Hermanson G. Bioconjugate Techniques, Second Edition

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1. Enzymatic Labeling of DNA

Enzymatic techniques can employ a variety of DNA or RNA polymerases to add controlled

amounts of modiﬁ ed nucleotides to an existing stand. However, the most common procedures

utilize either DNA polymerase I or terminal deoxynucleotide transferase. The polymerase is

used with a template to add modiﬁ ed nucleoside triphosphates to the end of a DNA molecule

or to various sites within the middle of a sequence. The terminal transferase can add modiﬁ ed

monomers to the 3  end of a chain without a template.

Three main procedures of enzyme labeling make use of a DNA polymerase: (a) random-

primed labeling, (b) nick translation, and (c) polymerase chain reaction (PCR). In random-

primed labeling, modiﬁ ed nucleoside triphosphates are added to a DNA template using a

random mixture of hexa-deoxynucleotides to serve as 3 -OH primers. The form of polymerase

used is the Klenow fragment which lacks the 5  -3  exonuclease activity of intact E. coli DNA

polymerase I (Feinberg and Vogelstein, 1983, 1984; Kessler et al., 1990). This method is a sim-

ple way of tagging probes prepared from a restriction digest template with randomly incorpo-

rated, labeled nucleotides.

Nick-translation labeling involves the use of a dual enzyme system acting on double-

stranded DNA (Rigby et al., 1977; Langer et al., 1981; Höltke et al., 1990). The enzymes

pancreatic deoxyribonuclease I (DNase I) and E. coli DNA polymerase I act in tandem

on a DNA helix to incorporate labeled nucleotides into the sequence. DNase I is capable of

breaking phosphodiester bonds in intact DNA double-stranded molecules. If it is used in the

presence of magnesium ions, it limits the hydrolysis caused by the enzyme to a single strand

at a time within the DNA helix. If DNase I is further restricted in the amount added to a

reaction, the number of breaks caused in the double helix can be controlled. The addition of

DNA polymerase I and the appropriate labeled and unlabeled nucleotide monomers causes the

breaks to be ﬁ lled as quickly as they form. Since a quantity of labeled nucleoside triphosphates

is present during the reaction, the labels get incorporated and the parent DNA strands are

modiﬁ ed.

Enzymatic labeling of DNA by use of PCR techniques perhaps provides the most powerful

way of not only adding a label, but also of amplifying the labeled polymer to produce numer-

ous copies of itself. First invented by Mullis (who went on to win the Nobel Prize; see Saiki

et al., 1985, 1988), PCR utilizes heat-stable forms of DNA polymerase, for example the com-

monly employed Taq polymerase isolated from thermophilic eubacterium ( Thermus aquaticus ).

The stability of the enzyme allows repeated elevated-temperature denaturations of target DNA,

followed by hybridization of two primers onto the single strands. Taq DNA polymerase then

creates a complementary sequence to the two single strands by elongation of the primers. If

repeated cycles of denaturation, hybridization, and elongation are done, the result is an expo-

nential ampliﬁ cation of the original DNA strands (for a review of PCR, see Innis et al., 1990).

Labeling of these ampliﬁ ed strands can be accomplished by one of two routes: using labeled

primers or using labeled deoxynucleoside triphosphates. Either way, the Taq polymerase incor-

porates the labels into the growing DNA copies of each PCR cycle.

Enzymatic labeling using any of these polymerase methods results in derivatized nucleoside

triphosphates being incorporated at numerous locations within an oligonucleotide strand.

These modiﬁ cations potentially can interfere with the hybridization of a probe to a comple-

mentary sequence, especially if the level of labeling is high. Enzymatic labeling using termi-

nal transferase is a way to avoid derivatization in the middle of a strand, and thus preserve

970 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

sequence or targeting speciﬁ city. The enzyme is able to add deoxyribonucleotides to the 3  -OH

ends of existing DNA probes without the need for a template. Since modiﬁ cation is limited to

a single end of the oligonucleotide, the probe sequence is not disturbed by labeling groups that

could possibly prevent hydrogen bonding interactions between base pairs.

