Part IV => DNA and RNA

4.2 NUCLEIC ACIDS

4.2a DNA Structure
4.2b RNA Structure
4.2c DNA Sequencing

Section 4.2a:
DNA Structure
I will climb up this
DNA ladder to see
where life takes me!

Synopsis 4.2a
- DNA is a double-stranded biopolymer comprised of four constituent
units or building blocks referred to as deoxyribonucleotidesthe base
components of which in general are adenine (A), cytosine (C), guanine
(G), and thymine (T)wherein A base pairs with T, and G with C
- DNA carries genetic information in the form of its sequence of
deoxyribonucleotideslinked together via phosphodiester bonds

- In the so-called DNA double helix, two antiparallel polynucleotide

strands wind around each otherthey are stabilized by van der Waals
forces (hydrogen bonding & stacking interactions), and ionic interactions
between opposite strands
- DNA double-helix can adopt three distinct conformations: A-DNA, BDNA, or Z-DNA
- DNA can be denatured by heating and renatured through annealing

Nucleic Acid Structure

Inorganic
phosphate
(PO43-)

N-glycosidic
bond

Hydrogen
phosphate
(HPO42-)

Phosphoryl
group

Phosphate
group

Phosphodiester
bond
Nucleic acid is a biopolymer of nucleotides
held together by phosphodiester bonds!

Comparison of Complex Bonds

Ester
[acyl group bonded to an R
moiety via an O atom]

Thioester
[acyl group bonded to an R
via an S atom]

Phosphoester
[phosphoryl group bonded to
an R moiety via an O atom]

O
||
R2COR1

O
||
-OCSR1
|
O-

Anhydride
[without hydride(H-)two acyl
groups bonded together via an O
atom]

Phosphoanhydride
[two phosphoryl groups bonded
together via an O atom]

O
O
||
||
OPOPO
|
|
OO-

ATP

(3)

O
||
-OPOR1
|
O-

O
||
Phosphodiester R2OPOR1
[phosphoryl group bonded to
|
two R moieties via O atoms]
O-

O
O
||
||
R1COCR2

(1)

(1)
(2)
(3)
(4)

(2)

Phosphoanhydride
Phosphoanhydride
Phosphoester
N-glycosidic

(4)

Schematic Representation of a Nucleic Acid Strand

- Vertical lines denote nucleosides

- Diagonal lines indicate phosphodiester linkages holding the nucleosides together
- Strand polarity is generally defined as running from the 5-C atom of (deoxy)ribose
at one end (the 5-end) to the 3-C atom at the other end (the 3-end)or simply
indicated as 5 3

Anti-Conformation of Bases Is Favored

- Nitrogenous bases can adopt two distinct steric conformations relative to the attached
sugar: syn (with) or anti (against)analogous to cis (same) and trans (across)
- In the nucleic acid biopolymers, the anti-configuration of bases is highly preferred in
order to avoid steric clashes with the sugars

The Double Helix Discovery (1953)

Rosalind Franklin
(1920-1958)

Maurice Wilkins
(1916-2004)

James Watson
(1928-date)

DNA Model (1953)

First formulated by Watson and Crick
but on the basis of x-ray crystallographic data
generated by Franklin (in collaboration with Wilkins)

Francis Crick
(1916-2004)

Double-Helix

Helical Axis

- The two strands (polynucleotides) wind around each

other along a principal (or helical) axis to form what
has come to be known as right-handed doublehelixwith a diameter of approximately 20
- The two strands run in opposite directions to each
otherthey are antiparallel!

12

- While the sugars linked together by phosphate groups

constitute the sugar-phosphate backbone, the bases
stack against each other lying roughly perpendicular to
the helical axis and pointing inwards into the core of
the double-helix
- The surface of the double-helix is consecutively
interspersed with two grooves of unequal width: minor
groove (12) and major groove (22)thus the double
helix harbors a pitch (rise/turn) of 34!

