Nucleic Acids From Wiley
Nucleic Acids From Wiley
Nucleic Acids From Wiley
Nucleic Acids
Genetic Engineering is a separate keyword.
Helmut Burtscher, Boehringer Mannheim GmbH, Penzberg, Federal Republic of Germany (Chaps. 1 6)
Sibylle Berner, Boehringer Mannheim GmbH, Tutzing, Federal Republic of Germany (Chap. 7)
Rudolf Seibl, Boehringer Mannheim GmbH, Penzberg, Federal Republic of Germany (Chap. 8)
Klaus Muhlegger, Boehringer Mannheim GmbH, Tutzing, Federal Republic of Germany (Chap. 9)
of evolution rst RNA and then DNA came into mon Watson Crick pairing (Fig. 3 A) or by
being [36, 37]. Hoogsteen base pairing (Fig. 3 B). The double-
stranded structure is further stabilized by hydra-
tion of the phosphate groups and hydrophobic
interactions between the aromatic ring systems
that result in stacking of the bases.
2. Structure
2.1. Structure of DNA
The joining of the DNA building blocks by 5 -
and 3 -phosphodiester bonds gives the molecule
polarity (Fig. 1); base sequences are always writ-
ten starting with the 5 -terminus, i.e., in the
5 3 direction. The specic base sequence
of DNA and its ability to form double-stranded
structures according to precisely dened rules Figure 2. Right-handed double-helix of DNA
are of utmost importance for the storage of ge- A = adenine, C = cytosine, G = guanine, T = thymine
netic information and for interactions with other
nucleic acids and proteins. In double-stranded DNA, the bases are
From X-ray analysis data, Crick and densely stacked and there is a cooperative ef-
Watson proposed a double-stranded structure fect between hydrogen bonding and stacking.
for DNA in 1953 in which two antiparallel (i.e., Internal bases can be continuously paired and
5 3 and 3 5 ) polynucleotide chains form unpaired; double-stranded regions open and
a right-handed helix (i.e., looking along the axis form single-stranded bubbles (breathing of
of the helix, the strands are coiled clockwise). DNA). Breathing is more frequent in regions rich
Naturally occurring DNA usually consists of in A T pairs and could be important for inter-
right-handed helices with a major and a minor actions with proteins. DNA helices can exist in
groove (Fig. 2). The hydrophobic bases are lo- various forms (A, B, C, D, and Z) [39] some of
cated inside the helix and the sugar phosphate which are interconvertible depending on the con-
backbone on the outside [38]. Bases that are centration and type of salts present. The helices
opposite each other are paired according to de- always exhibit a degree of microheterogeneity
ned rules as a result of hydrogen bond for- that plays an important part in genetic regula-
mation: adenine always pairs with thymine or tion mechanisms.
uracil and guanine with cytosine. Complemen- The DNA helices can exist in linear form
tary bases can be bound by the more com- (e.g., in the chromosomes of higher organisms)
Nucleic Acids 159
or as closed rings (e.g., in Escherichia coli); the quire recognition of nucleotide sequences in the
molecules can also be twisted (superhelicity or major groove of the B-DNA double helix.
supercoiling). In order to accommodate the large C-DNA helices can be observed at a relative
amount of DNA present in living cells, it must be humidity of 44 66 % in the presence of lithium
packaged as compactly as possible with the help salts. The helix is also right-handed and similar
of proteins and RNA. Proteins can recognize to the B form, but with 9.3 base pairs per turn.
specic binding sites on the DNA. The grooves D-DNA occurs in nature only in sequences
of the DNA helix are large enough to allow pro- with alternating adenine and thymine residues
teins to come into contact with the bases [40]. and in the DNA of the bacteriophage T 2 (T-
Dened regions in DNA can also be recognized DNA). The helix is also right-handed and has 8
with the help of the methylation pattern of the base pairs per turn.
bases (see Section 4.3). The left-handed conformation of Z-DNA has
an alternating sequence of pyrimidines and
purines and is formed in vitro at high salt con-
Forms of DNA. centrations (> 2 mol/L NaCl) or in the pres-
A-DNA can be observed in X-ray analyses at ence of divalent cations (Mg2+ > 0.7 mol/L).
66 % relative humidity. It has 11 base pairs per Unlike the right-handed helices (which have two
turn of the helix, the planes of the base pairs are grooves), this structure forms a single, very deep
tilted away from the vertical helical axis (19 ), groove that penetrates the helix axis. The sugar
the helix is right-handed and has a diameter of phosphate backbone assumes a zig-zag arrange-
ca. 2.3 nm. ment (therefore Z-DNA) with 12 base pairs per
B-DNA is the classical Watson Crick form. turn of the helix. Z-helices can form in vivo at
It represents the structure of DNA at a relative physiological salt concentrations. They are less
humidity of > 92 % and largely corresponds to stable than B-DNA, but are stabilized by su-
that found under physiological conditions. The percoiling, proteins, special ions, and methyla-
helix is also right-handed with about 10.2 10.4 tion [43]. Torsional stress of DNA in vivo can
base pairs per turn and a diameter of ca. 2 nm. favor the formation of Z-DNA [44]. Z-DNA
Single unpaired bases can be looped out of the and B-DNA are interconvertible; part of a DNA
helix and barely disturb the rest of the structure molecule may exist in the B form and another
[41,42]. Protein DNA interactions usually re- part in the Z form.
160 Nucleic Acids
Supercoiling. Circular DNA and DNA bet- Special Structural Elements. Short se-
ween xed sites can be twisted to supercoils. quences are frequently repeated in regulatory
The term supercoiling refers to the curvature of regions. Repeats can be recognized by DNA-
the double helix axis. Supercoiled (superhelical) binding proteins. Owing to DNA breathing,
DNA was discovered in the 1960s in polyoma double-stranded regions (hairpins or stem-loops
virus [45]. Rotation in the direction of winding and cruciform structures) can be formed at re-
is called positive supercoiling and rotation in the peats within a single strand (Fig. 4). This rarely
opposite direction is called negative supercoil- happens in double-stranded DNA because stem
ing. loops are energetically less favorable than lin-
Torsional stress due to negative supercoiling ear double strands. However, it is encountered
can be overcome by the formation of DNA struc- frequently in single-stranded DNA and RNA.
tures other than the B form. Negative supercoil- Supercoiling can promote the formation of cru-
ing is a strong driving force for the stabiliza- ciform structures, whereas transcription inhibits
tion of Z-DNA. Supercoiling makes DNA more it [55]. Hairpins can play a part in replication,
compact, which is very important in DNA pack- transcription, and RNA processing [56].
aging. Almost all naturally occurring superhe-
lical DNAs are underwound (i.e., have negative
superhelices) but overwound DNAs also exist
[46]. The strain produced by over- or underwind-
ing can be accommodated by the formation of
local single-stranded regions which tends to in-
crease with increasing temperature. Breathing
of the DNA (see third paragraph in Section 2.1)
plays an important part here too. A sequence
with > 90 % A T can exist permanently un-
paired in a superhelical molecule. This is impor-
tant for many reactions of DNA. Supercoiling
inuences transcription (see Section ) and vice
versa. Positive supercoils are formed in front of
the transcription apparatus and negative super-
coils behind it; these supercoils are controlled
by enzymes [47].
Homopyrimidine homopurine runs are fre- chromosome and has a single-stranded 3 -end
quently found in regulatory regions of eucary- containing 12 16 nucleotides. Telomeres can
otic genes and are especially sensitive to nucle- associate to form stable, parallel, four-stranded
ases [57]. There is a high tendency to form right- structures (G 4-DNA) [67].
handed structures other than B-DNA in such re-
gions [58].
Although the chains of double-stranded
DNA are normally antiparallel, parallel double-
stranded oligomers have also been found in vitro
[59]. They form a right-handed helix and are
even recognized by several enzymes. They are
less temperature-stable than the corresponding
oligomers.
At homopurine homopyrimidine sections
of the DNA, a homopyrimidine oligonucleotide
can attach itself parallel to the homopurine
strand in the major groove and form a triple he-
lix [58]. Structures of this type can be used for
specic strand cleavage with the help of coupled
ellipticine derivatives or metal chelates [60, 61].
Centromeres are important compact DNA
structures of the eucaryotic chromosome that are
rich in adenine and thymine. Their exact struc-
ture is not known but they are important for the
attachment of the spindle bers during mitosis.
The ends (telomeres) of linear chromosomes
(as in eucaryotic DNA) pose a special problem.
