Nucleic Acids: General Introductory article

Properties . Common Structures

Article Contents

Garrett A Soukup, Creighton University School of Medicine, Omaha, Nebraska, USA . Detection, Quantitation and Characterization
. Base Stacking and Base Pairing

Nucleic acids are biological polymers generally composed of four different monomer units . Unusual Base Pairing Patterns

termed nucleotides. The unit length of one molecule can be as much as one billion . Hairpins, Bends and Cruciforms

nucleotides. Although the biological roles that nucleic acids fulfil are diverse, the structure . Thermal Stability of Duplexes

and function of each molecule is elaborated from a common set of general properties. . Hydrolytic Degradation
. Deamination and Depurination
. Oxidative Reactions
Common Structures . Triplexes and Quadruplexes

The most recognized form of nucleic acids is the double

helix. In this form, two molecules twist around each other
to create a periodic structure. In order to appreciate how Nucleic acid polymers naturally exist as one of two
nucleic acids establish these intriguing forms, a fairly similar chemical forms: ribonucleic acid (RNA) or
detailed understanding of nucleic acid chemical composi- deoxyribonucleic acid (DNA) (Figure 1). The monomer
tion is required. units of nucleic acids are nucleotides, each of which is the
phosphate ester of a corresponding nucleoside. Generally,

Major groove

H
G•C
N
H (HO) 3′
H O
O N N
H O
deoxyguanosine N N O
5′ (guanosine) O
H cytosine deoxycytidine
N N N −
O (cytidine) O
(H)
guanine H P
O CH 3
O
O
5
4 6 (HO)
H H 1 H O
O H 3
N N 2 N
O (OH) H
N O
P 7 6 N O
− deoxyadenosine 5 1 5′
O (adenosine)
8
2 thymine O
9 4
3 (uracil)
N deoxythymidine
O 5′ χ N
O adenine (uridine)
4′ 1′
A•T(U)
2′
3′

O H
(OH)
3′ Minor groove

DNA
(RNA)

Figure 1 Chemical structure of DNA and RNA polymers. The bases are shown in blue while the phosphate and sugar backbone is shown in red. Chemical
differences that distinguish RNA structure from DNA structure are shown in parentheses. The nomenclature for each base and its corresponding
nucleoside is indicated. Atoms are numbered for one sugar, one purine base and one pyrimidine base. The glycosidic bond (w) of one nucleoside is
labelled, and the base of each nucleoside is shown in an anti conformation. Rotation about the glycosidic bond by 1808 would orient the base in a syn
conformation. A single phosphodiester linkage is shown between adjacent nucleosides on each strand. Shaded arrows highlight the antiparallel orientation
of each polynucleotide strand in a duplex. Dashed lines represent hydrogen bonds in each base pair, and the major and minor groove edges of each base
pair are indicated. Note that bond lengths are not proportional and may be exaggerated in the interest of clarity.

ENCYCLOPEDIA OF LIFE SCIENCES © 2001, John Wiley & Sons, Ltd. www.els.net 1
 Nucleic Acids: General Properties