22

- While transcription factors typically bind to the major

groove, therapeutic drugs generally target the minor
groove
- The right-handed double-helix formation is driven by
complementary base pairing: A/T and G/C pairing

20

Double-Helix: Physical Parameters

Helical diameter (20)
- the thickness of the helical rod

Helical axis

Helical pitch (34/turn)

- the length of one full helical turn (2 or 360 )
Helical density (10bp/turn)
- the number of bps per helical turn

Helical pitch

Helical rise (3.4/bp)

- the rise of helix per base pair (bp):
Helical rise = pitch/density
= (34/turn) / (10bp/turn)
= 3.4/bp
Helical Twist (36 /bp)
- the rotation of helix per bp:
Helical twist = /density
= (360 /turn) / (10bp/turn)
= 36 /bp
where is the angular distance (2 ) per turn
Base tilt (6 )
- the inclination of bps relative to the helical axis

Helical twist
Base tilt

Helical diameter

Double-Helix: Handedness

Counterclockwise
Spiral

Clockwise
Spiral

- In each case, the fingers curl in the direction the helix turnsprovided that the
thumb is pointing parallel to the principal axis of the helix!
- The handedness is retained irrespective of the orientation of the helixturning it
upside down has no effect!

Double-Helix: Complementary Base Pairing

- Adenine (A) always pairs with thymine (T) via TWO hydrogen bonds
- Guanine (G) always pairs with cytosine (C) via THREE hydrogen bonds
- DNA rich in GC base-pairing generally displays enhanced thermodynamic stability
ie it has a higher melting point (Tm)why?! Not due to an additional H-bond but
rather due to enhanced stacking interactions!

Double-Helix: Watson-Crick Base Pairs

- Within the double-helix, the A-T and G-C
complementary base pairs are of equal width of
approximately 11
- Thus, all combinations of base pairsA-T, T-A, GC, C-Gcan be interchanged without altering
the sugar-phosphate backbone or the
conformation of DNA as a whole!
- Owing to such geometric similarity between all
combinations of base pairs, DNA adopts a nearperfect symmetry (along its helical axis)made
up of similar parts that lie against each other
along a central axis giving it a symmetrical shape
(cf human body)
- The DNA symmetry is independent of its base
composition though base mismatching can result
in DNA distortion and bending
- The A-T and G-C base pairs as observed in DNA
double helix are referred to as Watson-Crick
base pairsso as to distinguish them from
other theoretical combinations of base pairs!
- Such Watson-Crick base pairing is primarily
driven by hydrogen bonding

Double-Helix: Thermodynamic Stability

DNA is stabilized by three major thermodynamic forces as follows:
(1) Hydrogen bondingalthough Watson-Crick base pairing is driven by Hbonding (a type of van der Waals force), such interactions only weakly
stabilize the DNA structurealso true of H-bonding in proteinsthis is in
part due to the fact the loss of such H-bonding (for example upon
denaturation of DNA) is by and large compensated by H-bonding
interactions of bases with water moleculesditto for proteins!
(2) Stacking Interactionsin addition to hydrogen bonding, the bases within
each strand of the double helix have the tendency to stack (due to dipoles
Stacking of bases
within the aromatic rings) on top of each other forming a layer of stacked
sheetsuch spatial alignment of bases is driven by the so-called stacking stabilizes DNA double helix
interactionsa special case of van der Waals forces!
(3) Ionic interactionssince DNA is a highly polar molecule due to the charged
sugar-phosphate backbone, metal ions (called counterions)such as Na+,
K+, Mg2+engage in ionic interactions with the charged phosphate groups
and, in so doing, further stabilize the DNA double helix
Avoid bad habitsmany scientists (and educators) erroneously refer to:
(a) ionic interactions (#3) as electrostatic interactionsall three types of
interactions outlined above have their basis in electrostatics!
(b) van der Waals forces (#1 & #2) as hydrophobic forceswhile the former
arise from direct interactions between electronic dipoles of (a)polar groups,
the latter constitute a phenomenon whereby water solvent is excluded from Negatively charged DNA
shielded with counterions
apolar groups (eg immiscibility of oil in water, protein folding)