DNA polymerases synthesize DNA from a DNA
template and always require an RNA primer to
start replication. Cleavage of this primer then re-
sults in a small 5 -gap which cannot be closed
by the polymerase. Under normal replication
conditions, the ends should therefore beocme Figure 5. The importance of telomeres
shorter with every cycle of DNA replication (see Without telomere addition (A) newly synthesized DNA
Section 4.1). Special enzymes (telomerases) are strands become shorter; with telomere addition by telom-
erase (B) constant length can be maintained.
responsible for adding telomere repeats to the
chromosome ends to maintain constant length
(Fig. 5): repeats can fold back and provide a 3 - 2.2. Structure of RNA
OH group which serves as a primer for copy-
ing the last segment of a linear DNA molecule. RNA is an unbranched single-stranded poly-
Disturbances in telomeres can lead to aging phe- mer with many intramolecular double-stranded
nomena [62, 63] and a role in carcinogenesis is sections that may account for 50 67 % of
also being discussed. Broken ends of chromo- the molecule. As in DNA, the backbone of
somes that are no longer protected by telom- RNA consists of 3 ,5 -phosphodiester bonds
eres are very susceptible to fusion with other (Fig. 1); however the sugar is ribose (and not de-
DNA ends and to degradation by nucleases [64]. oxyribose) and uracil replaces thymine. Double-
The antiparallel structure and function of telom- stranded RNA cannot form a B-helix because of
eres are highly conserved in all eucaryotes and steric hindrance caused by the 2 -OH groups of
are species specic. They consist of simple, ribose; helices of the A type are, however, pos-
tandemly repeated sequences with clusters of G sible.
residues [65, 66]. The G-rich strand is aligned The functional groups of the nucleotides in
in the 5 3 direction towards the end of the the major groove of the A type of double he-
162 Nucleic Acids
lix found in RNA are not easily accessible to disrupted is denoted the melting temperature. It
proteins [68]. Protein binding to RNA probably depends on the base composition (G C pairs are
occurs via interaction with single-stranded re- more stable than A T pairs). Double-stranded
gions. DNA ranging in size from 100 to > 100 000 base
Four functional RNA families exist: messen- pairs melts at ca. 90 C. In shorter double strands
ger RNA (mRNA), ribosomal RNA (rRNA), a gradual decrease in the melting temperature
transfer RNA (tRNA), and small nuclear RNA is observed. The melting temperature increases
(only in eucaryotes). The structure of tRNAs with increasing salt concentrations because the
has been studied most extensively; about half solubility of the bases decreases and hydropho-
of the ca. 75 90 nucleotides within the tRNA bic interactions are increased. Chemicals that
molecule are paired, resulting in a secondary compete with hydrogen bond formation, such
structure with a stem and three loops similar to as urea or formamide, lower the melting tem-
that of a cloverleaf [69]. perature of DNA. Methanol has a similar effect;
RNA has many different biological functions it increases the solubility of the bases and in-
and exhibits a spectrum of exible structures creases the interaction with water. The melting
that more closely resemble those of proteins of double-stranded DNA is also facilitated by
rather than those of the chemically related DNA solvents such as ethylene glycol, dimethylform-
[70]. RNAs have secondary structuresdouble- amide, dimethyl sulfoxide; low ionic strength;
stranded sections, hairpins, internal loops, and or extreme pH values. DNA can be denatured at
bulged bases. With unpaired nucleotides pro- an alkaline pH because the keto enol equilibria
nounced tertiary structures are formed in addi- of the bases are shifted preventing these groups
tion to the secondary structure. Examples of ter- from participating in hydrogen bonding.
tiary structure motives are pseudo knots [71], Since the stacked bases in the double-
produced by folding back in a hairpin and for- stranded helix are not as easily excited by
mation of a second stem loop structure [72]. UV light as in single strands, absorption at
Formation of DNA RNA hybrids is of im- 260 nm is lower for double-stranded DNA than
portance in the replication and transcription of for single strands. Increase in UV absorption
DNA and in the reverse transcription of viral can thus be used to measure DNA denatura-
RNA. Such hybrids can form secondary struc- tion. At 260 nm solutions containing 50 g/mL
tures but they are considerably more polymor- of double-stranded DNA, 50 g/mL of single-
phous than DNA alone [73]. stranded DNA, and 50 g/mL of free bases have
absorptions of ca. 1.00, 1.37, and 1.60, respec-
tively.
3. Properties Denaturation can also occur in the presence of
proteins that destabilize the helix (melting pro-
3.1. Physical and Chemical Properties teins). Such proteins are required to unwind the
helix during replication and to facilitate inter-
The size of naturally occurring DNA varies from action between single strands during genetic re-
a few thousand to 109 base pairs. The length combination.
of such molecules (micro- to centimeter range) The reassociation (renaturation) of thermally
can easily be measured under the electron mi- denatured DNA is a spontaneous process but
croscope. only occurs if the solution is cooled slowly be-
DNA absorbs UV light at 260 280 nm due to low the melting temperature. Renaturation can
its bases. Aqueous DNA solutions are very vis- take several hours, depending on the size of
cous; viscosity depends on DNA length, DNA the molecule, because it initially relies on ran-
concentration, and temperature. Heating to a dom base pairing (hybridization); it is, however,
critical temperature is accompanied by a de- a cooperative process. Rapid cooling of dena-
crease in viscosity because the hydrogen bonds tured DNA at salt concentrations > 50 mmol/L
responsible for base pairing are disrupted and the produces a very compact molecule in which
helix structure collapses. This process is called about two-thirds of the bases are hydrogen
thermal denaturation or melting of DNA. The bonded or stacked. At salt concentrations below
temperature at which one-half of the base pairs is
Nucleic Acids 163
10 mmol/L the DNA remains denatured even af- circular form and is organized in a series of su-
ter cooling. perhelical loops.
The length of RNA varies greatly: tRNA has The DNA of higher cells is enclosed within
a length of 75 90 nucleotides and mRNA can the nuclear membrane as morphologically dis-
be up to several thousand nucleotides long. De- tinct units of varying size (chromosomes); it is
naturation effects are rarely observed because associated with basic proteins called histones.
RNA has few truely double-stranded regions; it The number and size of the chromosomes are
is most likely to be observed in tRNA. species specic (karyotype). Two full turns of
Because they are extremely long, DNA the DNA double helix (146 base pairs) are
molecules are extremely sensitive to mechan- wound around a histone octamer (diameter ca.
ical inuences (shearing forces, e.g., vigorous 8.6 nm) to form a nucleosome. The width of the
stirring) and easily break into small fragments grooves varies due to the periodic arrangement
(ca. 1000 base pairs). Ultrasonic treatment of of A T trinucleotides on the inside and G C
DNA in solution produces fragments of ca. 100 trinucleotides on the outside of the nucleosome
500 base pairs owing to disruption of hydro- at intervals of about ten base pairs [53]. Nucle-
gen bonds and single-strand and double-strand osomes can become condensed into bers of 10
breaks in the sugar phosphate backbone [74]. or 30 nm (super superhelices, solenoids).
Nucleic acids are sparingly soluble in water (de- Eucaryotic cellular organelles (e.g., mito-
pending on the molecular mass). They are neg- chondria, chloroplasts) possess closed circular
atively charged and acidic at physiological pH DNA that is not associated with histones.
and form water-soluble alkali and ammonium
salts that can be precipitated with ethanol.
RNA and DNA are insoluble in cold acid. 4. Biosynthesis and Biological
DNA is more sensitive to acid hydrolysis than Function
RNA. At pH < 1, however, both DNA and RNA
break down into the free bases, phosphoric acid, 4.1. DNA Replication
and (deoxy)ribose. Acid hydrolysis can be used
to determine the base composition of nucleic The genetic information of all cellular organ-
acids (e.g., total hydrolysis can be achieved by isms is stored in double-stranded DNA (viruses
heating DNA in 90 % formic acid at 180 C for may, however, also have single-stranded DNA
30 min). The -glycosidic linkage between the or RNA, as well as double-stranded RNA). It
N-9 of purines and the C-1 of deoxyribose is se- is extremely important that the transfer of bi-
lectively cleaved at ca. pH 4, resulting in apurinic ological information in DNA (i.e., its base se-
sites. Anhydrous hydrazine cleaves the pyrimi- quence) occurs with a very high degree of ac-
dine residues. curacy. Because of perfected proofreading and
DNA is stable at pH 13, only 0.2 of 106 phos- repair mechanisms (see Section 4.5) DNA repli-
phodiester bonds are broken per minute at 37 C. cation has an error level of 108 1011 [77],
In contrast, RNA is rapidly hydrolyzed at alka- i.e., for every 108 1011 bases in newly syn-
line pH. thesized DNA only one is incorrectly incorpo-
DNA can be both specically and nonspecif- rated. The replication of DNA is carried out
ically cleaved by a variety of enzymes [de- by DNA polymerases which require a single
oxyribonucleases (DNases)]. RNA is cleaved by strand of DNA as a template and a short double-
ribonucleases (RNases). Some of these cleavage stranded piece of nucleic acid (formed with the
reactions are exploited for sequencing RNA [75, help of a primer) for initiation (Fig. 6; see also
76]. Enzymes, Chap. 6.2.).
