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STRUKTUR DAN SIFAT

KIMIA DNA/RNA
Genes and Genome
Helical turn:
10 base pairs/turn
3.4 nm/turn
Nucleic Acid Structure A, B and Z helices

A-form B-form Z-form


Nucleic Acid Structure
RNA structure

1. Normally occurs as single stranded


molecule
2. Secondary structure are formed some
time.
3. Globular tertiary structure are
important for many functional RNAs,
such as tRNA, rRNA and ribozyme
RNA

Forces for secondary and tertiary structure: intramolecular


hydrogen bonding and base stacking.
Secondary
tRNA structure

Tertiary
structure
Molecular Biology
Conformational variability of RNA is reflected in the
more diverse roles of RNA in the cell, when compared to
DNA.

Comparison of Structure and Function of protein and nucleic acids

Protein Nucleic Acids


Fibrous protein Globular Double Globular RNA
protein Helical DNA
Structural Enzymes, Genetic Ribozymes
proteins antibodies, material Transfer RNA
receptors etc (tRNA)
Signal
recognition etc.
Nucleic Acid Structure
Modified Nucleic Acids
The chemical modification of bases or nucleotides in
nucleic acids is widespread, and has a number of specific
roles. In cellular DNA, the modifications are restricted to
the methylation of the N-6 position of adenine and the 4-
amino group and the 5-position of cytosine. These
methylations have a role in restriction modification , base
mismatch repair and eukaryotic genome structure . A
much more diverse range of modifications occurs in RNA
after transcription, which again reflects the different
roles of RNA in the cell.
Chemical and Physical Properties of
Nucleic Acids
Stability of Nucleic Acids
1. Hydrogen bonding
Contributes to specific structures of nucleic acids or protein. For
example, a-helix, b-sheet, DNA double helix, RNA secondary structures
H-bonds within a structure does not normally confer the stability, that
is to say, it does not contribute the overall stability of these secondary
structures

2. Stacking interaction/hydrophobic interaction between


aromatic base pairs contribute to the stability of nucleic acids.
Even in single-stranded DNA, the bases have a tendency to stack on top of
each other, but this stacking is maximized in double-stranded DNA
It is energetically favorable to exclude water altogether from pairs of such
surfaces by stacking them together
Effect of Acid
In strong acid and at elevated temperatures: are
hydrolyzed completely to bases, ribose or
deoxyribose, and phosphate (e.g., perchloric acid
(HClO4) at > 100C)

In more dilute mineral acid, for example at pH 34,


the most easily hydrolyzed bonds are selectively
broken. E.g., glycosylic bonds attaching purine bases
to the ribose ring are broken by formic acid.
Application: the basis of the chemical DNA
sequencing method developed by Maxam and Gilbert.
Effect of Alkali-DNA

1. Increasing pH (> 7-8) has more subtle effects on DNA structure


2. The effect of alkali is to change the tautomeric state of the bases

keto form enolate form keto form enolate form

3. This affects the specific hydrogen bonding between the base pairs, with the
result that the double-stranded structure of the DNA breaks down; that is the
DNA becomes denatured .
Effect of Alkali-RNA
RNA are hydrolyzed at higher pH because of the present of 2-
OH group in RNA

HO

Alkali

2, 3-cyclic
phosphodiester

RNA is unstable at higher pH


Chemical Denaturation
A number of chemical agents can cause the denaturation of
DNA or RNA at neutral pH, e.g. Urea (H2NCONH2) is used in
denaturing PAGE; Formamide (HCONH2) is used Southern
and Northern blotting.