there are four different nucleosides that comprise RNA or tide strand in a duplex dictates the sequence of the
DNA and each contains a five-carbon (pentose) sugar and complementary strand. For example, an oligonucleotide
a nitrogen-containing base. In RNA the sugar is d-ribose, of sequence 5’-GGATTACCTT-3’ can bind or hybridize to
while in DNA the sugar is 2’-deoxy-d-ribose. In this a complementary oligonucleotide of sequence 5’-AAGG-
respect, RNA and DNA differ by the presence or absence, TAATCC-3’:
respectively, of a hydroxyl (OH) group at the second
carbon (C2’) of the sugar. The bases are categorized by 5 ′ - G G AT TA C C T T- 3 ′
their monocyclic or bicyclic nature and are respectively 3′ - C C T A AT G G A A - 5′
referred to as pyrimidines or purines. In DNA, the
pyrimidines are cytosine (C) and thymine (T), whereas Base pairing of nucleic acid polymers through hydrogen-
the purines are guanine (G) and adenine (A). RNA bond formation is commonly referred to as secondary
contains the same bases with the exception that uracil structure.
(U) is substituted for thymine. Therefore, another differ- Nucleic acids can achieve a number of helical conforma-
ence between DNA and RNA is the presence or absence, tions that are affected by the context or environment in
respectively, of a methyl (CH3) group at C5 of one which the molecules reside, or the chemical composition of
pyrimidine base. the polymer (RNA versus DNA). Three such helical
Despite the chemical distinctions between the monomer conformations are B-, A- and Z-form (Figure 2). These
units of RNA and DNA, the polymers are constructed forms differ with respect to various parameters, that
identically (Figure 1). Each base is joined to a sugar by a b- describe the three-dimensional conformation of the
glycosidic bond between a ring nitrogen on the base (N9 in double-helical structure (Table 1). Although duplex DNA
purines or N1 in pyrimidines) and C1’ of the pentose sugar has demonstrated the capability to take on each of these
to form a nucleoside. In this manner, the sugar is forms under appropriate conditions, B-form DNA is most
constrained in a furanose ring configuration in which C5’ biologically relevant as it persists under physiological
lies on the same side of the ring as the base. Each nucleoside conditions. In the B-form helix, base pairs stack one upon
derives its name from the constituent base and sugar. For the next virtually perpendicular to and centred on the helix
example, adenine and d-ribose constitute adenosine (A), axis. There are 10 base pairs per turn of the helix, and the
whereas adenine and 2’-deoxy-d-ribose constitute deox- structure has a wide major groove and narrow minor
yadenosine (dA). Nucleotides are the phosphate esters of groove that are established by which edge of the base pair is
nucleosides, where any hydroxyl group of the pentose presented (Figure 1). Furthermore, each sugar exhibits a
sugar can be esterified. For example, esterification of the C2’-endo conformation, meaning that C2’ lies above the
C5’ hydroxyl of adenosine by a single phosphate (PO4) plane of the furanose ring in the direction of the base and
group yields adenosine 5’-monophosphate (AMP). Such C5’. Duplex RNA, on the other hand, strictly conforms to
nucleotides are the monomer units of polynucleotides in an A-form helix, as the C2’ hydroxyl group present on each
which the C5’ and C3’ hydroxyls of adjacent nucleosides
are joined by phosphodiester linkages. Consequently, a
given polynucleotide has one 5’ terminus and one 3’
terminus, and the order of the bases establishes the
sequence of the polymer. Base sequence is a key feature
that distinguishes one nucleic acid molecule from another
and is typically referred to as primary structure.
It is the primary structure of nucleotide polymers that
dictates their specific interaction in the formation of
double-helical structures. Hydrogen bonds formed be-
tween bases on oppositely oriented or antiparallel poly-
nucleotide strands establish a predictable means of contact
(Figure 1). Hydrogen bonds are formed between appro-
priately positioned hydrogen bond donors and acceptors
presented on each base. The covalently bonded hydrogens
of amino (NH2) and imino (NH) groups serve as donors,
while the nonbonded electron pairs of oxygen and
ring nitrogen serve as acceptors. A set of hydrogen-bonded
bases positioned on opposing strands represents a
base pair. Interestingly, only pairing of guanine with
Figure 2 Three-dimensional space-filling models of B-, A- and Z-form
cytosine (G
C), and adenine with thymine (A
T) or uracil helices. Bases are coloured blue and the phosphate and sugar backbones
(A
U) maintains a regular geometry in a double-helical are coloured red. Major (M) and minor (m) grooves are indicated for each
structure. Consequently, the sequence of one polynucleo- double helix.