The DNA is synthesized from deoxyribonu-
cleotide triphosphates which are polymerized
3.2. Interaction with Proteins on the single-stranded DNA template with the
release of pyrophosphate; the cleavage of py-
In bacteria, DNA occurs as a complex with RNA rophosphate by a pyrophosphatase provides the
and proteins that is bound to but not surrounded energy required for DNA biosynthesis. The ad-
by a membrane. The DNA often has a closed dition of new nucleotides always takes place
164 Nucleic Acids
at the 3 -OH group of the sugar, therefore all Replication is probably a membrane-bound pro-
biologically synthesized nucleic acids grow in cess [79].
the 5 3 direction. Divalent cations such as Because DNA strands are antiparallel, one
Mg2+ or Mn2+ are important cofactors. Some strand (lagging strand) must be synthesized
polymerases contain 3 5 exonuclease activ- backwards to keep the replication complex
ity and can remove bases that have been incor- together. The direction of synthesis of this
rectly incorporated. The primer in vivo is usually strand is contrary to the direction of move-
RNA, which is synthesized by a special RNA po- ment of the replication fork and occurs discon-
lymerase (primase) and removed later by an ex- tinuously. Segments (Okazaki fragments) are
onuclease (e.g., DNA polymerase with 5 3 formed which are then joined by a DNA lig-
exonuclease activity). The various activities are ase. The strand, that is synthesized forward
combined in a multienzyme complex. is called the leading strand. Replication in eu-
caryotes starts at several points along the DNA
and the replication complexes are xed to the
nuclear matrix [80]. Viroids are the smallest
self-replicating structures known. These single-
stranded, circular, protein-free RNAs are a
few hundred nucleotides long and occur as
pathogens in higher plants [81].
of protein and small RNAs (snRNAs = small initiation complex is formed from ribosomes,
nuclear RNAs). The main components of the mRNA, a special tRNA, initiation factors, and
spliceosome are ribonucleoproteins (snRNPs = GTP. The mRNA start codon for protein biosyn-
small nuclear ribonucleoproteins) [90, 91]. In- thesis is usually AUG but GUG, UUG, or AUU
trons usually start with GU (5 -splice site) and may also be used [89]. After initiation of trans-
end with a pyrimidine-rich section followed by lation, the protein is synthesized step by step
AG (3 -splice site) [92]. The half-life of introns (elongation) from amino acids that are delivered
in mRNA varies from a few seconds to 10 and activated by tRNA. Each amino acid has a
20 min. Splicing always takes place in the nu- specic tRNA.
cleus. Unspliced RNA remains in the nucleus The tRNAs act as adapters between the
and is degraded. codons of mRNA and the corresponding ami-
The splicing mechanism is highly conserved: no acids. Apart from the usual bases, tRNA
a mammalian cell extract can process yeast contains several rare bases (e.g., 5-hydroxyme-
RNA. The number of possible transcription thylcytosine). Transfer RNAs have an amino
products can be increased by alternative splic- acid attachment site and a template recognition
ing (i.e., not all available exons of a gene are site (anticodon) for the mRNA. Amino acids are
used to create one mature mRNA). attached by an ester bond either to the 3 -or 2 -
In protozoa RNA is also edited, the pri- OH group of the terminal adenosine of tRNAs.
mary transcript is converted into its functional Loading the tRNAs with the correct amino acids
form not only by post-transcriptional removal is crucial for exact translation of genetic infor-
of certain bases, but also by insertion of others mation. This step is catalyzed by specic en-
[93,94]. zymes (aminoacyl-tRNA synthetases). Each of
In some RNA viruses (retroviruses), repli- these enzymes must be able to recognize a spe-
cation of the genetic material proceeds via a cic amino acid and its associated tRNA.
DNA intermediate in a process known as re- The amino acid sequence of the protein prod-
verse transcription. The information in RNA is uct is specied by the mRNA codons and thus
transcribed back to DNA with suitable enzymes ultimately by the base sequence of DNA. A stop
(reverse transcriptases). The DNA is then used codon (TAA, TAG, TGA) on the mRNA leads to
as a template for RNA synthesis. Reverse tran- termination of protein synthesis and the release
scription can also be used in vitro to produce of the protein. Differences between translation
intron-free complementary DNA (cDNA) from complexes in procaryotes and eucaryotes permit
eucaryotic mature mRNA. Telomerases are spe- the selective interruption of procaryotic protein
cial reverse transcriptases with an internal RNA synthesis (e.g., by some antibiotics).
template [95, 96].
This process (transposition) can lead to gene ac- tion (see Section 4.3) is also a recombination
tivation or inactivation. phenomenon [103].
Methylation of DNA is used by cells in differ-
ent ways [100, 101], e.g., to distinguish between
newly synthesized strands (unmethylated) and 4.5. DNA Repair
old strands (methylated). Methylation occurs at
Although changes in base sequences create the
dened sites: in bacteria at the N-6 position of
genetic variety required for evolution, these
adenosine and the C-5 position of cytosine, and
changes are only rarely benecial for individual
in eucaryotes, physiologically only at cytosine.
organisms. Despite many damaging inuences
The methylation pattern also differentiates bet-
(see Section 4.3), nucleic acids are replicated
ween endogenous and foreign DNA and is rec-
with amazing accuracy. This exact conservation
ognized by restriction modication systems (in
of genetic material is one of the fundamental re-
procaryotes): endogenous DNA is methylated
quirements for genetic success. Damaged DNA
at specic sites and foreign DNA is digested.
that is not repaired can lead to mutations and
Methylation can be used to switch genes on or
cell death, and give rise to tumors. Living organ-
off in eucaryotes. C-Methylation inuences the
isms have therefore developed a variety of en-
equilibrium between B- and Z-DNA [102] and
zymatic mechanisms for eliminating DNA dam-
can lead to altered DNA protein interactions by
age. Most damage is recognized by repair sys-
the modulation of DNA topology. Methylation
tems in the cell and repaired either by directly
patterns are stable and hereditary. Methylation
reversing the damage or by using the undamaged
can have a mutagenic effect: the deamination
second strand as a template. Escherichia coli has
of 5-methylcytidine to thymine occurs sponta-
been used as a model for studying repair mecha-
neously.
nisms [104]. The best studied mechanism is nu-
Degradation of nucleic acids can be effected
cleotide excision repair. This multistep process
in vivo and in vitro by a series of enzymes. Some
involves recognition of the error, nicking of the
DNases are specic for single strands and others
damaged strand, removal of nucleotides at the
for double strands, some cleave bonds located
damaged site, repair DNA synthesis, and liga-
within a double strand (endonucleases) and oth-
tion. It requires the participation of multienzyme
ers require a free DNA end (exonucleases). Ex-
complexes [105, 106].
onucleases act either in the 3 5 or the 5 3
direction; exonuclease activity is often associ-
ated with DNA polymerases for proofreading in 4.6. Nucleic Acids as Enzymes
DNA replication. Restriction endonucleases are
a special class of nucleases. RNases can also be Ribozymes are RNA molecules that act as
divided into exo- and endonucleases. RNase II is catalysts: sequence-specic RNA endonucle-
a bacterial exonuclease; RNase H is an endonu- ases, nucleotidyl transferases, phosphatases, ki-
clease that digests RNA in DNA RNA hybrids. nases, and glucanotransferases [37, 107]. The
sequence-specic cleavage of oligodeoxyri-
bonucleotides and single-stranded DNA can also
4.4. Recombination be catalyzed by ribozymes [108]. Previously,
only proteins (enzymes) were thought to have
In recombination two DNA molecules interact activities of this kind. Ribozymal properties
and exchange segments to produce two new were discovered in the early 1980s in the self-
molecules that contain genetic information from splicing introns of Tetrahymena [109] and ri-
both parental DNAs. DNA recombination is by bonuclease P [110], which cleaves tRNA precur-
no means limited to genetic engineering, but oc- sors and contains RNA as the catalytic subunit.
curs widely in nature. This process is catalyzed In eucaryotes, small nuclear RNAs (snRNAs)
by enzymes and usually requires the presence are required for the splicing of pre-mRNA; they
of homologous regions (homologous recombi- are part of the spliceosome, an RNA protein
nation). In rarer cases, nonhomologous, illegit- complex [90].
imate recombination can take place. Transposi- The smallest RNA structure that exhibits cat-
alytic (cleavage) activity is 19 nucleotides long
168 Nucleic Acids
and forms a hammer head structure with its tar- rication of eucaryotic mRNA [118] taking ad-
get [111]. No pure DNA catalysts have been vantage of its poly(A) tails.
discovered yet [112]. However, since a few of Nucleic acids can also be puried by ion-
the ribonucleotides in ribozymes can be replaced exchange chromatography (e.g., by HPLC)
by deoxyribonucleotides, the three-dimensional [119]. Suitable separating media are hy-
structure that permits nucleic acid catalysis may droxyapatite and anion exchangers. Reversed
not necessarily be limited to RNA [113, 114]. phase methods and gel ltration are also used.
