•Molecular Biology Course

Chemical and Physical Properties of Nucleic

Acids
1. Stability of Nucleic Acids

2. Effect of Acid & applications Chemical properties

3. Effect of alkali & applications

4. Chemical denaturation

5. Viscosity & applications

Physical properties
6. Buorant density & application

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C. Properties of nucleic acids

Stability of Nucleic Acids

1. Hydrogen bonding
• Does not normally contribute the stability of nucleic acids or protein
• Contributes to specific structures of these macromolecules. For example, a-helix,
b-sheet, DNA double helix, RNA secondary structure

2. Stacking interaction/hydrophobic interaction between

aromatic base pairs/bases contribute to the stability of nucleic acids.
• It is energetically favorable for the hydrophobic bases to exclude waters and stack on
top of each other
• This stacking is maximized in double-stranded DNA

Fig
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C. Properties of nucleic acids

Effect of Acid

Strong acid + high temperature: completely hydrolyzed to

bases, riboses/deoxyribose, and phosphate

pH 3-4 : apurinic nucleic acids [glycosylic bonds

attaching purine (A and G) bases to the ribose ring are
broken ], can be generated by formic acid

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C. Properties of nucleic acids

Effect of Alkali & Application

1. High pH (> 7-8) has subtle (small) effects on DNA structure
2. High pH changes the tautomeric state of the bases

enolate form
keto form enolate form keto form

Base pairing is not stable anymore because of the change of tautomeric states of the
bases, resulting in DNA denaturation
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C. Properties of nucleic acids

RNA hydrolyzes at higher pH because of 2’-OH groups in

RNA

2’, 3’-cyclic
phosphodiester

alkali OH free 5’-OH

RNA is unstable at higher pH 5

C. Properties of nucleic acids

Chemical Denaturation
Urea (H NCONH ) : denaturing PAGE
2 2

Formamide (HCONH ) : Northern blot

2

Disrupting the hydrogen bonding of the bulk water

solution

Hydrophobic effect (aromatic bases) is reduced

Denaturation of strands in double helical structure

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C. Properties of nucleic acids

Buoyant density (DNA)

1.7 g cm-3, a similar density to 8M CsCl
Purifications of DNA: equilibrium density gradient centrifugation

Protein floats

RNA pellets at the bottom

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•Molecular Biology Course

Spectroscopic and Thermal

Properties of Nucleic Acids
1. UV absorption:
• nucleic acids absorb UV light due to the aromatic bases
• The wavelength of maximum absorption by both DNA and RNA is 260 nm (lmax =
260 nm)
• Applications: detection, quantitation, assessment of purity (A 260/A280)

2. Hypochromicity: decrease in absorbance caused by the fixing of the bases in

a hydrophobic environment by stacking (formation of dsDNA molecules), which makes
these bases less accessible to UV absorption. dsDNA, ssDNA/RNA, nucleotide.
The hyperchromicity (increase in absorbance) of DNA  occurs when the DNA duplex is
denatured. The UV absorption is increased when the two single DNA strands are being
separated, either by heat or by addition of denaturant or by increasing the pH level.
The opposite, a decrease of absorbance is called hypochromicity, occurs when renaturation
occurs
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C. Properties of nucleic acids

3. Quantitation of nucleic acids

Extinction coefficients: or mass attenuation coefficient 
The mass attenuation coefficient is a measurement of how strongly a 
chemical species or substance absorbs or scatters light at a given wavelength, per
unit mass.
 
At a wavelength of 260 nm, the average extinction coefficient for double-stranded
DNA is 0.020 (μg/ml)-1 cm-1, for single-stranded DNA it is 0.027 (μg/ml)-1 cm-1, for
single-stranded RNA it is 0.025 (μg/ml)-1 cm-1 and for short single-stranded
oligonucleotides it is dependent on the length and base composition.

The values for ssDNA and RNA are approximate

(1) The values are the sum of absorbance contributed by the different bases (e : purines
> pyrimidines)
(2) The absorbance values also depend on the amount of secondary structures due to
hypochromicity.

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4. Purity of DNA
A260/A280:
dsDNA--1.8
pure RNA--2.0
protein--0.5

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C. Properties of nucleic acids

5. Thermal denaturation/melting: heating leads to the destruction of

double-stranded hydrogen-bonded regions of DNA and RNA.

RNA: the absorbance increases gradually and irregularly

DNA: the absorbance increases cooperatively.
melting temperature (Tm): the temperature at which 40% increase in
absorbance is achieved.
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C. Properties of nucleic acids

6. Renaturation:
Rapid cooling: only allow the formation of local base paring. Absorbance
is slightly decreased
Slow cooling: whole complementation of dsDNA. Absorbance decreases greatly
and cooperatively.

Annealing: base paring of short regions of complementarity within or between DNA strands.
(example: annealing step in PCR reaction)
Hybridization: renaturation of complementary sequences between different nucleic acid molecules.
(examples: Northern or Southern hybridization) 13