2
 Nucleic Acids: General Properties

Table 1 Average structural parameters for various helical forms

Parameter B-form A-form Z-form
Helix handedness right right left
Base pairs/turn 10 11 12
Base pairs/repeating unit 1 1 2
w orientation anti anti anti, syn
Sugar conformation C2’-endo C3’-endo C2’-endo, C3’-endo
Rise/base pair (Å)a 3.4 2.6 3.7
Major groove width (Å) 11.7 2.7 8.8
Major groove depth (Å) 8.8 13.5 3.7
Minor groove width (Å) 5.7 11.0 2.0
Minor groove depth (Å) 7.5 2.8 13.8
a
One angstrom (Å) is equal to 10 2 10 m.

d-ribose sugar is not accommodated in the B-form Methods for detecting nucleic acids are varied. Histori-
conformation. cally, nucleic acids have been identified based on their
In the A-form helix, base pairs are stacked around and ability to bind to basic dyes. This principle of rendering
are tilted with respect to the helix axis, effectively leaving a nucleic acid molecules ‘visible’ through interaction with a
hole down the centre of the helix. There are 11 base pairs second compound also includes fluorescent dyes. Basic
per turn, the major groove is narrow and deep, the minor dyes exploit the negative charge of the polymer and bind
groove is wide and shallow, and each sugar has a C3’-endo through electrostatic interactions, thereby ‘colouring’ the
conformation. However, both A- and B-form helices are polymer. Fluorescent dyes such as ethidium bromide are
right-handed, indicating the direction in which the typically planar polycyclic compounds similar to the bases.
phosphate and sugar backbone of each strand turns They interact with nucleic acids through hydrophobic
around the helix axis. Additionally, the orientation of interactions by intercalating or stacking between adjacent
each glycosidic bond (w) is anti, meaning that the base is base pairs. In this environment the dye fluoresces with
positioned away from the sugar rather than over it (syn). greater intensity when excited with the appropriate
The Z-form helix is unique in that the glycosidic bond wavelength of light. Another means of visualizing nucleic
orientation and sugar conformation alternate regularly acid polymers is through the incorporation of radio-
between anti and syn, and C2’-endo and C3’-endo, nuclides such as 32P, a radioactive form of phosphorus.
respectively, from one nucleoside to the next in each Nucleic acids containing 32P are easily detected because
strand. Each base pair of the duplex is made up of they emit radiation that exposes photographic film. These
nucleotides in alternate conformations. These structural techniques for visualizing the whereabouts of nucleic acid
characteristics not only give the double helix a reverse or polymers are particularly useful when coupled with
left-handed turn, but they cause the phosphate and sugar another method termed gel electrophoresis. Here, the
backbone to trace a zigzag course. The Z-form helix negative charge of the polymer is utilized to affect its
requires 12 base pairs per turn, and has a major groove that migration through a gelatinous matrix placed in a high-
is wide and shallow and a minor groove that is narrow and voltage electric field. Nucleic acid molecules are thus
deep. Duplex DNA can acquire a Z-form conformation separated based on their charge, length and shape.
under conditions of high salt or helical stress caused by Consequently, gel electrophoresis is widely used to
physically constraining the molecule in an underwound or characterize nucleic acid species.
supercoiled state. A widely useful characteristic of nucleic acid polymers is
that the bases absorb ultraviolet (UV) light of a specific
wavelength in a manner that is proportional to their
Detection, Quantitation and concentration. This spectroscopic property can therefore
be exploited both to detect and to quantitate nucleic acids.
Characterization Moreover, UV absorption can be used to characterize
nucleic acid secondary structure using a variety of
Analyses of nucleic acid polymers can be relatively simple techniques. As will be discussed later, the temperature-
or highly complex. However, each method of analysis relies dependence or thermal stability of duplex DNA or RNA
on some general chemical property of the polymer or can be monitored directly by UV absorption. Further-
biophysical principle. Some of the methods for detecting, more, other techniques such as optical rotary dispersion
quantitating and characterizing nucleic acids are briefly (ORD) and circular dichroism (CD) utilize UV absorption
considered.

