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3
...
2
...

Electronegative O and N atoms with free lone pairs are potential hydrogen bond
acceptors
...
Many of the
oxygen, nitrogen, and hydrogen atoms in the nitrogenous bases are very effective
hydrogen bond donors and acceptors, as illustrated in Figure 3
...
Adenine (A) and
thymine (T) form base pairs via hydrogen bond donors and acceptors, and the A-T
base pair has two hydrogen bonds between the bases
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2a)
...
4–1
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Moreover, the nucleic acids have polar groups that
form a complex network of hydrogen bonds with the surrounding water molecules
(see Chapter 4)
...
The hydrogen bonds due to hydration
depending on the structure also affect nucleic acid stability
...


O
Thymidine (T)

A

T

G

C

(a)

(b)

Figure 3
...
The hydrogen
bonds are shown in dashed lines
...
Stacking interactions are shown in arrows
...
2
...
The individual bases make strong
stacking interactions with neighboring bases, which are major contributors to
duplex stability (Figure 3
...
The stacking interactions are much more prevalent in
duplexes than in single strands
...
The degree of stabilization afforded by base stacking depends on the DNA
sequence
...
The values of free energy change at 37 ∘C(−ΔG37∘ ) due to stacking interactions during duplex formation are 0
...
6 kcal mol−1 [3]
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The DNA duplex stability increases
with increasing salt concentration
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2
...
3
...
The duplex formation is favorable for enthalpy
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chain
...
1)
−−
−−
−
− B⋅C
B+C←
−−
→
DNA
...
2)

respectively
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0
0
...
6
0
...
2
Tm
0

0

(a)

20
40
60
Temperature (°C)

80

Absorbance at 260 nm

1
...
8
0
...
4
0
...
3)
where Dss and Df indicate the single-stranded (unfolded) and folded DNA structures,
respectively
...
(3
...
(3
...
4)
where Ct is the total strand concentration, s has a value of 1 for self-complementary
duplexes and 4 for non-self-complementary duplexes, and 𝛼 is the fraction of strands
in a duplex:
Ct = [A] + 2 [A] (self − complementary duplex)

(3
...
6)

For a unimolecular transition,
Kobs = 𝛼∕(1 − 𝛼) = [Df ]∕[Dss ]

(3
...
8)
Figure 3
...
At low temperatures, the strands are in duplex form and the
absorbance is low
...
The UV absorbance of the duplex is increased by dissociation of the duplex,
and the increment of absorbance is referred to as hyperchromicity
...
) For self-complementary
or non-self-complementary duplexes with equal concentrations of each strand,
the melting temperature, T m (in degrees Kelvin), is the point at which the concentrations of strands in duplex and in single strands are equal (Figure 3
...

The steepness of the transition indicates the cooperativity of the transition
...
T m is
most accurately measured by fitting the lower and upper baselines
...
The van’t Hoff equation relates
∘
∘
the T m (in degrees Kelvin), Ct , ΔH , and ΔS :
∘
∘
∘
−1
Tm = R ln(Ct ∕s)∕𝛥H + 𝛥S ∕𝛥H
(3
...
987 cal K−1 mol−1 or 8
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The slope
∘
∘
∘
of the van’t Hoff plot gives the ΔH , and the y-intercept gives the ratio of ΔH to ΔS
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10)
To increase the accuracy of these parameters, data analysis can be performed by
curve fitting as shown below
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11)
ds

(3
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13)
∘

∘

Kobs = exp(−𝛥H ∕RT + 𝛥S ∕R)

(3
...
4 Factors Influencing Stabilities of the Non-canonical Nucleic Acids

where R is the gas constant and mds and bds or mss and bss represent the slope and
intercept of the upper baseline or lower baseline for the melting curve of a duplex
∘
dissociation (Figure 3
...
The six variables (𝜀ds , 𝜀ss , bds , bss , ΔH , and
∘
ΔS ) can be calculated by the curve fitting using Eqs
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11–3
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These calculations,
which are performed using a PC equipped with the curve fitting software such as
IGOR Pro, KaleidaGraph, or ORIGIN, derive the thermodynamic parameters from
the shape of the melting curve
...
(3
...
15)

T

∘

∘

where RΔH ∘ ,ΔS∘ is the correlation coefficient between ΔH and ΔS
...
The errors of 𝜎
Tm
t
ΔH ∘ , 𝜎 ΔS∘ , and 𝜎𝛥G∘
percent of each thermodynamic value

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