In all proven examples of RNA catalysis, the Microgram amounts of DNA fragments can
substrate attacked by the ribozyme is itself a nu- easily be isolated and puried by techniques
cleic acid. However, there are indications that such as electroelution from agarose gels, bind-
rRNA also attacks other molecules (i.e., is an ing to anion-exchange paper, dialysis, or binding
RNA enzyme). RNase P is the only known nat- to glass milk (powdered glass suspension). After
urally occurring ribozyme that does not cleave purication, DNA can easily be concentrated by
itself, but attacks other molecules [72, 115]. precipitation with ethanol or 2-propanol.
DNA can be transferred relatively easily in
vivo between related organisms (especially in
5. Isolation, Purication, and bacteria) via endogenous mechanisms (e.g., con-
jugation, some viruses). Several other methods
Transfer are available for the transfer of DNA in the lab-
oratory: application as a CaCl2 precipitate, ex-
Chromosomal DNA from bacteria or eucary-
posure to an electric eld (electroporation), and
otes has a very high molecular mass and is ex-
the use of viral vectors (see also Genetic En-
tremely fragile. Thus to isolate DNA, cells must
gineering).
be gently lysed with enzymes (pronase, protease
K, lysozyme) and mild detergents. Accompa-
nying RNA can be removed by treatment with
enzymes which digest RNA or by gel ltration 6. Analysis of Nucleic Acids
[116]. Proteins associated with DNA are dena- Sequencing. Computer data banks such as
tured with phenol and extracted [117]. EMBL [120] and GenBank [121] contain se-
Plasmid DNA is relatively small and has a quence information in the order of millions of
closed circular and supercoiled form. It is more nucleotides.
stable than chromosomal DNA when subjected Single or double-stranded DNA can be se-
to shearing forces and can be renatured much quenced [122]. The most widely used methods
more easily after denaturation; isolation and pu- are the Sanger method [123] and the Maxam and
rication of plasmid DNA is achieved relatively Gilbert method [124] ( Genetic Engineering,
easily (e.g., by equilibrium centrifugation in ce- Chap. 3.3.). In the Maxam and Gilbert method
sium chloride ethidium bromide gradients at the DNA is cleaved with chemicals statistically
150 000 400 000 g for 5 48 h). Kits are also and specically at one of the four bases. The
available which permit the isolation of pure plas- Sanger method employs enzymatic synthesis
mid DNA in a short time without ultracentrifuga- of a complementary copy of a single-stranded
tion using binding to glass powder (glass milk) DNA template and random chain termination
and/or gel ltration. Similar methods are used with dideoxynucleotides. Both methods produce
for the isolation of RNA. However, degradation DNA fragments that are separated by gel elec-
by ribonucleases must be prevented (RNA is trophoresis.
much more sensitive to nucleases than DNA). Both methods initially made use of radioac-
Either proteases or chloroform are employed to tively labeled nucleotides and subsequent expo-
remove protein; with chloroform protein pre- sure on X-ray lms for identication of the frag-
cipitates at the phase boundary. Phenol is not ments. More recent variations use uorophores
normally used because a small part of the RNA [uorescein, tetramethylrhodamine, Texas red,
also goes into the phenol phase. Oligo(dT) cel- 4-chloro-7-nitrobenzo-2-oxa-1-diazole (NBD)]
lulose (cellulose-bound oligonucleotides con- which are more stable, easier to use, less dan-
taining only thymine) can be used for the pu- gerous, and permit the automatic detection of
Nucleic Acids 169
all four nucleotides on a single track. Sequences acted with avidin and visualized by electron mi-
of > 450 bases can be determined per gel run and croscopy.
track [125128]. An automatic sequencing ma-
chine allows determination of sequences of up Scanning tunneling microscopy (STM) al-
to 10 000 bases per day. Further acceleration by lows the observation of single-stranded DNA
combination with automatic DNA preparation adsorbed on graphite with atomic resolution. It
in robotic workstations may be possible [129]. opens up the possibility of structural studies on
More recent techniques aim at faster se- DNA and of direct sequencing [141143]. The
quence determinations (several hundred bases proles show excellent correlation with the re-
per second [130]) with smaller amounts of DNA. sults of X-ray analysis.
Variations of the Sanger method permit determi-
nation of sequences up to 10 000 bases without Centrifugation. Ultracentrifugation in ce-
subcloning [131]. sium chloride gradients can be used to measure
The Sanger method can also be used for se- the molecular mass of DNA and also for the
quencing RNA [132] and sequences up to 150 experimental identication of covalently closed
bases can be determined in a single run. Re- circles. Sedimentation at alkaline pH (> 11.3)
verse transcriptase is used to synthesize DNA causes disruption of the hydrogen bonds and
with RNA as a template. The RNA sequence is unwinding of the DNA molecules. In the case
then deduced from the DNA sequence. Another of linear DNA, two single strands are ob-
method for RNA sequencing involves the use of tained which have a higher sedimentation coef-
specic RNases. cient than native DNA at salt concentrations
> 0.3 mol/L. Covalently closed circles cannot
X-Ray Analysis. X-ray analysis is of consid- be denatured into single strands, they collapse
erable importance for elucidating the structure of and have a sedimentation coefcient that is three
nucleic acids and their building blocks [44]. Use- times higher than that of native DNA.
able crystals can be obtained from aqueous solu- In equilibrium centrifugation in cesium chlo-
tions for molecules up to the size of tRNA (75 ride, ethidium bromide is rmly bound to DNA
90 base pairs). Nucleic acids of higher molecular in salt solutions by intercalating between base
mass are too heterogeneous to produce crystals, pairs. This lowers the density of the DNA by
quasi-crystalline bers drawn from concentrated about 0.15 g/cm3 and the DNA unwinds with
aqueous solutions are used instead. increased binding of ethidium bromide. Unlike
linear DNA, covalently closed molecules have
Nuclear Magnetic Resonance (NMR). The a limited absorption capacity for ethidium bro-
best method for determining the structure of rel- mide. Linear DNA is consequently lighter than
atively short (10 20 bases or base pairs) DNA covalently closed circular DNA. Use of a cesium
or RNA fragments in solution is 1 H-, 13 C-, 15 N-, chloride density gradient with ethidium bromide
or 31 P-NMR (one- or two-dimensional) [133, therefore permits the separation of supercoiled
134]. NMR is also used to study short triple and linear (relaxed) DNA of the same size.
helices [135], DNA RNA hybrids in solution
[136], DNA drug complexes, mismatches, and Staining and Quantication. Nucleic acids
intercalating agents. Torsion angles and the mo- can be stained with ethidium bromide [144],
bility of base pairs and the sugar phosphate back- Stainsall, 1-ethyl-2-[3-(1-ethylnaphtho[1,2 d ]
bone can be determined. thiazoline-2-ylidene)-2-methyl-1-propenyl]-
naphtho[1,2 d ] thiazolium bromide [145], or
Electron Microscopy. Electron microscopy silver [146148]. DNA stained with ethidium
permits the determination of the length of DNA. bromide can be quantied in electrophoresis
DNA heteroduplexes, binding of large protein gels after being photographed [144, 149]. Sil-
factors [137], and supercoiled DNA [138] can ver staining followed by a densitometer scan is
be visualized. A special application is DNA- equally sensitive (1 2 ng of DNA can be de-
hybridization electron microscopy [139, 140]: tected) [146]. A quick and sensitive estimation
biotinylated DNA probes are hybridized to se- of the amount of nucleic acid can be obtained by
lected regions of DNA or RNA and then re-
170 Nucleic Acids
measuring the UV absorption at 260 nm: an ab- tric eld can be observed with a uorescence
sorption of 1.00 corresponds to a concentration microscope [155].