3
 Nucleic Acids: General Properties

to characterize secondary structure. Such techniques can contributes significantly to the overall stability of double
be used to distinguish one helical conformation from helices. Additionally, the stacking energy between two
another, or to analyse transitions between single-stranded bases is sequence-dependent due to the different electronic
random coils and double-stranded helices. Another properties of individual bases. In a single-stranded
method used to characterize nucleic acid structure is polynucleotide, a purine–purine stack is generally more
ultracentrifugation. Like gel electrophoresis, centrifuga- stable than a purine–pyrimidine stack, which in turn is
tion can be used to separate polynucleotide species based more stable than a pyrimidine–pyrimidine stack. How-
on their physical parameters. For a given molecule, the rate ever, in double-stranded helices, adjacent base pairs exhibit
of sedimentation by centrifugation is largely affected by the different stacking energies depending upon the identity of
mass and shape of the molecule and the viscosity of the each base. For example, the stacking energy is greater for
solute. 5’-AT-3’ pairing to 5’-AT-3’ than for 5’-TA-3’ pairing to 5’-
While the aforementioned techniques can provide a TA-3’. Therefore, base stacking and base pairing con-
wealth of information concerning polynucleotide confor- tribute to sequence-dependent variations in helix stability.
mation, they usually afford a low-resolution description of
nucleic acid structure. Some methods used to provide high-
or atomic-resolution structural data involve nuclear
magnetic resonance (NMR) spectroscopy and X-ray Unusual Base Pairing Patterns
diffraction. NMR spectroscopy is based on the absorption
of radiofrequency electromagnetic radiation by atomic Hydrogen bonds are only weakly directional and are
nuclei. Absorption frequency is dependent upon nuclei approximately 30 times weaker than covalent bonds.
type, context and proximity to other nuclei. X-ray Consequently, hydrogen bonds can be bent or stretched
diffraction exploits the ability of atoms arranged in a to a certain degree. The flexibility of hydrogen bonds
regular or crystalline array to diffract electromagnetic permits a multitude of base-pairing interactions. In
radiation of wavelengths in the angstrom (Å) range. The addition to the G
C, A
T and A
U pairs typical of B- and
regular or periodic spacing of atoms within the crystal A-form helices, there are 25 possible base-pairing interac-
diffract X-rays, and distances in the resulting diffraction tions that contain at least two hydrogen bonds. A number
pattern are inversely proportional to distances between of these base pairs have been found to exist in certain
atoms. Any information concerning the positioning of nucleic acid structures.
atoms in space, such as that collected by NMR spectro- In double-stranded nucleic acids, a number of unusual
scopy or X-ray diffraction, can be used to generate a three- base pairs or mismatches can be accommodated without
dimensional model of a molecule. greatly perturbing the three-dimensional structure of the
double helix (Figure 3). For example, the overall geometry
of the G
T or G
U pair closely resembles that of the G
C
pair. However, the G
T(U) pair is less stable because it
Base Stacking and Base Pairing forms only two hydrogen bonds and is aligned slightly
differently. Another interaction that can be accommo-
As previously mentioned, two nucleic acid molecules can dated in a double helix is G
A pairing. The G
A pair has a
associate to form double-stranded helical structures. The similar geometry with adenosine in either a syn or anti
helix is mainly stabilized by base stacking and base-pairing conformation. Two hydrogen bonds are formed in either
interactions. The formation of hydrogen bonds between case, although N1 or N7 of adenosine alternately serves as
bases can be quite versatile. However, in the case of the B- a hydrogen bond acceptor. The repertoire of potential
or A-form helix, only base pairs composed of G
C, A
T or base-pairing interactions is expanded under conditions in
A
U maintain a regular geometry. Three hydrogen bonds which hydrogen bond donors and acceptors are deproto-
are formed between appropriately positioned donor and nated or protonated, respectively (Table 2). Under acidic
acceptor groups in a G
C base pair, and two hydrogen conditions, N1 of adenosine is converted from a hydrogen
bonds are formed in the A
T(U) pair (Figure 2). Therefore, bond acceptor to a donor by protonation. In this form,
G
C pairs afford greater stability to a double helix than do adenosine can form two hydrogen bonds with cytidine to
A
T(U) pairs. establish a C
A 1 pair.
Base stacking occurs between adjacent bases in a The biological role of DNA as the storage medium for
nucleotide polymer. This interaction is largely driven by heritable or genetic information necessitates that the
a hydrophobic effect wherein each heterocyclic base canonical G
C and A
T base pairs be strictly enforced.
decreases the extent of unfavourable solvent interaction Any mismatches or degeneracy in the molecular code that
by interacting with another. In other words, one planar directs how genetic information is replicated would be
base stacks upon the next to reduce the solvent-exposed detrimental to an organism’s survival. Therefore, repair
surface area of each individual base. Base stacking occurs mechanisms are in place to ensure the integrity of the DNA
in both single- and double-stranded polynucleotides and double helix. Such strict constraints are not imposed upon