Double-Helix: DNA Denaturation

max

- When DNA is heated above the so-called melting temperature or Tm, its native doublehelical structure collapses such that the two strands peel apart and become disordered
such a phenomenon is referred to as DNA denaturation

- Denaturation of DNA is accompanied by changes in its physicochemical properties:

(1) Viscositythe viscosity of native DNA undergoes dramatic decrease due to the
loss of a highly rigid rod-like double helix that is otherwise resistant to deformation
(2) Hyperchromicitylight absorbance in the ultraviolet region (due to the conjugated
pi bonds in bases) becomes enhanced with a particular increase in the absorbance
maximum ( max) @ 260nm in a phenomenon termed the hyperchromic effect
this is due to the disruption of electronic interactions between bases (or due to
their enhanced solvent exposure)

Double-Helix: DNA Melting Curve

- Monitoring changes in absorbance @ 260nm ( 260)
as a function of temperature shows that DNA
denaturation occurs dramatically over a narrow
260
temperature range yielding a sigmoidal curve
- Such a curve is referred to as the melting curve
and the temperature at its midpoint is called the
melting temperature or Tm
- The stability of DNA (hence its Tm) is dependent
upon external factors such as solvent and pH as well
as its base compositionGC-rich sequences tend to
have higher Tm due to their enhanced stacking
interactions rather than due to an additional
hydrogen bond!
- The sigmoidal behavior resulting from the melting
of DNA is indicative of a cooperative process
simply put, DNA denaturation occurs in a
cooperative mannerwhat does this mean?!

- A cooperative process is rather like a domino

effectthe DNA structure resists external abuse in
the form of rising temperature until one of its parts
finally gives in, and when it does, it destabilizes the
remainder of DNA such that it rapidly falls from
grace into pieces (disordered single strands)!

Double-Helix: DNA Renaturation

- DNA denaturation is a reversible process in
that lowering the temperature to around
50 C fully restores the double-helical
conformationthis process is referred to as
DNA renaturation
- But, there is one caveatwhen denatured
DNA is rapidly cooled below its Tm, it attains a
partially base-paired structure in lieu of
returning to its native double-helical
conformationthis is because the
complementary strands do not have
sufficient time to find each other
- In order to avoid such a kinetic trap, DNA is
incubated at a temperature no more than
about 25 C below the Tmsuch incubation
ensures that sufficient thermal energy is
available for partially base-paired regions to
rearrange or anneal by melting and
reforming
- Once fully renatured, the DNA can then be
cooled and frozen without altering its
double-helical conformation

Partially renatured DNA

DNA Conformations

- Owing to its conformational flexibility (albeit somewhat restricted relative to proteins), DNA adopts
distinct structural conformations in solution depending on the solvent and base composition
- Three major conformations of DNA are referred to as the A-DNA, B-DNA, and Z-DNAof these three
conformations, B-DNA is thermodynamically most stable and hence the biologically most common form

- The discussion on double helix has hitherto been solely focused on B-DNAhow does such biologically
most prevalent form of DNA differ from others?

DNA Conformations: Sugar Puckers

C3-endo (deoxy)ribose [A-DNA]

C2-endo (deoxy)ribose [B-DNA]

- Owing to their greater flexibility, 5-membered sugar rings (furanoses) such as those of
(deoxy)ribose are rarely coplanar in that certain atoms protrude or pucker out of the plane
of the ring in order to minimize steric clashes between attached substituents
- Such non-planar conformation of sugar rings is referred to as ring puckeringrecall 1.2 (the
chair conformations of 6-membered glucopyranose)
- In the case of furanoses, ring puckers are designated according to whether the atom n is out of
plane on the same side (n-endo) or on the opposite side (n-exo) as the C5 moietythus C3endo indicates that the C3 atom is on the same face as C5 moiety, whereas C3-exo indicates
that the C3 atom is on the opposite face of C5

- C2-endo and C3-endo (deoxy)ribose puckers are in thermodynamic equilibrium and form the
basis of conformational differences in A-DNA, B-DNA, and Z-DNA (unstable/transient)