of ca. 50 g/mL of double-stranded DNA. Pulse-eld electrophoresis is used to sepa-
DNA quantication in the picogram range rate larger linear DNA molecules. Here the di-
has been reported with a silicon sensor, that re- rection of the electric eld is changed at in-
sponds to the interaction between DNA-binding tervals. The DNA molecules therefore have to
proteins and DNA [150]. Quantication in the change their orientation, the time required for
picogram range is also possible after specic hy- this change depends on the size of the DNA
bridization (see Chap. 8). [44, 156]. Unlike conventional electrophoresis
Fluorescence imaging of DNA in picogram (duration 30 60 min), a pulse-eld run takes
to nanogram amounts is made possible by 20 140 h. Usually, 1.5 % agarose gels are used
laser excitation of a DNA ethidium homod- and 10 20 g of DNA can easily be separated
imer [5,5 -diazadecamethylene) bis(3,8-diami- at eld strengths of 2.5 10 V/cm. Separation
no-6-phenylphenanthridinium) dichloride di- can be achieved up to ca. 12106 base pairs
hydrochloride] complex at 488 nm [151]. [157]. Separation is affected by DNA topology
and sequence, pulse time (seconds to minutes),
Electrophoresis. Since DNA is negatively eld geometry, eld strength, gel composition,
charged because of its phosphate groups, it mi- sample concentration, temperature, and running
grates in an electrical eld (usually at a pH of ca. time [154, 158].
7). The electrophoretic mobility of DNA frag-
ments in solution is independent of the molecu-
lar mass (constant linear charge density). How- 7. Chemical Synthesis
ever, electrophoresis of DNA in gel matrices
(agarose or polyacrylamide) at a constant eld The chemical synthesis of oligonucleotides
strength is an effective method of separating began in 1955 when A. M. Michelson and
DNA molecules according to molecular mass. A. R. Todd prepared the rst dinucleoside
The mechanism of separation is not well under- monophosphate [159]. They used the so-called
stood [152]. triester method to show that oligonucleotides
Important experimental parameters are gel of dened sequence can be prepared by purely
concentration, eld strength, temperature, and chemical methods. In 1956 Khorana et al. suc-
running time. Gels of 0.5 2 % are usually em- cessfully synthesized the same nucleotide by
ployed for the separation of 50 20 000 base the diester method [160]. This process domi-
pairs at eld strengths of 2 3 V/cm [153]. The nated oligonucleotide synthesis until the mid-
mobilities of linear and supercoiled DNA of 1970s, and was also used by Khorana and
equivalent molecular mass are different. Estima- coworkers for the rst total synthesis of a gene
tion of the molecular mass of supercoiled DNA (alanine-specic tRNA from yeast) [161]. The
molecules based on linear standards is therefore importance of synthetic oligonucleotides was
not possible. generally underestimated because the synthesis
Polyacrylamide gels (4 12 %) give a sharp was time-consuming and expensive. It was the
separation of polynucleotides up to a length of progress in genetic engineering that resulted in
about 600 base pairs. Special gels can sepa- a dramatic increase in the demand for synthetic
rate polynucleotides with up to 2500 base pairs oligonucleotides, which, in turn, led to the de-
[148]. Mobility varies logarithmically with the velopment of new synthesis concepts and to the
size of the DNA at eld strengths below 1 V/cm automation of DNA and RNA synthesis.
[154]. Separation efciency is limited in the
range of 10 000 50 000 base pairs, there is no
resolution above 50 000 base pairs. 7.1. Synthesis Strategy
Denaturing gels (e.g., with urea) are used in
The chemical synthesis of DNA consists of three
the sequencing and analysis of single-stranded
steps:
nucleic acids. Under special conditions, the be-
havior of individual DNA molecules in an elec- 1) Separate synthesis of the two complementary
strands
Nucleic Acids 171
synthesis of oligonucleotides can be divided into 2 -OH group of ribose (in RNA synthesis). In-
the following steps: termediary protecting groups protect the 5 -OH
group of the sugar and are cleaved after each
1) Preparation of monomer units that are either condensation step.
protected or can be activated The most important protecting groups are
2) Functionalization of the support depicted in Figure 9. Adenine and cytosine
3) Stepwise synthesis of the desired nucleotide are usually converted to the corresponding acid
sequence by one of the three methods listed amides with excess benzoyl chloride, the amide
above of guanine is obtained with isobutyric anhydride.
4) Cleavage of the protecting groups and the Base-protecting groups are introduced with a
support method developed by Ti et al. (transient pro-
5) Isolation and purication of the desired se- tection) [165]. After termination of the syn-
quence thesis, the protecting groups are removed with
32 % aqueous ammonia at 50 60 C in 4 5 h.
In long, guanine-rich sequences, the O-6 po-
sition of guanine can also be protected [166
7.2. Protecting Groups 169], usually with a 4-nitrophenylethyl residue
[170]. This is especially important for attaining
In order to avoid unwanted side reactions dur- good yields in the triester process because other-
ing oligonucleotide synthesis, functional groups wise sulfonation with the condensing agent eas-
that do not participate in the linking reaction ily occurs. More labile amidine or acyl base-
must be suitably protected. Only a few protective protecting groups are of interest for the syn-
groups have gained acceptance and are based on thesis of phosphate-modied oligonucleotides
the work of Khorana et al. [164]. because they are more sensitive to hydroly-
Two types of groups can be distinguished: sis than their diester analogues and treatment
permanent and intermediary protecting groups. with ammonia at 60 C can cause considerable
Permanent protecting groups are maintained strand breakage [171173]. Important examples
throughout the synthesis and protect the exo- of such groups are the 4-phenoxyacetyl residue
cyclic amino functions of adenine, cytosine, and (Pac) for adenine and guanine and the isobutyryl
guanine; the OH group of phosphorus; and the residue for cytosine.
The tert-butyldimethylsilyl residue is pre- the DMT residue. The resulting orange solu-
ferred as the protecting group for the sugar 2 - tion containing the dimethoxytrityl cation [190]
OH group in the solid-phase synthesis of olig- can be determined by measuring the absorbance
oribonucleotides [174]. at 495 nm. The average loading is usually 15
The 5 -OH group of deoxyribose or ribose 25 mol per gram of CPG.
is usually blocked with the 4,4 -dimethoxytrityl
group [164], which can easily be removed after
each coupling step by mild acid hydrolysis with 7.4. Methods of Synthesis
3 % di- or trichloroacetic acid in dichlorometh-
ane [175]. The three main methods of oligonucleotide
In the triester method the 2-chlorophenyl synthesis are the phosphate triester, the phos-
group is used to protect the phosphorus [176], phoramidate (phosphite), and the H-phospho-
while in the phosphoramidite method, the - nate methods (see Fig. 8). The phosphoramidite
cyanoethyl group is preferred to the originally method is the most widely used (Fig. 10, see next
used methyl group [177, 178]. The -cyanoethyl page). The reaction cycle involves
group can easily be removed at the end of the
synthesis by -elimination with aqueous am- 1) Cleavage of the 5 -OH DMT group
monia [179]. No internucleotide phosphorus- 2) Condensation of the next monomer building
protecting group is required in the H-phospho- block (chain elongation)
nate process. 3) Masking of 5 -OH groups that have not re-
acted completely (capping)
4) In the phosphoramidite and H-phosphonate
7.3. Functionalization of the Support processes, an additional oxidation step is re-
quired after each condensation step or the end
Polystyrene [180], polyacrylamide [181], cel- of the synthesis respectively to obtain the de-
lulose [182], and silica [183] have been used sired internucleotide phosphodiester.
for solid-phase synthesis of oligodeoxyribonu-
cleotides. The preferred support material is con- Chain elongation in all processes occurs in
trolled pore glass (CPG) [184, 185] functional- the 3 5 direction.