4
 Nucleic Acids: General Properties

(H)
CH 3
O
H N
N N N N R
H R H
O O N N
O

N H N H
N N
H H
N N N N N N
R R H
H

G•T(U) G•A

H N
N N
H N
H H H
N N R
N H O N N
R
N N N H
N H + N
H
N O N N N
R R H

+
C•A G•A(syn)

Figure 3 Unusual base pairs that may be accommodated in double-helical structures. Hydrogen bonds are indicated by dashed lines and R groups
represent the continuation of polynucleotide structure through the phosphate and sugar backbone. All bases are in an anti configuration unless otherwise
specified.

Table 2 pKa values for bases in nucleosides at zero ionic Hairpins, Bends and Cruciforms
strength
In the formation of a double-helical structure, a poly-
Nucleoside Site of protonation pKaa nucleotide may interact with itself rather than a second
Adenosine N1 3.5 polynucleotide. Structures formed by base pairing between
Cytidine N3 4.2 complementary regions of the same polynucleotide are
Guanosine N1 9.4 termed hairpins (Figure 4a). Almost any RNA polynucleo-
Uridine N3 9.4 tide can ‘fold’ to form one or many hairpin structures. The
Thymidine N3 9.9 stability of a hairpin is dependent upon the sequence and
a length of the base-paired stem and the single-stranded loop
pKa is the pH at which half of the sites are protonated and half are
deprotonated.
that connects each segment of the stem. Of course, longer
stem regions or those having a greater proportion of G
C
pairs are more stable. However, certain loop sequences
greatly enhance the formation and stability of RNA
hairpin structures. Such sequences conform to either a 5’-
RNA polynucleotides, which exhibit great versatility with GNRA-3’ or 5’-UNCG-3’ motif, where N represents any
respect to base-pairing interactions. In fact, unusual base- base identity and R indicates purines. These tetraloop
pairing interactions abound in biologically relevant RNA sequences are stabilized by specific hydrogen bonding and
structures such as transfer RNAs (tRNAs), ribosomal base-stacking interactions that facilitate a turn in the
RNA (rRNAs), and catalytic RNAs (ribozymes). Further- phosphate and sugar backbone.
more, they contribute significantly to the unique structure While hairpin formation is a mainstay in RNA structure,
and activity of each RNA. it does not play such a pivotal role in DNA structure.
However, double-stranded DNA containing an inverted

