Hermanson G. Bioconjugate Techniques, Second Edition

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Protocol

1. Prepare diazotized -aminobenzoyl biocytin by using the protocol outlined in Chapter 11,

Section 5, but instead of starting with 2 mg the biotinylation reagent dissolved in 40 l of

1 N HCl, use 9 mg in 180 l of 1 N HCl. Proportionally scale up the other reactant quanti-

ties used in the protocol. After the reaction is complete, immediately adjust the pH of the

ﬁ nal solution to 9.

2. Add 1  g of single-stranded DNA to the above solution.

3. React for 30 minutes at room temperature.

4. Purify the biotinylated DNA probe by ethanol precipitation, gel ﬁ ltration, n -butanol

extraction, or dialysis as discussed in previous sections.

Reaction of Biotin–BMCC with Sulfhydryl-Modiﬁ ed DNA

Biotin–BMCC is a sulfhydryl-reactive biotinylation reagent containing a maleimide group at

the end of an extended spacer arm. The long spacer (32.6 Å) provides enough distance between

modiﬁ ed oligonucleotides and the bicyclic biotin end to allow efﬁ cient binding of (strept)avidin

probes, even when hybridized to target sequences. The reagent may be used to add a biotin

label to DNA or RNA molecules after they have been modiﬁ ed to contain thiol groups. For

instance, cystamine labeling at the 5  phosphate group of DNA via carbodiimide-mediated

phosphoramidate formation followed by disulﬁ de reduction (Section 2.2, this chapter) can cre-

ate the required sulfhydryl groups. Subsequent reaction with biotin–BMCC results in a deriva-

tive labeled only at an end of the DNA probe ( Figure 27.12 ), thus avoiding the potential for

hydrogen bonding interference in hybridization assays.

Since maleimide groups are highly speciﬁ c for coupling to thiols in the pH range of 6.5 to

7.5, side reaction products can be avoided. The reaction is complete within two hours at room

temperature.

Protocol

1. Prepare 10–20 g of a sulfhydryl-containing oligonucleotide in 200 l of 50 mM sodium

phosphate, 10 mM EDTA, pH 7.2 (the methods outlined in Section 2.2 of this chapter

can be used to form the thiol group).

2. Dissolve biotin–BMCC in DMSO at a concentration of 5 mg/ml. Prepare fresh.

3. Add 50  l of the biotinylation solution to the oligo. Mix well.

4. React for 2 hours at room temperature.

5. Isolate the biotinylated probe by ethanol/salt precipitation as described in Section 1 for

nick translation (this chapter).

Biotin–Hydrazide Modiﬁ cation of Bisulﬁ te-Activated Cytosine Groups

Biotin–hydrazide is the hydrazine derivative of D-biotin prepared using its valeric acid carboxy-

late (Chapter 11, Section 3). The hydrazide group typically is used to react with aldehyde and

ketone groups to give hydrazone linkages. However, the hydrazide compound also can undergo

transamination reactions with cytosine residues via catalysis with bisulﬁ te (Section 2.1, this chap-

ter) ( Figure 27.13 ). DNA or RNA probes containing cytosine groups may be modiﬁ ed to contain

biotin labels using a simple, one-step procedure (Reisfeld et al., 1987). The detection limit of

DNA probes biotinylated using this technique can be less than 1 pg on blots, when analyzed

990 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

using a streptavidin–HRP conjugate with chemiluminescent detection. A longer chain analog of

biotin–hydrazide, biotin-LC-hydrazide, may be used to create an extended spacer between the

oligonucleotide and the bicyclic biotin group, thus increasing the binding efﬁ ciency of avidin or

streptavidin conjugates. Alternatively, a hydrophilic biotin–PEG–hydrazide compound may be

used in this reaction to provide greater water solubility for the label (Chapter 18, Section 3).

Leary et al. (1983) reported that increasing the spacer arm length from 4 to 11 atoms when

biotinylating DNA probes can increase the detectability of the target DNA approximately 4-fold.

The following method is adapted from Reisfeld et al . (1987).

Protocol

1. Prepare 50 g of a single-stranded DNA probe in 300 l of 50 mM sodium acetate, pH 4.5.

2. Dissolve biotin–hydrazide in water at a concentration of 10 mg/ml.

3. Add 300  l of the biotin–hydrazide solution to the DNA solution.

4. Add sodium bisulﬁ te to obtain a ﬁ nal concentration of 1 M in the reaction medium.

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 991

Figure 27.12 Biotin–BMCC can be used to modify a reduced, cystamine derivative of DNA, forming a thioether

linkage.

5. React for 24 hours at 37 °C.

6. Remove excess reactants by dialysis against water at 4 °C.

2.4. Enzyme Conjugation to DNA

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed

in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme

molecule provides detectability for the oligonucleotide through turnover of substrates that can

produce chromogenic or ﬂ uorescent products. Enzyme-based hybridization assays are perhaps

the most common method of nonradioactive detection used in nucleic acid chemistry today.