Mechanism
Disrupting the hydrogen bonding of the bulk water
solution

Hydrophobic effect (stacking interaction) is reduced

Denaturation of the strands


Viscosity

Consequence of the DNA high viscosity


1. A high axial ratio (2 nm in diameter, and a length
of micrometers, millimeters or even several
centimeters in the Eukaryotic chromosomes)
2. Relatively stiff

Applications:
Long DNA molecules can easily be damaged by
shearing force, or by sonication.
Pay attention to avoid shearing problem when need to
isolate intact very large DNA molecule.
Buoyant density
DNA and 8M CsCl has a similar density, around 1.7 g cm-3
Purifications of DNA: equilibrium density gradient centrifugation

DNA=1.66+0.098(G+C)
Mole fraction

Protein floats

RNA pellets at the bottom


Spectroscopic and Thermal
Properties of Nucleic Acids
UV absorption

Nucleic acids absorb UV light due to the conjugated


aromatic nature of the bases
The wavelength of maximum absorption of light by
both DNA and RNA is 260 nm (lmax = 260 nm)
Applications: can be used for detection, quantitation
and assessment of purity (A260/A280)
Hypochromicity

Caused by the fixing of the bases in a hydrophobic environment


by stacking, which makes these bases less accessible to UV
absorption. A260 value: dsDNA<ssDNA or RNA<nucleotide

Quantitation of nucleic acids


Extinction coefficients: 1 mg/ml dsDNA has an A260 of 20;
The corresponding value for ssDNA and RNA is approximately 25
The values for ssDNA and RNA are approximate for two reason:
(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 in a given molecule.
Purity of DNA
The approximate purity of dsDNA preparations may be
estimated by determination of the ratio of absorbance
at 260 and 280 nm (A260/A280).

A260/A280:
dsDNA = 1.8
pure RNA = 2.0
Protein <1, 0.5 or so
Thermal denaturation/melting

Heating also 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 the mid-point of the
smooth transition, which has a 20% increase in absorbance. 80-100 C for
long DNA molecules
Renaturation

Rapid cooling: allows only the formation of local region of base


paring. The decreased in A260 is rather small.
Slow cooling: allows time for whole complementation of dsDNA.
Absorbance in 260nm decreases greatly and cooperatively.

Note: Relative concepts


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)
DNA Supercoiling

1. Closed-circular DNA: Almost all DNA molecules in


cells can be considered as circular
2. Supercoiling: Most natural DNA is negatively
supercoiled, that is the DNA is deformed in the
direction of unwinding of the double helix.
3. Topoisomer: A circular dsDNA molecule with a
specific linking number which may not be changed
without first breaking one or both strands.
Molecular Biology

Learn about several concepts:


1, Linking Number (Lk): the number of double-helical turns in
the DNA molecule.
2. The level of supercoiling may be quantified in terms of the
change in linking number (Lk) from that of the
unconstrained (relaxed) closed-circular molecule (Lk).
2, Twist:the local winding up or unwinding of the
double helix
3, writhe) : the coiling of the helix axis upon itself.
Twist and writhe are interconvertible according to the equation
Lk = Tw + Wr.
A rubber tubing model for
DNA supercoiling, which
illustrates the change in
DNA conformation. The
changes in linking number
are indicated

Conformational flexibility
in supercoiled DNA as
modeled by rubber tubing.
The changes in twist and
writhe at constant linking
number are shown
Molecular Biology

Topoisomerases exist in cell to regulate the level of


supercoiling of DNA molecules.
Type I topoisomerase: breaks one strand and change the
linking number in steps of 1.
TypeII topoisomerase: breaks both strands and change the
linking number in steps of 2.
Gyrase: introduce the negative supercoiling (resolving the
positive one and using the energy from ATP hydrolysis.
The mechanisms of (a) type I and (b) type II
topoisomerases
Intercalator
Ethidium bromide locally unwinding of bound
DNA, resulting in a reduction in twist and
increase in writhe.

(a) Ethidium bromide; (b)


the process of
intercalation, illustrating
the lengthening and
untwisting of the DNA
helix.
Replication Origins and
Palindromes
High concentration of palindromes
exists around replication origins of
other herpesviruses
Locating clusters of palindromes
(above a minimal length) on CMV
genome sequence might reveal
likely locations of its replication
origins.
Palindromes in Letter
Sequences
Odd Palindrome:
A nut for a jar of tuna
remove spaces and capitalize

ANUTFORA JAROFTUNA

Even Step on no pets


Palindrome:

STEPON NOPETS