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chapter 23
Structure and Properties of DNA
2. The rise in ,4
2 6 0
occurs over a range of 6-8°C.
3. The maximum A
2 6 0
is about 37% higher than the
starting value.
The hypothetical state of a DNA molecule in different
regions of the melting curve is also shown in Figure 23-8.
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 num-
ber of broken base pairs increases with temperature. A
convenient parameter to characterize a melting transition
is the temperature at which the rise in A
2 5 0
is half com-
plete. This temperature is called the
melting temperature,
Tm.
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 hydro-
gen 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 temper-
ature 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 for-
mer type of substance is methanol, which increases the
solubility of the bases. Sodium trifluoracetate is an exam-
ple 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 mini-
mizes contact between bases and water and maximizes the
contact of the highly soluble phosphate group with wa-
ter. 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 struc-
ture. If one interaction is eliminated, the other is weak-
ened, which explains why
Tm
drops so markedly following
addition of a reagent that destroys either type of interac-
tion. 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 disentan-
glement 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°C, the value
of A
2 6 0
increases by 37% and the solution consists en-
tirely 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 A
2 6 0
drops significantly because random intrastrand hy-
drogen 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 re-
pulsion 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 concen-
tration and in a high salt solution, interstrand hydrogen
bonding competes with the intrastrand bonding just men-
tioned. This effect can be used to re-form native DNA from
denatured DNA.
23.4 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, A26o drops immediately to
the initial, undenatured value and the native structure is
restored. Thus, if strand separation is not complete 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 cor-
rect 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.