Terminal transferase labeling was originally developed using radiolabeled (typically

nucleoside triphosphates (Roychoudhury et al., 1979; Tu and Cohen, 1980). Later, the tech-

nique was extended to the use of nonradioactive nucleotide derivatives (Kumar et al ., 1988).

Regardless of the type of enzymatic labeling used, it is important that the label be incorpo-

rated into the nucleoside triphosphates or primers in a way that does not affect enzyme rec-

ognition and activity. Thus, every enzymatic labeling procedure for modifying RNA or DNA

probes must start with chemical derivatization of individual nucleotides. Of the many chemical

procedures that can be used to modify a nucleoside triphosphate monomer, there are only a

few that will result in a derivative still able to be enzymatically added to an existing oligonucle-

otide strand.

Of the purine nucleosides, dATP may be derivatized at its N-6 position using a long linker

arm terminating in a detectable group without losing the ability to be enzymatically incorpo-

rated into DNA probes. By contrast, if modiﬁ cation is done at the C-8 position of purine bases,

DNA polymerase cannot by used to add the labeled monomer to an existing strand. C-8 deriv-

atives, however, can be added at the 3  terminal using terminal transferase enzyme.

The pyrimidine nucleosides dUTP or dCTP can be modiﬁ ed at their C-5 position with

a spacer arm containing a tag, such as a biotin group, and still remain good substrates for

DNA polymerase. Enzymatic labeling with a biotin-modiﬁ ed pyrimidine nucleoside triphos-

phate is one of the most common methods of adding a detectable group to an existing DNA

strand.

Figure 27.1 illustrates some of the common nucleoside triphosphate derivatives that can be

used in enzymatic labeling processes. The following sections describe procedures for enzymatic

labeling using nick translation, random prime labeling, and 3  tailing with terminal transferase.

For a review of these methods in greater detail, see Kricka (1992), or Keller and Manak (1989).

See Section 2 (this chapter) for a discussion of the chemical methods that can be used to label

individual nucleic acids for incorporation into oligonucleotides by enzymatic means.

Protocol for the Labeling of DNA by Nick Translation

1. In a tube kept cold on ice, add 10 l of 10  nick-translation buffer (0.5 M Tris, 0.1 M

MgCl

, 0.08 M 2-mercaptoethanol, pH 7.5, containing 0.5 mg/ml bovine serum albumin,

BSA), 0.5 g of double-stranded probe DNA to be labeled, 1 l of DNase I at a con-

centration of 2 ng/ml, 1 l each of 3 types of unmodiﬁ ed deoxynucleoside triphosphates

(dNTPs at 100 M concentration), 1 l of a labeled dNTP (at 300 M), 32 l water. Then

add 1  l of DNA polymerase containing 5–10 units of activity.

2. React for 1 hour at 15 ° C.

3. Quench the reaction by the addition of 4 l of 0.25 M EDTA, 2 l of 10 mg/ml tRNA,

and 150  l of 10 mM Tris, pH 7.5.

4. Purify the labeled DNA from excess reactants by precipitation. Add 20 l of 4 M LiCl

and 500  l of ethanol (chilled to  20 ° C). Mix well.

5. Store at  20 ° C for 30 minutes, and then separate the precipitated DNA by centrifuga-

tion at 12,000 g.

1. Enzymatic Labeling of DNA 971

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides

by enzymatic means. The ﬁ rst two are biotin derivatives of pyrimidine and purine bases, respectively, that can

be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modiﬁ cation of

DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The

third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal

transferase. The modiﬁ ed oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to

create a detectable probe.

972 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

6. Remove the supernatant and wash the pellet with 70 and 100 percent ethanol, centrifug-

ing after each wash.

7. Re-dissolve the labeled DNA pellet in water and store at  20 ° C until used.

Protocol for the Labeling of DNA by Random Priming

1. Denature 1 g of probe DNA (single stranded) with 5 g of random hexanucleotide prim-

ers by boiling for 5 minutes and then rapidly chill on ice. Incubate at least 10 minutes to

allow the primers to hybridize to random sites within the probe DNA.