DNA Conformations: Structural Comparison

DNA Conformations: View Perpendicular to the Helical Axis

DNA Conformations: View Along the Helical Axis

A-DNA
Hollow core
Wide and compact

B-DNA
Semi-solid core
Thin and elongated

Z-DNA
Solid core
Thin and compact

- The three forms are in equilibrium exchange with each with the B-DNA being the
dominant structure under physiological conditions
- Dehydrating conditions (eg desiccation or dehydration of B-DNA upon binding of
certain proteins) may favor the A-DNA conformation
- Chemical modifications of B-DNA (eg methylation of bases such as cytosine and
adenine) may favor the Z-DNA conformation

Exercise 4.2a
- Draw the complete structure of a nucleoside triphosphate before and after
it becomes incorporated into a polynucleotide chain
- Describe the structural differences between A-, B-, and Z-DNA, including
handedness, diameter, and presence of grooves
- Explain why most nucleotides adopt the anti-conformation
- In what nucleic acid conformation(s) does the ribose pucker with C2 endo?
C3 endo?
- Describe the forces that stabilize nucleic acid structure. Which is most
important?
- Explain the molecular events of nucleic acid denaturation and renaturation

Section 4.2b:
RNA Structure

Synopsis 4.2b
- RNA (ribonucleic acid) is a single-stranded biopolymer comprised of four
constituent units or building blocks referred to as ribonucleotidesthe
base components of which in general are adenine (A), cytosine (C), guanine
(G), and uracil (U)tRNA also contains hypoxanthine (I)
- Like DNA, phosphodiester bonds link ribonucleotide residues in RNA
- Unlike DNA, RNA molecules are usually single-stranded but may adopt
secondary structure by virtue of their ability to engage in intra-strand
base pairswherein A base pairs with U, and C with G/I
- Of the many forms that it adopts, messenger RNA (mRNA) is the only coding
RNAie it is translated into proteins!
- In addition to mRNA, RNA also exists in the form of so-called noncoding
RNA or ncRNA species involved in orchestrating numerous cellular
functions such as RNA translation, RNA splicing, and gene regulation
- Two most abundant forms of ncRNA in cells include:
(1) Ribosomal RNA (rRNA)a constituent of ribosomes
(2) Transfer RNA (tRNA)a component of translational machinery

Single-Stranded RNA (ssRNA)

- RNA differs from DNA in two major respects:

(1) Deoxyribose (2-H) is replaced with ribose (2-OH)
(2) Thymine (5-methyluracil) is replaced with uracil (5-H)
- The fact that the RNA is predominantly a single-stranded (ssRNA) polymer is largely
due to the ribose sugarthe steric clashes between 2-OH moieties on the ribose
sugar of ribonucleotides prevent the formation of a double helix!

Double-Stranded RNA (dsRNA)

loop

- RNA can also adopt secondary structure via

intramolecular base-pairing of long stretches
of complimentary ribonucleotides within the
same strand to form double-stranded (dsRNA)
structures

- Such intra-strand base-pairing (due to A-U

and G-C base pairs)as opposed to interstrand base-pairing in DNAresults in the
formation of RNA structures referred to as
stem-loop or hairpin loop fold

stem

RNA-DNA Double Helix

DNA
strand

In the presence of complementary DNA

strands, RNA can also assume double helix
conformation that usually resembles that
of A-DNA or a mixture of A-DNA/B-DNA

Short stretches of such hybrid RNA-DNA

double helices are required during the:
(1) Initiation of DNA replication, where
they serve as RNA primers (see 4.3)
(2) Transcription of RNA on DNA templates
(see 4.4)

RNA
strand

Exercise 4.2b

- List the structural differences between DNA and RNA

- What structures can RNA adopt alone and in complex with
complementary strands of DNA?