5
 Nucleic Acids: General Properties

5’– G G A C G
U ized sequence motif known to cause an intrinsic bend in
U
5’– G G A C G U U C G C G U U C –3’ I · I I I
C duplex DNA is an A-tract (Figure 4c). A-tracts are segments
3’– C U U G C
(a) G of five or six consecutive A
T pairs in which base stacking
relative to the helix axis differs slightly from that of base
5’– C A A G T A G A C C T T T T G G T C T C G T A A –3’
I I I I I I I I I I I I I I I I I I I I I I I I pairs in the adjacent helical segments. Curvature results
(b) 3’– G T T C A T C T G G A A A A C C A G A G C A T T –5’
either from cumulative incremental bends throughout the
A-tract, or from bends that occur at each junction between
the A-tract and adjacent helical segments. Either way, the
bend is considered as centred on the A-tract and the helix
TT axis is deflected 208. If multiple A-tracts are phased with
T T the helical repeat of DNA, the bends occur on the same side
C–G
C–G of the helix and their effects are additive. Many other
A–T sequences such as 5’-RGCY-3’, where R is a purine and Y is
G–C
a pyrimidine, can similarly cause DNA bending. There-
5’– C A A G T A – T C G T A A –3’
I I I I I I I I I I fore, the precise local structure of double-helical DNA
3’– G T T C A T – A G C A T T –5’
does not always conform to the ideal helix.
C–G
T–A
G–C
G–C
A A
AA Thermal Stability of Duplexes
5’– G A T G C A A A A A C A T C C A A A A A G T T C G –3’ Duplex nucleic acid structures can be disrupted or
I I I I I / / / / / I I I I I / / / / / I I I I I
3’– C T A C G T T T T T G T A G G T T T T T C A A G C –5’ denatured at high temperature. The so-called ‘melting’ of
double helices can be monitored easily because base
stacking and base-pairing interactions significantly reduce
UV absorption. As the temperature of a solution contain-
ing a polynucleotide duplex is raised, the absorption of UV
(c) light will greatly increase around the temperature at which
Figure 4 Specific double-helical structures. (a) Hairpin formation. An RNA
the two strands separate into single-stranded polynucleo-
hairpin with a UNCG tetraloop sequence is shown. Self-complementary tides. The transition in UV absorption typically occurs
regions are indicated by open and filled wedges. (b) Cruciform formation. over a 48C to 88C range. The midpoint of the transition or
A DNA duplex containing an inverted repeat sequence is shown. (c) melting curve identifies the melting temperature (Tm) at
Intrinsic DNA bending. A sequence containing two A-tracts phased with which half of the polynucleotide species is duplex and half
the helical repeat of B-DNA is shown. Arrows indicate bends centred on
each A-tract. A schematic representation of the double helix illustrates the
is single-stranded. In general, DNA duplexes are relatively
cumulative bend affected by each phased A-tract. Thick lines trace the path less stable to thermal denaturation than RNA duplexes or
of each phosphodiester backbone, while the thin line denotes the helix axis. RNA
DNA duplexes.
The Tm for a given duplex is otherwise affected by a
number of factors. The major determinants include
repeat sequence can reorganize into a cruciform structure sequence length, G
C content and salt concentration. As
under conditions that promote unwinding of the helix previously mentioned in regard to hairpin formation,
(Figure 4b). The local unfolding of the double helix affords longer duplexes or those having a higher proportion of G
C
each strand of the inverted repeat an opportunity to base pairs are more stable and therefore will have a higher Tm.
pair to itself rather than the complementary strand. In this Monovalent cations, such as sodium (Na 1 ) and potassium
manner, the double-stranded helix is interrupted by two (K 1 ), stabilize double helices by partially alleviating
hairpin structures, one on each strand, to form a four-helix negative charge along the phosphodiester backbone.
junction. The formation of cruciform DNA is promoted by Consequently, higher salt concentration will also increase
supercoiling, a natural phenomenon in which the double the Tm of a given polynucleotide duplex. Of course, the
helix is underwound. However, whether cruciform DNA inclusion of mismatched bases in a duplex will generally
serves a significant biological role is unknown. have a destabilizing effect and decrease Tm. In this regard,
Although very long double-helical structures may gently thermal stability has been an important means of assessing
curve or bend, segments of double-stranded DNA shorter the extent to which alternate base-pairing arrangements or
than 150 base pairs in length are characteristically straight other perturbations affect nucleic acid helices. These and
and rigid. However, localized bends in duplex DNA can be other thorough investigations of the sequence dependence
induced by external factors such as proteins that bind of helix stability have made it possible to predict
DNA, or they may be intrinsic structural features thermodynamically favourable secondary structures from
attributable to specific base sequences. A well-character- sequence alone.

6
 Nucleic Acids: General Properties

Hydrolytic Degradation inherently positioned in proximity to the phosphorus

centre in the phosphodiester backbone. Furthermore, the
Both RNA and DNA can be degraded through cleavage of reaction is promoted either by deprotonation of the C2’
the phosphodiester backbone. However, the chemical hydroxyl or protonation of the C5’ oxygen. Consequently,
stability of each polymer is drastically different. RNA is RNAs are easily degraded in alkaline or acidic solutions.
uniquely subject to a transesterification reaction in which A similar mechanism of phosphodiester cleavage that is
the C2’ oxygen serves as a nucleophile for attack on the relevant to both RNA and DNA polymers is one in which a
adjacent phosphorus (Figure 5a). The reaction proceeds second molecule serves as the attacking nucleophile
through a pentacoordinate phosphate intermediate where (Figure 5b). The reaction is similarly promoted by
the bond formed between the C2’ oxygen and the deprotonation of the attacking group and protonation of
phosphorus centre is in line with the bond to be broken the leaving group. However, the products terminate with a
between the phosphorus centre and the C5’ oxygen. The C5’ phosphate and C3’ hydroxyl. Water can serve as a
reaction products terminate with a C5’ hydroxyl and C2’, nucleophile in the spontaneous hydrolytic degradation of
C3’-cyclic phosphate. This cyclizing mechanism of RNA RNA or DNA polymers, although the reaction is
transesterification occurs easily because the nucleophile is approximately 100 000 times less likely to occur than