2. Add to a tube on ice, 5 l of 10  random priming buffer (0.5 M Tris, 0.1 M MgCl

, 10 mM

2-mercaptoethanol, pH 6.6, containing 0.5 mg/ml BSA), 1 l each of 3 types of unmodiﬁ ed

deoxynucleoside triphosphates (dNTPs at 100 M concentration), 1 l of a labeled dNTP (at

100 M), plus 48 l of water. Then add 2 l of DNA polymerase (5–10 units).

3. Combine the probe DNA/hexanucleotide preparation with the reaction solution and

incubate for 2 hours at 37 ° C.

4. Quench the reaction by the addition of 2 l of 0.5 M EDTA, 2 l of 10 mg/ml tRNA, and

150  l of 10 mM Tris, pH 7.5.

5. Purify the labeled probe by ethanol precipitation according to steps 4–7 of the protocol

previously described for nick translation.

Protocol for Labeling DNA at the 3  End Using Terminal Transferase

1. Prepare 1 g of puriﬁ ed DNA probe, either by restriction digestion or by synthetic means.

2. Add to the puriﬁ ed probe, (a) 20 l of 0.5 M potassium cacodylate, 5 mM CoCl

, 1 mM

dithiothreitol (DTT), pH 7.0, (b) 100 M of a modiﬁ ed deoxynucleoside triphosphate,

4  l of 5 mM dCTP, and 100  l of water. Mix.

3. Add terminal transferase to a ﬁ nal concentration of 50 units in the reaction mixture.

4. React for 45 minutes at 37 ° C.

5. Isolate the labeled probe by alcohol precipitation as described previously for nick

translation.

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides

The chemical modiﬁ cation of nucleic acids at speciﬁ c sites within individual nucleotides or

within oligonucleotides allows various labels to be incorporated into DNA or RNA probes.

This labeling process can produce conjugates having sensitive detection properties for the locali-

zation or quantiﬁ cation of oligo binding to a complementary strand using hybridization assays.

Some form of chemical labeling process must be used regardless of whether the ﬁ nal oligo

conjugate is created by enzymatic or strictly chemical means. If enzymatic modiﬁ cation is to

be done, the initial label still must be incorporated into an individual nucleoside triphosphate,

which then is polymerized into an existing oligonucleotide strand (Section 1, this chapter).

Fortunately, many useful modiﬁ ed nucleoside triphosphates are now available from commer-

cial sources, often eliminating the need for custom derivatization of individual nucleotides.

Chemical modiﬁ cation also may be used to label directly an oligonucleotide, eliminating the

enzymatic step altogether. The chemical modiﬁ cation of nucleic acids can encompass several

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 973

strategies. The initial derivatization only might be done to add a spacer arm to a particular

reactive group on the nucleotide structure. The spacer typically contains a terminal functional

group, such as an amine, that can be used to couple another molecule. A secondary modiﬁ -

cation might be to add a ﬂ uorescent tag to the end of the spacer, thus creating a detectable

complex. The spacer also may be used to react with a crosslinking agent, such as a heterobi-

functional compound (Chapter 5), that can facilitate the conjugation of a protein or another

molecule to the modiﬁ ed nucleotide. It should be noted that if enzymatic methods are used to

incorporate a small spacer into an oligonucleotide, subsequent chemical conjugation steps still

will be needed to add a small label or protein tag.

In some cases, if an oligonucleotide contains the appropriate functional group, a label may

be directly incorporated into it using chemical methods. For instance, certain ﬂ uorescent mole-

cules or biotin tags can be used to modify nucleotides without going through an initial derivati-

zation step with a spacer arm. Such labels usually contain nucleophilic or photoreactive groups

that can couple directly to the oligo using an intermediate activating agent or by photolyzing

with UV light, respectively.

Many of the chemical derivatization methods employed in these strategies involve the use of

an activation step that produces a reactive intermediary. The activated species then can be used

to couple a molecule containing a nucleophile, such as a primary amine or a thiol group. The

following sections describe the chemical modiﬁ cation methods suitable for derivatizing indi-

vidual nucleic acids as well as oligonucleotide polymers.