Section 4.2c:
DNA Sequencing

Synopsis 4.2c

In the laboratory, nucleic acids can be cleaved at specific

sequences by restriction enzymes

Nucleic acid fragments can be separated on the basis of

their size using gel electrophoresis

After separation, DNA fragments can be sequenced via a

method referred to as the chain-terminator method

The human genome contains ~23,000 genes,

corresponding to about 1.2% of its 3 billion nucleotides

Restriction Endonucleases: Restriction Sites

Restriction
Enzyme

Restriction
Site

Microorganism

Restriction endonucleases are protein enzymes that cleave doublestranded DNA (dsDNA) at specific sites called the restriction sites

Restriction Endonucleases: DNA Cleavage

Sticky ends

Blunt ends

- Restriction endonucleases generally cleave DNA at palindromessequences that are identical

on both strands and related by two-fold symmetry (they read the same when rotated by 180
about an axis perpendicular to the plane of the page)
- Words such as ROTOR and RADAR are palindromesthey read the same forward and backward!
- Restriction endonucleases can cleave DNA in a staggered manner to generate dsDNA fragments
with overhangs or sticky ends
- Restriction endonucleases can also cut DNA straight through or at the symmetry axis to produce
dsDNA fragments with blunt ends

Agarose Gel Electrophoresis

Gel Apparatus
Separation of DNA fragments on a gel after
treatment with a restriction enzyme

Electrophoretogram
Analysis of separated DNA
fragments on the gel

- After treatment with restriction endonucleases, DNA fragments can be separated on agarose gel
electrophoresisa method that primarily separates molecules on the basis of their size

- Following their separation, the gel can be stained with DNA-binding fluorescent dyes (eg ethidium
bromide) and the DNA fragments can be visualized and purified for sequencing

Chain-Terminator Method: DNA Polymerase

- DNA polymerase converts single-stranded DNA (ssDNA) template into double-stranded (dsDNA)
- ssDNA template can be generated by heating dsDNA to break up the hydrogen bonds holding
individual strands together

- DNA polymerase adds nucleotides to the 3-end of a nascent chain base-paired to the
complementary strandthat is it requires the priming of the template strand!
- Such priming is achieved using a short polynucleotide (typically 20-40bp long) complementary in
sequence to the 3-end of the ssDNA templatethis is called the primer
- DNA polymerase elongates the 3-end of such a primer by stepwise addition of complementary
nucleotides

Chain-Terminator Method: Sanger Sequencing

ddATP

ddGTP

2,3-Dideoxynucleoside triphosphate (ddNTP)

ddCTP

ddTTP

- In the classical Sanger method, DNA template is incubated with:

(a) DNA polymerase
(b) Complementary primer
(c) 4 x deoxynucleoside triphosphates (dNTPs)
(d) 4 x differentially-tagged fluorescent ddNTPs
- When the ddNTP is incorporated into the complementary strand in place of dNTP
analog, chain growth is terminated due to the lack of a free 3-OH group!
- This generates a series of truncated DNA fragments (that differ by one
nucleotide) with differential fluorescent-tagged 3-ddNTP
- Gel electrophoresis separates differentially tagged fluorescent DNA fragments on
the basis of size
- The first complete map of human genome in 2001 was obtained using the Sanger
method

Frederick Sanger
(1918-2013)

Chain-Terminator Method: Automation

- In the modern automated chainterminator method, electrophoresis is
conducted in a capillary tube in order
to achieve higher sample throughput
- In this so-called capillary
electrophoresis (CE), negatively
charged DNA fragments migrate
through an electrolyte (eg a solution
containing hydroxyethyl cellulose) in
lieu of a traditional polyacrylamide gel

- After fractionation via CE, DNA

fragments (differing by one nucleotide)
are detected with a LASER on-the-fly
ie as they exit the capillary tube instead
of being analyzed later!
- This generates a series of colored peaks
that are automatically recorded and
converted to a chromatogram for
further analysis

Chain-Terminator Method: Data Analysis

- Each colored profilecomprised of successive peaksrepresents the sequence

pattern of a DNA fragment harboring one of the four ddNTPs

- Each peakwithin a colored profiledenotes the 3-end sequence of that pattern

- The sequence indicated is complimentary to the original template strand

Exercise 4.2c
-

Explain how restriction enzymes generate either sticky ends or blunt

ends

Why do the smallest fragments of DNA move the farthest during

electrophoresis?

List all the components and explain their purpose in the reaction
mixture used for the dideoxy DNA sequencing method