The A260 remains constant up to temperatures well above those encountered by living cells in nature.

2. The rise in A260 occurs over a range of 6-8 C
3. The maximum A260 is about 37% higher than the starting value.

DNA Melting curve

                 Before the rise begins, the molecule is fully double stranded. In the region of rapid denaturation, base pairs in various segments of the molecule are broken; the number of broken base pairs increases with temperature. A convenient parameter to characterize a melting transition is the temperature at which the rise in A260 is half complete. This temperature is called the melting temperature, Tin. The value of Tm varies both with base composition and experimental conditions. In particular, Tm increases with increasing percent G + C, which is a result of the hydrogen bonds in a GC pair (three) versus an AT pair (two). A higher temperature is required to disrupt a GC pair than an AT pair. Reagents such as urea and formamide, which can hydrogen-bond with the DNA bases, reduce Tm. These denaturing agents maintain the unpaired state at a temperature at which a broken base pair would normally pair again, so that permanent melting of a section of paired bases requires less thermal energy. Other reagents either enhance the interaction of weakly soluble substances (such as the nucleic acid bases) with water or disrupt the water shell; such substances should weaken hydrophobic interactions. 

                 An example of the former type of substance is methanol, which increases the solubility of the bases. Sodium trifluoracetate is an example of the second type. The addition of both these reagents greatly reduces Tm because hydrophobic interactions are also important in stabilizing the DNA structure. In fact, the three-dimensional structure of DNA is one that minimizes contact between bases and water and maximizes the contact of the highly soluble phosphate group with water. Minimization of base-water contact is accomplished by stacking of the bases, which occurs even in single stranded DNA. The bases of double-stranded DNA are more stacked than those in single-stranded DNA because of the hydrogen bonds between the two strands. Both hydrogen bonds and hydrophobic interactions are weak and easily disrupted by thermal motion. Maximum hydrogen bonding is achieved when all bases are oriented in the right direction. Similarly, stacking is enhanced if the bases are unable to tilt or swing out from a stacked array. Clearly, stacked bases are more easily hydrogen-bonded, and correspondingly, hydrogen-bonded bases, which are oriented by the bonding, stack more easily. Thus, the two interactions act cooperatively to form a very stable structure. 

                 If one interaction is eliminated, the other is weakened, which explains why Tm drops so markedly following addition of a reagent that destroys either type of interaction. When hydrogen bonds and hydrophobic interactions are eliminated, the helical structure of DNA is disrupted and the molecule loses its rigidity. This collapse of the ordered structure is accompanied by complete disentanglement of the two strands. At high pH, the charge of several groups engaged in hydrogen bonding is changed and base pairing is reduced. At a pH greater than 11.3, all hydrogen bonds are eliminated and DNA is completely denatured. When a DNA solution is heated above 90° the value of A260 increases by 37% and the solution consists entirely of single strands whose bases are unstacked. 

              If the solution is then rapidly cooled to room temperature and the salt concentration is greater than 0.05 mol/L, the value of A260 drops significantly because random intrastrand hydrogen bonds re-form between distant short tracts of bases whose sequences are complementary (or nearly so). After cooling, about two-thirds of the bases are either hydrogen bonded or in such close proximity that stacking is restored and the molecule is very compact. In contrast, if the salt concentration is 0.01 mol/L or less, the electrostatic repulsion due to unneutralized phosphate groups keeps the single strands sufficiently extended that the bases cannot approach one another. Thus, after cooling, no hydrogen bonds are re-formed. At a sufficiently high DNA concentration and in a high salt solution, interstrand hydrogen bonding competes with the intrastrand bonding just mentioned. This effect can be used to re-form native DNA from denatured DNA.

Renaturation of DNA:

             If a DNA solution is heated to a temperature at which most (but not all) hydrogen bonds are broken and then cooled slowly to room temperature, A260 drops immediately to the initial, undenatured value and the original structure is restored. Thus, if strand is not completely separated and denaturing conditions are eliminated, the helix rewinds. A related observation is that if two separated strands come in contact and form even a single base pair at the correct position in the molecule, the native DNA molecule will re-form. This phenomenon is called renaturation, or reannealing. Two requirements are necessary for renaturation to occur.
1. The salt concentration must be high enough that electrostatic repulsion between the phosphates in the two strands is eliminated-usually 0.15-0.50 M NaCl is used.

2. The temperature must be high enough to disrupt the random, intrastrand hydrogen bonds described above. However, if the temperature is too high, stable interstrand base pairing will not occur or be
maintained. The optimal temperature for renaturation is 20-25°C below the value of Tm.

Cot curves

             Renaturation is slow compared with denaturation. The rate-limiting step is not the rewinding of the helix but the precise collision between complementary strands such that base pairs are formed at the correct positions. Since two molecules participate in the rate-limiting step, renaturation is a concentration-dependent process requiring several hours under typical laboratory conditions. In particular, the kinetics of association follows a simple second-order rate law with the association rate increasing with DNA concentration. The kinetics are conveniently described by an equation that relates the fraction, f , of single-stranded (dissociated) DNA and the time elapsed after exposing a DNA sample to renaturing conditions (high salt concentration, elevated temperature):

                                         f = 1/ (1+kcot)

in which Co is the initial DNA concentration, t is the elapsed time in seconds, and k is a rate constant. A plot of f versus the logarithm of the product Cot yields a sigmoid curve commonly called a Cot ("cot") curve. A notable feature of these curves is that the renaturation rate is related to the molecular weight of the DNA. A useful index for characterizing these curves is Co/t1/2, with the value of Cot corresponding to renaturation of half of the DNA ( f = 1/2). Comparison of the Co/t1/2 values for E. coli DNA (M.W. = 2.7 X 109) and T4 DNA (M.W. = 1.1 X 108) shows that T4 DNA renatures roughly 50 times faster than E. coli DNA. 

             The reason is that if the two DNA samples have equal molar concentrations of nucleotides, the T4 sample will contain many more DNA molecules than the E. coli sample. Cot analysis is not-usually done with intact DNA molecules but rather with fragments having lower molecular weights. This breakage does not affect the relative values of Co/t1/2 since the number of different kinds of fragments of T4 DNA is smaller and hence their concentration larger than the corresponding number of fragments in the E. coli sample. Studies of a variety of prokaryotic DNAs show that the value of Co/t1/2 is directly related to the total size of the DNA of the organism (the genome size). 

             However, this relation does not apply to the DNA of eukaryotes because of the presence of highly repetitive sequences. The fragmentation of the DNA molecules allows a new feature of base sequences to be seen. If the unbroken denatured DNA were allowed to renature, the Cot curve would be a smooth curve in which Co/t1/2 would be proportional to the size of the DNA. However, if the DNA were broken into small pieces, several fragments from each molecule would contain the repeated sequence, and these fragments would renature more rapidly than the bulk of the sequences. Approximately 30% of human DNA contains sequences of bases that are repeated 20 times or more.