2.1. Diamine or Bis-Hydrazide Modiﬁ cation of DNA

One of the more useful chemical modiﬁ cations that can be done on nucleic acids or oligonucle-

otides is to add an amine-terminal spacer arm using a diamine compound. The resultant amine

derivative can be targeted by numerous amine-reactive crosslinkers or modiﬁ cation reagents to

create a detectable conjugate. A similar approach can be used to modify a DNA probe with a

bis-hydrazide compound (Chapter 4, Section 8) to produce terminal hydrazide group. The oli-

gonucleotide derivative then can be coupled with aldehyde-containing molecules to form con-

jugates. The following methods utilize activation reagents which transform a particular site on

nucleic acids into an amine-reactive or hydrazide-reactive intermediate. Coupling a diamine or

bis-hydrazide compound to these activated species results in the formation of an alkyl spacer

arm terminating in a primary amine group or a hydrazide functional group, respectively.

Conjugation via Bisulﬁ te Activation of Cytosine

Single-stranded DNA molecules can react with sodium bisulﬁ te, adding a sulfonate group

across the 5,6-double bond of cytidine bases and creating 6-sulfo-cytosine derivatives. The reac-

tion catalyzes the deamination of cytosine to uracil by loss of the 4-amino group. Subsequent

loss of HSO



effectively forms uracil bases ( Figure 27.2 ). This reaction sequence was recog-

nized in the early 1970s as potential evidence for the mutagenicity of bisulﬁ te (Shapiro et al .,

1973, 1974). Shapiro and Weisgras (1970) demonstrated that the bisulﬁ te reaction also can

cause transamination to occur at the N-4 position of cytosine. In the presence of an amine-

containing molecule, such as a diamine, sodium bisulﬁ te will cause the exchange of the

N-4 amine for another amine-containing compound, effectively forming a new covalent linkage

974 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

with release of ammonium ion ( Figure 27.3 ). Draper and Gold (1980) used this reaction to

produce primary amine groups on a poly(C) oligonucleotide by coupling 1,3-diaminopropane

to a limited number of the cytosine residues. The amine derivative subsequently could be used

to couple a ﬂ uorescent probe to the polymer, allowing sensitive studies of messenger RNA.

Figure 27.2 Treatment of cytosine bases with bisulﬁ te results in a multi-step deamination reaction, ultimately

leading to uracil formation.

Figure 27.3 The reaction of cytosine with bisulﬁ te in the presence of an excess of an amine nucleophile (such

as a diamine compound) leads to transamination at the N-4 position. This process is a route to adding an amine

functional group to cytosine residues in oligonucleotides.

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 975

Bisulﬁ te-catalyzed transamination also can be used to label oligonucleotide probes for appli-

cation in nonradioisotopic hybridization assays. Viscidi et al. (1986) described a method for

derivatizing cytosine groups in DNA probes using the short spacer, ethylenediamine. Other

diamine molecules also may be used, such as 1,3-diaminopropane, 1,6-diaminohexane, or 3,3  -

iminobispropylamine. The use of the long, hydrophilic Jeffamine molecules (Texaco Chemical

Co; see Chapter 1, Section 4.3) may be especially well suited for this type of modiﬁ cation due

to the presence of a hydrophilic polyethylene glycol (PEG)-based spacer. Longer spacer arms

may provide better steric accommodation for larger detection components without interfering

substantially in the probe ’s ability to hybridize to a complementary DNA strand.

If an amine-containing ﬂ uorescent probe or hydrazide-containing compound is transami-

nated onto an oligonucleotide using bisulﬁ te, the labeling of nucleic acids can be done in a

single step. An example of this approach is the coupling of biotin hydrazide (Chapter 11,

Section 3) to cytosine residues, resulting in a biotinylated oligonucleotide suitable for

(strept)avidin-based detection systems (Reisfeld et al ., 1987) (Chapter 23).