ized with a long-chain alkylamine spacer. The
material is relatively rigid, does not swell, and Phosphate Triester Method. This method
is inert to all reagents used in the synthesis. In ad- was rst used to synthesize oligonucleotides
dition, it withstands the high mechanical stress from suitable building blocks in solution [191
encountered in the large number of reactions per- 194]. It can also be employed for the solid-phase
formed in automatic synthesizers. The narrow synthesis of nucleotides up to a chain length of
pore-size distribution gives more uniform diffu- about 20 bases. The monomer building block
sion parameters and, consequently, more repro- is a protected nucleoside phophodiester deriva-
ducible ow rates and availability of functional tive (Fig. 8) [193] which reacts with the free
support sites. 5 -end of a support-bound nucleoside to give
The support is functionalized with an al- a neutral phosphotriester. Various aromatic sul-
kylamine by silanization with 3-aminopro- fonic acid derivatives [192, 195, 196], primarily
pyltriethoxysilane [183, 186]. Heating and sub- mesitylene sulfonylnitrotriazole (MSNT), serve
sequent treatment with methanol result in cross- as condensing agents. The reaction can be accel-
linkage of the silyl groups and hydrolysis of erated by using catalysts such as N-methylimi-
outer, less strongly xed groups [187]. The nu- dazole [175]. This method is not widely used any
cleoside at the 3 -OH end of the DNA to be longer due to disadvantages such as long reac-
synthesized is linked to the support via a suc- tion times, a large number of side reactions, and
cinyl spacer [183, 188, 189]. Amino groups of the frequently observed nicks caused by incom-
the support that have not reacted are capped plete cleavage of the internucleotide protecting
with acetic anhydride [188]. To determine the group.
loading of the support with nucleoside material,
an aliquot is treated with strong acid to remove
174 Nucleic Acids
can also be used in the synthesis are described solution of the appropriate amine in carbon tetra-
in [216, 217]. chloride [213].
The 2 -O-methylribonucleotides (9) are well
established modied nucleosides that are used
as monomer building blocks for oligonucleotide
synthesis [224]. The synthesis of the corre-
sponding nucleoside phosphoramidites is de-
scribed in [225]. Oligonucleotides with -2 -
deoxynucleosides (-DNA) form -DNA with
natural DNA. This form has parallel strands and
is also nuclease resistant [226, 227].
To increase the binding of an oligonucleotide
to the target, an intercalating agent (e.g., 2-
methoxy-6-chloro-9-aminoacridine [228, 229]
or phenazine [230]) can be covalently attached
to the oligonucleotide. These agents insert in-
ternally between the base pairs of the double
helix, increasing duplex stability.
A second method involves binding of pho-
toreactive groups (e.g., psoralen [231233],
azidophenacyl or azidoproavin derivatives,
proavins, and porphyrins [234238]) that are
activated by UV or visible light and can cross-
link with the target DNA or RNA. Sequence-
Figure 11. Modied oligonucleotides specic articial endonucleases [239] are
oligonucleotides that bear a group which can
Like native DNA, phosphorodithioate ana- induce specic cleavage of the target nucleic
logues (6) are stereochemically uniform prod- acid after binding to the target. Such groups
ucts and can be prepared by the phos- are the above-mentioned photoreactive groups:
phoramidite method with the corresponding subsequent treatment with piperidine allows
thiophosphoramidite monomers [218]. cleavage of the target sequence at the cross-
Methylphosphonates (5), phosphoramidates linking site. Direct light-induced cleavage at
(7), and phosphotriesters (8) are nonionic and neutral pH is achieved with ellipticine and di-
have a more hydrophobic character than nat- acapyrene derivatives [238]. Metal chelates at-
urally occurring nucleic acids. The synthe- tached to oligonucleotides (e.g., iron EDTA
sis of oligonucleoside methylphosphonates is [240242], copper phenanthroline [243246],
described in [219222], and is best carried or iron porphyrin [247]) lead to relatively spe-
out with nucleoside 3 -methylphosphonous acid cic cleavage of the target sequence. A nonspe-
imidazolide, which in turn is prepared from cic DNase or RNase bound to a complemen-
methylphosphonous acid bisimidazolide and tary oligonucleotide can also destroy the target
the fully protected nucleoside. Alternatively, nucleic acid [248, 249].
a H-phosphonate is reacted with tert-butyldi- Nonradioactive labeling of synthetic oligonu-
methylsilyl chloride to give silyl phosphite cleotides has become an important technique
and subsequently oxidized with alkyl or aryl in molecular biology and several chemical pro-
halides [223]. All of the early investigations on cedures have been developed. Oligonucleotides
the biological activity of these oligonucleotide are covalently attached to marker groups such
analogues (which were named matagen, an as uorescent dyes, biotin, and other biologi-
acronym for mashing tape for gene expression) cally active groups (see also Section 8.2). These
were conducted by Ts o and Miller [222]. reporter groups can be attached to an oligonu-
Phosphoramidates (7) can be prepared by re- cleotide at the 5 -OH terminus, the 3 -OH ter-
acting H-phosphonate oligonucleotides with a minus, the phosphate backbone, or one of the
bases. Attachment at the 5 -OH terminus via pro-
Nucleic Acids 177
tected aminoalkyl or thioalkyl phosphoramidites often necessary for the detection of changes in
is most common. These amino- or thiolinkers nucleic acids (point mutations, deletions, inser-
are added in the last synthesis step and produce tions, translocations, expression level or number
a free amino or thiol terminal after cleavage of of copies of the genetic information) in the di-
the protecting groups [250252]. Subsequent re- agnosis of hereditary diseases [24], or in the in-
action with the activated marker molecule (e.g., vestigation of tumors [25,26]. This chapter will
biotin-N-hydroxysuccinimide) gives the labeled be limited to describing the uses of nucleic acids
oligonucleotide. in probe technology but other applications will
A large number of bifunctional linkers (e.g., rst be briey summarized.
maleimidohexanoyl-N-hydroxysuccinimide es- The second important eld of application for
ter) have been described which allow coupling of nucleic acids is genetic engineering ( Genetic
enzymes [253], peptides, and proteins to linked Engineering) which allows the production of
oligonucleotides via amino groups of the amino proteins in bacteria, fungi, animal or plant cell
acids or thiol groups of cysteine. Attachment at cultures, or in animals or plants. The nucleic acid
the 3 -OH terminus can be carried out by linking that codes for the desired protein is introduced
a ribose unit with T 4 RNA ligase, followed by into the organism and expressed.
periodate cleavage of the ribose ring, and sub- Antisense technology
sequent reductive amination [254, 255]. Label- is a third eld of application of nucleic acids
ing via a 3 -terminal thiol group is described which, however, still only exists in model sys-
in [256]. Also a modied support is commer- tems. This technology aims to prevent the ex-
cially available which produces an aliphatic pri- pression of nucleic acid sequences that have
mary amino group at the 3 -terminus [257]. At a negative effect on an organism. This can
the internucleotide bond reporter groups can be be achieved by the interception and binding
attached, e.g., via a phosphoramidate linkage of mRNA with synthetic antisense oligonu-
[258260]. cleotides (see Section 7.6) or with an antisense
The attachment of reporter groups to mod- RNA from a recombinant expression system.
ied bases usually occurs at the C-5 position The mRNA forms double strands with the an-
of pyrimidines or the C-8 position of purines tisense RNA by specic base pairing and can-
[261]. During oligonucleotide synthesis, a fully not be translated [270, 271]. Another method of
protected deoxyuridine phosphoramidite [262] stopping expression is the specic cleavage of
modied with a triuoroacetylaminopropenyl the target nucleic acid by catalytic RNA (ri-
residue is inserted at the desired position in the bozymes) [270, 271]. The target nucleic acid
sequence (usually at C-5); after cleavage of the is recognized by specic base pairing with the
protecting groups, the group to be coupled is ribozyme. Antisense technology may be used
bound via the resulting amino function. Nu- to treat infections caused by viruses, bacteria,
merous other labeling positions have been de- or parasites by cleaving or preventing the ex-
scribed (e.g., the C-4 position [263265] of 2 - pression of the genetic information of these
deoxycytidine and the N-6 position of the ade- pathogens. Another possibility is the treatment
nine ring [266] or the exocyclic amino function of diseases resulting from overexpression of an
of the guanine residue [267]). endogenous gene or its expression in an erro-
neous target cell.
