This document discusses the stability of nucleic acids and how differential scanning calorimetry (DSC) can be used to characterize it. DSC directly measures the stability and unfolding of biomolecules like DNA and RNA as they are heated. It determines values like the transition midpoint temperature (Tm), enthalpy (ΔH), and heat capacity change (ΔCp) associated with unfolding. DSC data provides information on factors influencing nucleic acid stability, including sequence effects, environmental conditions, and structure formation.

1. STABILITY OF
NUCLEIC ACIDS

2. WHY????
 Nucleic acid structures are
stabilized by non-covalent
intramolecular interactions
between the bases.
 All biological processes
involving DNA and RNA
require these structures to
be in the stable and in the
appropriate conformation.
 It is important to know how
nucleic acids form their
biologically active states
and how these active
states are stabilized.

3. IMPORTANCE OF
STABILITY
 There have been rapid
advances in structural biology
and relating structure to
biochemical function and
mechanism.
 However, knowledge of
nucleic acid structure alone
does not ensure accurate
prediction of stability, function
and biological activity.
 The complete characterization
of any biomolecule requires
stability determination and the
forces which lead to stability
and correct folding.

4. HOW TO CHARACTERISE
PHYSICAL STABILITY
 The stability of DNA is characterised
by applying heat upon the DNA/RNA
samples (Thermal Stability).
 Differential Scanning Calorimetry
(DSC) is used as one of the
equipment to characterise the stability
of nucleic acids.

5. DIFFERENTIAL SCANNING
CALORIMETRY
 Differential Scanning
Calorimetry (DSC) is a
powerful analytical tool
which directly measures
the stability and unfolding
of biomolecules.
 In DSC, the sample is
heated at a constant rate,
and there is a detectable
heat change associated
with thermal denaturation.

6. PRINCIPLE OF DSC
 Detection of phase transitions.
 The basic principle underlying this
technique is that when the sample
undergoes a physical
transformation such as phase
transitions, more or less heat will
need to flow to it than the
reference to maintain both at the
same temperature.
 Whether less or more heat must
flow to the sample depends on
whether the process
is exothermic or endothermic.
 For example, as a solid
sample melts to a liquid, it will
require more heat flowing to the
sample to increase its temperature
at the same rate as the reference.
 This is due to the absorption of
heat by the sample as it
undergoes the endothermic phase
transition from solid to liquid.

7. WHAT IT DETERMINES????
 A single DSC experiment can
determine:
Transition midpoint (Tm)
Enthalpy (∆H) and heat capacity
change (∆Cp) associated with
uncoiling
Presence of multiple melting site
domains

8. TRANSITION MIDPOINT
 Denaturation midpoint of a DNA is defined as
the temperature (Tm)or concentration of
denaturant (Cm) at which both
the folded and unfolded states are equally
populated at equilibrium.
 Tm is often determined using a thermal shift assay.
 If the widths of the folded and unfolded wells are
assumed to be equal both these states will have
identical free energies at the midpoint.
 This would mean that the free energy of the
folded state is lower at the denaturation midpoint
than the unfolded state. In such a scenario, the
temperature at which both the wells have identical
free energies is termed the characteristic
temperature (To)

9. DNA STRUCTURAL
TRANSITIONS
 Double stranded DNA is a dynamic
structure and should never be
considered as a static entity. The two
strands are held together by non-
covalent interactions (hydrogen bonding
and base stacking).
 The energy of these interactions is such
that the helix can come apart quite easily
at physiological temperatures. If this
were not so, gene expression would not
be possible at the temperature of living

10.  DNA can be heated and, at a certain
temperature, the two strands will come apart.
We say that the DNA helix has melted or
denatured.
 This transition can be followed by the increase
in the absorption of ultraviolet light by the
molecule as it goes from helix to random coil
(the denatured form). This is called
hyperchromicity.• The increase in UV
absorbance at 260 nm
for the denatured (coil)
is used to follow the
transition from helix to
coil.

11. ENTHALPY OF DNA
 The free
energy of
DNA
supercoiling
is enthalpy.
 Enthalpy
(∆H) and
heat capacity
change
(∆Cp)
associated
with
uncoiling

12.  As the temperature
increases, you start to get
local unwinding of the
double-stranded DNA.
 This unwinding occurs
preferentially in regions
where the two strand are
held together less strongly.
 In these regions the strands
separate to form bubbles of
single-stranded regions.

13.  The DNA sequence in these regions is
enriched in A/T base pairs because the
interactions between the two strands are
weaker in A/T rich regions.
 In G/C rich regions strands are held
together more strongly so they don't
unwind until higher temperatures.
 DSC measures ∆H due to heat
denaturation. Nucleic acid unfolding is
typically endothermic. During the same
experiment, DSC also measures the
change in heat capacity (∆Cp) for
denaturation.

14. MULTIPLE MELTING
POINTS
 The melting point of DNA is around 60
ºC. Note that ‘melting’ in this sense is
not a change of aggregate state, but
simply the dissociation of the two
molecules of the DNA double helix.
 The melting temperature of DNA
refers to the temperature at which
50% of DNA in a sample has
denatured from double-stranded DNA
(dsDNA) to single-stranded DNA
(ssDNA).

15.  Sensitive
measurement of the
melting curve of a
sample of DNA can be
used to detect single
nucleotide differences
between two DNA
samples.
 This technique is
possible because
guanine-cytosine (GC)
pairs contribute
greater stability to
dsDNA than
adenosine-thymine
(AT) pairs.

16. FACTORS INFLUNCING
STABILITY
 Many factors are responsible for
the Nucleic Acid Stability,
including
hydrogen bonding,
conformational entropy,
physical environment (pH,
buffer, ionic strength,
excipients, etc.)

17.  DSC data, either used alone or in
conjunction with stability and structural
data, can provide information on:
Effects of DNA and RNA sequence
Effects of buffer, pH, salt, additives
Duplex, triplex and quadrauplex
structures
Formation of RNA and DNA complexes
Formation of nucleic acid-protein
complexes
Effects of small molecule drugs on
nucleic acid stability