Since the site of modiﬁ cation on cytosine bases is at a hydrogen bonding position in double

helix formation, the degree of bisulﬁ te derivatization should be carefully controlled. Reaction

conditions such as pH, diamine concentration, and incubation time and temperature affect the

yield and type of products formed during the transamination process. At low concentrations

of diamine, deamination and uracil formation dramatically exceed transamination. At high

concentrations of diamine (3 M), transamination can approach 100 percent yield (Draper and

Gold, 1980). Ideally, only about 30–40 bases should be modiﬁ ed per 1,000 bases to assure

hybridization ability after derivatization.

Bisulﬁ te modiﬁ cation of cytosine residues also can be used to add permanently a sulfone

group to the C-6 position. In this scheme, the sulfone functions as a hapten recognizable by

speciﬁ c anti-sulfone antibodies. At high concentrations of bisulﬁ te and in the presence of methyl-

hydroxylamine, cytosines are transformed into N

-methoxy-5,6-dihydrocytosine-6-sulphonate

derivatives (Herzberg, 1984; Nur et al., 1989). Labeled antibodies can then be used to detect

the hybridization of such probes.

Protocol for Labeling Nucleic Acids by Bisulﬁ te-Catalyzed Transamination

1. Prepare single-stranded DNA (denatured) at a concentration of 1 mg/ml.

2. Prepare bisulﬁ te modiﬁ cation solution consisting of: 3 M concentration of a diamine (i.e.,

ethylenediamine), 1 M sodium bisulﬁ te, pH 6. The use of the dihydrochloride form of

the diamine avoids having to adjust the pH down from the severe alkaline pH of the

free-base form. Note: The optimum pH for transaminating biotin–hydrazide to cytosine

residues using bisulﬁ te is 4.5 (see Section 2.3, this chapter).

3. Add 20  l of the DNA to 180  l of bisulﬁ te modiﬁ cation solution. Mix well.

4. React for 3 hours at 42 °C.

5. Dialyze the solution against water overnight at 4 °C to remove excess reactants.

6. The modiﬁ ed DNA may be recovered by alcohol precipitation according to the method in

Section 1 (this chapter) described previously for nick-translation modiﬁ cation. Alternatively,

dialysis or gel ﬁ ltration may be done to remove excess reactants.

Conjugation via Bromine Activation of Thymine, Guanine, and Cytosine

The nucleotide bases of DNA and RNA can be activated with bromine to produce reactive

intermediates capable of coupling to nucleophiles (Traincard et al., 1983; Sakamoto et al .,

976 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

1987; Keller et al., 1988). Bromination occurs at the C-8 position of guanine residues and the

C-5 of cytosine, yielding reactive derivatives which can be used to couple diamine spacer mol-

ecules by nucleophilic substitution ( Figure 27.4 ). Other pyrimidine derivatives also are reac-

tive to bromine compounds, but adenine residues are more resistant. However, even AMP can

be immobilized through the introduction of an aminohexyl spacer at the C-8 position using

bromination (Lowe, 1979). Either an aqueous solution of bromine or the compound N -bromo-

succinimide can be used for this reaction. The alkaline modiﬁ cation proceeds rapidly, but may

be too severe for RNA molecules. Coupling of amine-containing molecules is done at elevated

temperatures (50 °C) to assure good incorporation. Both amine-bearing spacers and probes may

be coupled using this strategy. Moreover, the sites of derivatization using bromine activation

are not involved in hydrogen bonding during base pairing, thus maintaining hybridization abil-

ity in the ﬁ nal conjugate.

Optimal bromination of a DNA probe is in the range of 30–35 bases per 1,000 bases, a

level which can be controlled by the amount of N-bromosuccinimide added. Over labeling can

prevent speciﬁ c interactions with target DNA, even if the point of initial modiﬁ cation is not a

hydrogen bonding site.

Figure 27.4 Reaction of guanine bases with N-bromosuccinimide causes bromination at the C-8 position of the

ring. Amine nucleophiles can be coupled to this active derivative by nucleophilic displacement. Reaction of diamine

compounds results in amine-terminal spacers that can be further modiﬁ ed to contain detectable components.

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 977

The major disadvantage with bromination is the extreme toxicity of bromine. Use a fume

hood for all operations. Avoid the breathing of fumes or contact with skin or eyes. Protective

clothing and gloves are recommended.