Finally, nucleic acid technology offers great
8. Uses promise in research. As a result of the dramatic
improvements in cloning and sequencing large
The direct use of nucleic acids in nucleic acid sections of DNA, DNA sequences or genes are
probe technology is becoming increasingly im- increasingly being used as a starting point for the
portant. Here, labeled nucleic acids are used as analysis of a biochemical mechanism. Nucleic
probes to detect other nucleic acids by means of acid sequences are also used to study evolution-
hybridization [21,23]. Identication of the spe- ary relationships between different organisms by
cic nucleotide sequence of a pathogen (virus, comparing sequences of conserved genes.
bacterium, parasite) can be used to diagnose an
infection [268, 269]. More complex analyses are
178 Nucleic Acids
8.1. Hybridization Techniques for or tissue is cut into ultrathin layers and xed. Hy-
Nucleic Acid Detection bridization takes place on the slide between the
labeled probes and the nucleic acids in the sam-
Nucleic acid hybridization is based on the fact ple material. Examination under the microscope
that two complementary nucleic acid strands reveals which and how many cells contain the
(DNA DNA, RNA RNA, DNA RNA) asso- nucleic acid of interest, as well as its subcellu-
ciate to form a double strand according to the lar location. In situ hybridization permits differ-
rules of base pairing (see also Genetic Engi- entiation between individual cells, whereas in
neering, Chap. 3.1.). The nucleic acid to be de- solid-phase blotting methods the nucleic acids
tected must be present as a single strand, i.e., from many cells are mixed together. In situ hy-
denaturation of double strands is required be- bridization also permits the assignment of a nu-
fore hybridization. The nucleic acid probe must cleic acid sequence to a chromosome by analyz-
contain a sequence complementary to that of the ing cells that are in the metaphase of cell division
nucleic acid under investigation and have a la- [276, 277]. The areas of the chromosomes con-
bel to permitidentication of the double strands taining the complementary sequences are specif-
formed by hybridization. ically labeled by the nucleic acid probe.
nucleic acid of interest is present in the solu- cleic acid of interest at adjacent positions. Spa-
tion and forms a bridge between the two probes. tial proximity of the two labels is only guaran-
The capture probe can be covalently coupled to teed if the nucleic acid of interest is present and
the solid phase [280], or can be labeled with a produces a signal that can be measured (e.g., en-
molecule that is distinct from the detection label zymatic activity, light of a certain wavelength)
and is bound to the solid phase via a xed antag- [284].
onist. Examples are biotin-labeled probes with a
streptavidin- or avidin-loaded solid phase [281]
or antigen-loaded probes with an appropriate an- 8.2. Labeling and Detection Systems
tibody on the solid phase.
Strand displacement is another method for Radioactive labeling is losing importance due to
detecting hybrids formed in solution (Fig. 13) the development of sensitive, nonradioactive la-
[282, 283]. Here, the probe is a hybrid of a short, bels. Nucleic acids are usually radioactively la-
labeled oligonucleotide and a longer nucleic beled by enzymatic incorporation of nucleotides
acid strand forming a partially single-stranded, containing 3 H, 35 S, or 32 P isotopes with poly-
partially double-stranded molecule. The longer merases. Alternatively, the 5 -OH terminus of
strand is complementary to the nucleic acid of DNA can be labeled with 32 P by polynucleotide
interest. If the nucleic acid of interest is present kinase [21]. Radioactive nucleic acids are de-
in the assay, double strands are formed with the tected by exposure to X-ray lm or a photoemul-
single-stranded region of the probe. This double- sion (in situ hybridization) or by measurement
stranded region of the unlabeled probe strand in a scintillation counter.
and the nucleic acid of interest is longer than the In nonradioactive methods, the nucleic acid
double-stranded region formed between the two can be directly labeled with a uorescent or lu-
probe strands. Therefore complete hybridization minescent dye [285], bound to an enzyme (e.g.,
occurs between the longer probe strand and the -galactosidase, horseradish peroxidase, alka-
nucleic acid of interest. As a result the short, line phosphatase) or coupled with biotin or a
labeled probe strand is released and can be de- hapten (antigenic determinant) [284]. The hap-
tected. ten (or biotin) is detected by the binding of an ap-
propriate antibody (or streptavidin/avidin). The
antibody and streptavidin/avidin are, in turn, di-
rectly coupled to a dye molecule or to an enzyme
[21]. Additional chemical methods for labeling
nucleic acids are described in [21, 286288] and
in Section 7.6.
Photolabeling is a simple and fast labeling
method [21, 289]. Biotin or a hapten is cou-
pled to a photoreactive group (photobiotin [289],
photodinitrophenyl phosphate (DNP) [290], or
photodigoxigenin [291]) which reacts with the
amino groups of the nucleotides. On irradiation
with light the photoreactive group is activated re-
sulting in the formation of a covalent bond bet-
ween biotin or the hapten (DNP, digoxigenin)
and the nucleic acid.
Nucleic acids from biological sources (e.g.,
Figure 13. Strand displacement plasmids) are usually labeled with the aid
ss = single-stranded; ds = double stranded of enzymes. Modied nucleotides are in-
Finally, there are a series of modications of serted with polymerases [21]; DNA poly-
a hybridization principle which does not require merases are used to insert the deoxyri-
the separation of bound from unbound probe. bonucleotide triphosphate derivatives (e.g., bi-
Here, two labeled probes hybridize with the nu- otin dUTP, biotin-dATP, digoxigenin dUTP,
bromo dUTP, and uorescein dUTP) in nick
180 Nucleic Acids
translation ( Enzymes, Chap. 6.2.1.) or by ran- fying (i.e., increasing the amount of) the nucleic
dom primed labeling. The 3 -terminus of nucleic acid of interest [21].
acids can be labeled with these nucleotides by
terminal transferase. Modied ribonucleotides Polymerase Chain Reaction (PCR). The
(e.g., biotin UTP and digoxigenin UTP) are polymerase chain reaction is an established tech-
inserted into RNA probes by in vitro tran- nique [15, 27, 292, 293] which is used to amplify
scription with RNA polymerases, particularly a particular nucleic acid from a complex mixture
with phage-coded RNA polymerases [21, 22] of sequences. The amplied DNA can be subse-
( Enzymes, Chap. 6.3.). quently sequenced and/or cloned using genetic
engineering techniques, or can be used to in-
crease the sensitivity of pathogen detection. The
sensitivity can be increased to such an extent
that, for instance, one genome from the human
immunodeciency virus (HIV) integrated in the
DNA of a single cell can be detected in the pres-
ence of many uninfected cells.
The PCR is based on the repetition of three
partial reactions in 20 50 cycles (Fig. 14):
1) An oligonucleotide primer is annealed to
each of the two denatured complementary
strands of the DNA sequence to be ampli-
ed.
2) Primer extension catalyzed by a DNA poly-
merase converts the region to be amplied
into two double strands.
3) The double strands are denatured by heating.
Theoretically the number of templates can be
doubled in each cycle, so that 2n templates
would be created in n cycles; 25 cycles would
result in an amplication rate of 3.4107 . How-
ever, the partial reactions are not perfectly ef-
cient, and amplication rates of ca. (1 3)106
are obtained in 25 cycles. Implementation of the
PCR is facilitated by the use of thermostable
DNA polymerases because otherwise new po-
lymerase would have to be added at the start
of each cycle. Typical PCR cycles consist of a
few minutes of primer annealing at ca. 55 C, a
few minutes of elongation at ca. 70 C, and 1
2 min of denaturation at 95 C. These steps can
be carried out automatically in programmable
thermocyclers.
Figure 14. Polymerase chain reaction (PCR) RNA can also be amplied by PCR if a DNA
template is created in the rst step with the help
of reverse transcriptase. The PCR product can be
detected by electrophoresis with or without pre-
8.3. Amplication Systems vious cleavage with restriction endonucleases,
and with or without hybridization with a probe.
The sensitivity limit of nucleic acid probe tech- The PCR product can be labeled directly by us-
nology can be increased by specically ampli- ing labeled primers [294] or by incorporating la-
beled nucleotides (Section 8.2) [295, 296]. Con-
Nucleic Acids 181
tamination is a serious problem in PCR because ods described above, the target sequence itself
of the enormous sensitivity of the method. For is not multiplied, instead the probe is amplied
example, contamination of a sample with a sin- after it has located the target sequence (signal
gle molecule of the DNA of interest can lead to amplication). Nonspecically bound and con-
an incorrect positive result. taminating probe molecules cause background
problems that can be alleviated by repeating the
Other Amplication Systems. In tran- hybridization in cycles (reversible target cap-
scription-based amplication oligonucleotide ture) [303].
primers are annealed to the nucleic acid of in- Another method of signal amplication in-
terest which, in addition to complementary se- volves the formation of probe networks in which
quences to the target, also contain a promoter the target-specic probe has target sequences for
(binding site) for RNA polymerase [297]. After secondary probes. A Christmas tree of labeled
primer elongation RNA polymerases are used to probe sequences is produced for each original
produce RNA transcripts starting from the pro- target molecule [304306].
moters. The transcripts can be converted to DNA
with reverse transcriptase and used again as tar-
gets for the oligonucleotide primers. 8.4. Applications of Probe Technology
The ligase chain reaction uses a ligase instead
of a polymerase in temperature cycles analogous Detection of Infectious Microorganisms.