R O O Base R O Base R O Base

O O

O O H B
O O H B O O H B
P P P
O− Base O O− Base O H −
Base O O O O
O O O− O
H H
A
A A
H O O H O O H O O

(a) R R R

R O O Base R O R O
O Base O Base

O O−
A H O H (OH) H (OH) H (OH)
A H O A H O
O − P
P O− O
Base O P Base O
Base O
O
O
O O− O
O R
O R
R H B H B
(HO)H O H B (HO)H O
(HO)H O
(b) R R
R

H H H H O O H H O
N N N
H H
− + H H −
H H
N
HSO3
H N –NH3 H N −HSO3 N N HNO2 N
pH 5 H H2O H pH 8
N O N O –O S N O N O N O N O
−
O3S 3
(c) R R R R (d) R R

O H O O
N H +N H N H
N N N
R O N H R O N H H2O R O OH N H
O N N O N N O N N
H pH<2 H H H

O H(OH) O H(OH) O H(OH)

(e) R R R

O O O O
H H H3C
N N H3 C H H
N •OH N N •OH HO N
HO
N H N H HO
N N N N N O N O
H
(f) R H R H (g) R R

Figure 5 Reactions affecting nucleic acid structure. (a) Cyclizing mechanism of RNA transesterification. (b) Hydrolysis of RNA or DNA. (c) Deamination of
cytosine by bisulfite. (d) Deamination of cytosine by nitrous acid. (e) Depurination of guanosine. (f) Hydroxyl radical conversion of guanine to 8-
hydroxyguanine. (g) Hydroxyl radical conversion of thymine to thymine glycol. The participation of a base (B) or acid (A) is shown in the first two reactions.
R groups represent the continuation of polynucleotide structure through the phosphate and sugar backbone.

7
 Nucleic Acids: General Properties

RNA transesterification. Therefore, the phosphate and

sugar backbone of DNA possesses relatively immense
chemical stability by virtue of the fact that each sugar lacks
a C2’ hydroxyl group. Indeed, DNA is the most stable of
the biological polymers: an attribute that ensures the
integrity of genetic information.

Deamination and Depurination

The bases in polynucleotides are also subject to various
reactions that specifically change the chemical nature of the
polymer. In biological systems, any chemical alteration of
a base might lead to a permanent change or heritable
mutation in sequence. For example, bisulfite and nitrous
acid promote deamination or removal of an amino group
from cytosine (Figures 5c and 5d, respectively). Each
reaction results in conversion of cytosine to uracil. In
DNA, cytosine deamination will ultimately lead to
conversion of a G
C base pair to an A
T base pair if left
uncorrected. Additionally, the glycosidic bonds of purine
nucleosides are susceptible to acid hydrolysis by protona-
tion at N7 of guanosine or adenosine (Figure 5e). Depur-
ination of DNA is more favourable than that of RNA,
although the formation of an abasic site in the polynucleo-
tide results in either case. A complete loss of sequence
information represented by an abasic site in DNA can
likewise cause a permanent mutation. These aspects of
nucleic acid chemistry underlie the importance of biologi-
cal DNA repair mechanisms.