Protocol for Labeling Nucleic Acids by N -Bromosuccinimide Activation

Bromination

1. Mix in a microfuge tube, 20 g of the DNA probe to be labeled, 20 l of 1 M sodium

bicarbonate, pH 9.6, and 196  l of water. Chill on ice.

2. In a fume hood, dissolve N-bromosuccinimide (Thermo Fisher) in water at a concentra-

tion of 1.42 mg/ml.

3. Add 4 l of the N-bromosuccinimide solution to the DNA solution (makes an 8 mM ﬁ nal

concentration of brominating reagent). Mix well.

4. React on ice for 10 minutes. Use the bromine-activated DNA immediately.

Coupling a Diamine-Containing Spacer or Probe

1. Dissolve a diamine spacer (i.e., ethylenediamine or 1,6-diaminohexane—Thermo Fisher)

in water at a concentration of 80–100 mM. Caution: Amine-containing molecules such

as diamines are highly corrosive if they are in the free-base form (not the dihydrochlo-

rides). Wear gloves and other protective clothing. The pH of an aqueous solution of free-

base diamine will be pH  12 and may fume. The solution also may generate heat upon

dissolution of the amine. Keeping it in an ice bath will help maintain a cool solution with

less fuming. Using a dihydrochloride form of a diamine, if available, will avoid the prob-

lems associated with corrosiveness, heat, and fuming.

2. Add 25 l of the diamine solution to the bromine-activated DNA solution prepared in

the Bromination section, above.

3. React for 1 hour at 50 °C.

4. The diamine-modiﬁ ed DNA may be isolated from excess reactants by ethanol precipi-

tation according to steps 4–7 of the protocol described previously for nick translation

(Section 1, this chapter). Alternatively, dialysis or gel ﬁ ltration may be done to remove

excess reactants.

Conjugation via Carbodiimide Reaction with the 5  Phosphate of DNA

(Phosphoramidate Formation)

The water-soluble carbodiimide EDC (Chapter 3, Section 1.1) rapidly reacts with carboxylates

or phosphates to form an active complex able to couple with amine-containing compounds.

The carbodiimide activates an alkyl phosphate group to a highly reactive phosphodiester

intermediate. Diamine spacer molecules or amine-containing probes then may react with

this active species to form a stable phosphoramidate bond. Alternatively, bis-hydrazide com-

pounds (Chapter 4, Section 8) may be coupled to DNA using this protocol to result in terminal

hydrazide groups able to react with aldehyde-containing molecules Ghosh et al., 1989). Speciﬁ c

labeling of DNA probes only at the 5  end is possible using these techniques.

Carbodiimide modiﬁ cation of the phosphomonoester end groups on DNA molecules was

ﬁ rst used in Khorana ’s lab to determine nucleotide sequences (Ralph et al., 1962). That early

978 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

work used the water-insoluble reagent N , N  -dicyclohexylcarbodiimide (DCC) (Chapter 3,

Section 1.4) in an organic/aqueous solvent system to effect the conjugations.

Chu et al. (1983, 1986) and Ghosh et al. (1990) describe modiﬁ ed carbodiimide protocols

using the water-soluble reagent, EDC, instead of DCC. They also incorporate a second reactive

intermediate, a phosphorimidazolide, created from the reaction of the phosphomonoester at

the 5 -terminus of DNA with EDC in the presence of imidazole. A reactive phosphorimida-

zolide will rapidly couple to amine-containing molecules to form a phosphoramidate linkage

(Figure 27.5 ). The chemistry had been used previously to effect the formation of phosphodi-

ester linkages between short DNA strands (Shabarova et al ., 1983).

Figure 27.5 Oligonucleotides containing a 5 -phosphate group can be reacted with EDC in the presence of

imidazole to form an active phosphorimidazolide intermediate. This derivative is highly reactive with amine

nucleophiles, forming a phosphoramidate linkage. Diamines reacted with the phosphorimidazolide result in

amine-terminal spacers that can be modiﬁ ed with detectable components.

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 979