to the PCR [298, 299]. Two oligonucleotides are Detection of nucleic acids can be used to iden-
annealed adjacent to one another on the tem- tify pathogenic viruses, bacteria, or parasites.
plate strand and joined by a ligase. The ligated In comparison with the detection of proteins
strand is then removed by denaturation and two or antigens, nucleic acid detection has the ad-
oligonucleotides can anneal again in the next cy- vantage that it allows identication of infectious
cle. organisms that frequently change their antigens
Other amplication systems do not require (e.g., HIV) or that exist in a variety of serotypes.
cycles with different temperatures but proceed Furthermore, viral infections can be detected
isothermally. In self-sustained sequence repli- at the latent stage when no detectable synthe-
cation (3 SR) [300] and nucleic acid sequence sis of viral proteins occurs (e.g., HIV or her-
based amplication (NASBA) [301], three en- pes viruses). DNA probe technology can be
zymes act simultaneously at a constant temper- more sensitive than immunological techniques
ature to amplify the target sequence. Reverse due to the possibility of nucleic acid amplica-
transcriptase synthesizes DNA on an RNA tem- tion. Although immunological tests are still eas-
plate by extending oligonucleotide primers that ier, faster, and cheaper, competitive nucleic acid
contain promoter sequences for RNA polymer- tests are to be expected in the future.
ases. The RNA strand of the resulting DNA Detection of the nucleic acid of a pathogen
RNA hybrid is digested with RNase H allow- can also serve as a direct measure of the in-
ing double-stranded DNA to be produced by re- fectiousness of a patient or test material [307].
verse transcriptase with a second primer. RNA Quantitative determination of the pathogenic nu-
polymerase then transcribes the double-stranded cleic acid can be used to follow the progress of
DNA starting at the promoters, and the resulting treatment.
RNA is again converted to DNA with reverse Detection of viruses is possible with a
transcriptase. broad spectrum of nucleic acid probes, rang-
Another isothermal amplication technique ing from synthetic oligonucleotides to the com-
is based on the replication of RNA genomes plete cloned viral genome. Infections caused by
with the help of Q replicasea RNA-dependent some virus groups (e.g., papilloma viruses or en-
RNA polymerase from phage Q [302, 58]. Nat- teroviruses [308]) can be diagnosed with a probe
ural substrates of Q replicase are characterized containing group-specic sequences identical in
by RNA sequences forming a special secondary all members of the group and by using probes
structure. For nucleic acid detection these se- with nucleic acid sequences specic for each
quences are modied by the insertion of short member.
probe sequences. Unlike the amplication meth-
182 Nucleic Acids
For the detection of bacteria, the fact that each sive oncogenes (tumor suppressor genes) [314,
bacterium contains approximately 10 000 ribo- 315], the loss of gene expression results in a tu-
somes and thus 10 000 copies of rRNAs can be mor; for example, the loss of the retinoblastoma
used to increase the sensitivity level. The rRNA gene results in an eye tumor, retinoblastoma. An-
sequences are relatively well conserved and can other area of tumor diagnosis is the detection of
used to establish relationships between differ- clonality of a leucocyte population caused by the
ent organisms in phylogenetic trees. Tests for transformation of a white blood cell [316]. Since
individual bacterial species, for groups, or even all lymphocytes have their own characteristic re-
for a wide range of bacteria can be carried out arrangement of immunoglobulin genes or T-cell
with appropriate oligonucleotide probes [309]. receptor genes, the appearance of a dened pat-
Another group of nucleic acid probes contains tern in many lymphocytes indicates proliferation
sequences of bacterial pathogenicity factors and of a single cell.
allows differentiation between pathogenic and Methods that use the genetic diversity of hu-
apathogenic types of a species (e.g., Escherichia mans to study individuals have wide applica-
coli) [268, 269]. Analogously, detection of resis- bility. Determination of the genes of the ma-
tance genes permits detection of antibiotic re- jor histocompatibility locus (HLA) can be used
sistance and simultaneous identication of the both for diagnosis (transplantation medicine,
pathogen [310]. Bacterial identication is also prediction of risk of disease, autoimmune dis-
applied in the detection of pathogens in food eases) and for differentiation between individ-
(e.g., Salmonella). uals [317]. Probes which hybridize with repet-
itive sequences that occur in each individual in
Detection of Human DNA Sequences. Nu- a slightly different, genetically determined ar-
cleic acid probe technology can be used for rangement can be used for nger printing [28].
the diagnosis of hereditary diseases and ge- Here nucleic acids cleaved by restriction en-
netic counseling [24]. If the genetic basis of zymes are separated electrophoretically and hy-
the disease is known, the alteration in the nu- bridized with these probes in the Southern blot
cleic acid can be detected by hybridization with technique (Section 8.1). Several fragments of
the appropriate region. Such alterations include different sizes, each containing these repetitive
point mutations (sickle-cell anemia), deletions sequences, produce a signal. Each individual in-
(Duchennes muscular dystrophy), or variations herits half the sequences from the father and
in expression (forms of thalassemia). If the ge- half from the mother, and therefore has a unique
netic cause of the disease is unknown, genetic fragment pattern [318320]. This technique is
markers that are inherited together with the de- used in forensic medicine to identify criminals
fect gene are analyzed, e.g., recognition sites for by DNA isolated from cells (hair, blood etc.).
restriction enzymes (restriction fragment length Other elds of application are in the determina-
polymorphism, RFLP) [311]. tion of paternity and family relationships.
The analysis of human sequences can also
be applied in tumor diagnosis [25, 26]. Some
human tumors are associated with characteris- 9. Nucleosides and Nucleotides
tic chromosomal changes or alterations in genes
(oncogenes) [312]. Suitable nucleic acid probes 9.1. Nucleosides
can be used for the highly sensitive detection
of DNA sequences of different chromosomes The term nucleosides was originally applied to
connected by chromosomal translocations or for the ribose derivatives of purines that can be iso-
the detection of mutations in oncogenes that lated from an alkaline RNA hydrolysate. Later,
are associated with tumors. For instance, the this name was given to all purine and pyrim-
Philadelphia chromosome (a translocation bet- idine N-glycosides of d-ribose and 2-deoxy-
ween chromosome 9 and 22) is characteristic for d-ribose. Today, this term refers to all natu-
chronic myelogenous leukemia [313]. Point mu- ral or synthetic compounds which consist of a
tations in the ras oncogene are associated with heterocyclic nitrogen-containing base (aglycon)
several tumors, and amplication of the N-myc and a carbohydrate residue (glycon). The nitro-
gene with neuroblastomas. In the case of reces- gen atom (N-nucleoside) or carbon atom (C-
Nucleic Acids 183
Other derivatives which contain only a The nucleosides are obtained by modica-
part of the ribofuranosyl residue (e.g., acy- tion of existing nucleosides or total synthesis.
clonucleosides) also show antiviral activ- Total synthesis can be carried out in a linear or
ity. This class of substances includes acy- convergent manner. Due to the chiral centers on
clovir (25), ganciclovir (26) and phospho- the glycon residue, preparation of optically pure
nylmethyl analogues [e.g., (S)-9-(3-hydroxy- nucleosides requires stereoselective methods of
2-phosphonylmethoxypropyl) adenine (27)]. synthesis, especially for the glycosidation of the
Nucleosides with a halogen substituent in aglycons. The most important methods of to-
the heterocyclic moiety are important an- tal synthesis will be described briey. Detailed
tiviral drugs (e.g., 5-iodo-2 -deoxyuridine, 5- surveys of the chemical synthesis of and on nu-
bromovinyl-2 -deoxyuridine (28), or 2-chloro- cleoside derivatives are given in [3135].
2 -deoxyadenosine).
Synthesis of N-Nucleosides. The rst syn-
theses date back to the work of Fischer and
Helferich who condensed acetyl-protected -
d-glucopyranosyl halides with the silver salts
of heterocycles. The analogous chemical syn-
thesis of the natural nucleosides adenosine and
guanosine was described by Todd and cowork-
ers [321]. This method was improved after the
introduction of the chloromercury purine deriva-
tives [322] and the use of mercury cyanide
(which avoids the tedious preparation of the
heavy metal salts of the nucleobases) [323].
The Hilbert Johnson method was developed
for the preparation of pyrimidine nucleosides
[324]. This method starts with 2,4-dialkoxypy-
rimidines, which yield the corresponding nu-
cleosides on reaction with O-acetyl-protected
Nucleic Acids 185
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