Oxidative Reactions
Other insults to the chemical integrity of DNA come from
strong oxidizing agents such as hydroxyl radicals gener-
ated by ionizing radiation or incomplete metabolic
reduction of oxygen. The incomplete reduction of oxygen Figure 6 Three-dimensional space-filling models and hydrogen-bonding
(O2) to water (H2O) produces superoxide ion, hydrogen patterns for triple and quadruple helices. For the triplex, canonically
peroxide or hydroxyl radicals, the latter of which are paired bases and their phosphate and sugar backbones are coloured
extremely reactive. Hydroxyl radicals can nonspecifically blue and red, respectively, while the bases and backbone of the third strand
are coloured gold and green, respectively. For the quadruplex, bases are
damage DNA by breaking the phosphodiester backbone. coloured blue and the backbone is coloured red. Hydrogen bonds are
Strand scission occurs by a number of mechanisms, each of indicated by dashed lines and R groups represent the continuation of
which produces polynucleotide fragments with different 5’ polynucleotide structure through the phosphate and sugar backbone.
and 3’ terminal products. Alternatively, hydroxyl radicals
can attack and chemically modify the bases of nucleic
acids. Two examples are the production of 8-hydroxygua-
nine and thymine glycol (Figures 5f and 5g, respectively). Triplexes and Quadruplexes
These lesions in the sequence of DNA are suspected to
contribute to mutations that ultimately lead to cancer. As previously discussed, nucleic acids are capable of
Therefore, ionizing radiation and other compounds that forming a multitude of base-pairing interactions beyond
cause DNA damage are typically carcinogenic or cancer- the canonical G
C and A
T(U) pairs characteristic of
causing. duplexes. This diversity of hydrogen-bonding arrange-
ments can be extended to include base triplet and
quadruplet interactions (Figure 6). In much the same way

8
 Nucleic Acids: General Properties

that contiguous base-pairing interactions between two biological activity of naturally occurring proteins that bind
polynucleotide strands form a double-helical structure, DNA.
triplexes or quadruplexes are formed by extended stacks of Quadruplexes are formed by the self-assembly of
base triples or quadruples, respectively. These three- and guanosine-rich polynucleotides. Base quadruples com-
four-stranded helices are therefore stabilized by the same posed entirely of guanosine residues are commonly
interactions as typical duplex polynucleotides. referred to as G-quartets (Figure 6). Stacking of one G-
Triplexes are formed by the interaction of a third quartet upon the next leads to the formation of a
polynucleotide strand with a duplex and are generally monotonous quadruple-helical structure that is typically
stabilized by cations that partially neutralize the negative stabilized by potassium ions (K 1 ). Either RNA or DNA
charge of each phosphodiester backbone. Bases in the third can form quadruplexes, and the orientation of each strand
strand stack in the major groove and form hydrogen bonds with respect to the next can vary. Quadruplex structures
with purine bases in one strand of the duplex. In this may resist both thermal and chemical denaturation and
manner, triple helices can accommodate a number of therefore can have immense structural stability. For this
distinct base combinations that are dependent upon reason, G-quartet formation is believed to have a
polynucleotide type and orientation in the major groove. biological role in maintaining the integrity of the single-
For example, G
G
C and T
A
T (or A
A
T) base triples can stranded G-rich ends of telomeric DNA.
be formed between duplex DNA and a third strand
composed of DNA (Figure 6). The third strand is oriented
antiparallel with respect to the purine strand of the duplex
and may not be composed of RNA. Alternatively, triple Further Reading
helices can be formed between duplex DNA and a third Blackburn GM and Gait MJ (eds) (1990) Nucleic Acids in Chemistry and
strand composed of RNA through the formation of Biology. New York: Oxford University Press.
C 1
G
C and U
A
T base triples. Here, the third strand Bloomfield VA, Crothers DM and Tinoco I (eds) (2000) Nucleic Acids:
binds parallel to the purine strand of the duplex, and the Structures, Properties and Functions. Sausalito, CA: University
complex is more stable under acidic conditions that favour Science Books.
Cantor CR and Schimmel PR (1980) Biophysical Chemistry. New York:
protonation of cytosine residues. In either of these
W.H. Freeman and Company.
manners, DNA or RNA oligonucleotides can be targeted Guschlbauer W (1976) Nucleic Acid Structure. New York: Springer-
to polypurine sites in duplex DNA. This type of site- Verlag.
specific recognition of DNA has been exploited to direct Saenger W (1984) Principles of Nucleic Acid Structure. Springer
DNA cleaving agents to unique sites or to inhibit the Advanced Texts in Chemistry. New York: Springer-Verlag.