The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic

acids, thus eliminating the need for radioactivity in most assay systems.

The conjugation reactions involved in DNA–enzyme crosslinking are not unlike the methods

used to form antibody–enzyme conjugates (Chapter 20, Section 1). Bifunctional crosslinkers

can be used to couple a modiﬁ ed oligonucleotide to an enzyme molecule using the same basic

principles effective in protein–protein conjugation. The only requirement is that the DNA mole-

cule be modiﬁ ed to contain one or more suitable functional groups, such as nucleophiles like

amines or sulfhydryls. The modiﬁ cation process used to create these functional groups can use

enzymatic (Section 1, this chapter) or chemical (Section 2, this chapter) means and it can result

992 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

Figure 27.13 Biotin–hydrazide may be incorporated into cytosine bases using a bisulﬁ te-catalyzed transamina-

tion reaction.

in random incorporation of modiﬁ cation sites or be directed exclusively at one end of the DNA

molecule, such as in 5  phosphate coupling.

The following sections describe some of the more common procedures of preparing DNA–

enzyme conjugates.

Alkaline Phosphatase Conjugation to Cystamine-Modiﬁ ed DNA Using Amine- and

Sulfhydryl-Reactive Heterobifunctional Crosslinkers

A cystamine group added to the 5  phosphate of DNA molecules using a carbodiimide reaction

(Section 2.2, this chapter) can be used in a heterobifunctional crosslinking scheme to conju-

gate with alkaline phosphatase. Crosslinking agents containing an amine-reactive portion and

a sulfhydryl-reactive part work best in forming this type of conjugate. Perhaps one of the most

common heterobifunctional reagents used for DNA–enzyme formation is SPDP (Chapter 5,

Section 1.1). SPDP contains an NHS ester on one end able to create an amide bond linkage

with amino groups on protein molecules. After modiﬁ cation of alkaline phosphatase with

this crosslinker, the enzyme is activated to contain pyridyl disulﬁ de groups for coupling to the

sulfhydryls on a cystamine-modiﬁ ed DNA probe ( Figure 27.14 ). The reaction forms disulﬁ de

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 993

Figure 27.14 An oligonucleotide modiﬁ ed with cystamine and reduced to generate a free sulfhydryl may be

conjugated with an SPDP-modiﬁ ed enzyme, forming a disulﬁ de linkage.

bonds between the oligonucleotide and the alkaline phosphatase enzyme. Since the crosslink

occurs only at the 5  end of the DNA strand, the presence of an enzyme molecule does not

adversely affect the ability of base pairing and hybridization to a target sequence.

The following protocol assumes that the labeling process used to create a sulfhydryl-modi-

ﬁ ed DNA probe already has been done according to the method of Section 2.2 (this chap-

ter). The modiﬁ cation procedure for activating alkaline phosphatase with SPDP may be done

according to the protocol described in Chapter 5, Section 1.1. To obtain efﬁ cient labeling of

all the alkaline phosphatase added to the reaction medium, the modiﬁ ed oligo is reacted in

a 10-fold molar excess. Reaction of the DNA probe in excess allows easy separation of not-

coupled oligo from conjugated probe, thus eliminating any potential interference in hybridi-

zation assays due to unlabeled oligonucleotide.

Protocol

1. Dissolve a 5  -sulfhydryl-modiﬁ ed oligonucleotide in water or 10 mM EDTA at a con-

centration of 0.05–25  g/ l. Calculate the total nmoles of oligo present based upon its

molecular weight.

2. Prepare SPDP-activated alkaline phosphatase in 50 mM sodium phosphate, 0.15 M NaCl,

10 mM EDTA, pH 7.2. Add to the oligo solution, an amount of the activated enzyme

representing a 10-fold molar excess over the calculated amount of DNA present.

3. React at room temperature for 30 minutes with gentle mixing.

4. The alkaline phosphatase–DNA conjugate may be puriﬁ ed away from excess oligo by

dialysis, gel ﬁ ltration, or through the use of centrifugal concentrators. A simple way of

removing unreacted oligo is to use Centricon-30 concentrators (Amicon) which have a

molecular weight cutoff of 30,000. Since the enzyme molecular weight is approximately

140,000 and the conjugate is even higher, a relatively small DNA probe will pass through

the membranes of these units while the conjugate will not. To purify the conjugate using

Centricon-30 s, add 2 ml of the phosphate buffer from step 2 to one concentrator unit,

then add the reaction mixture to the buffer and mix. Centrifuge at 1,000 g for 15 minutes

or until the retentate volume is about 50 l. Add another 2 ml of buffer and centrifuge

again until the retentate is 50 l. Invert the Centricon-30 unit and centrifuge to collect

the retentate in the collection tube provided by the manufacturer.

Alkaline Phosphatase Conjugation to Diamine-Modiﬁ ed DNA Using DSS

Disuccinimidyl suberate (DSS) is a homobifunctional crosslinker containing an amine-reac-

tive NHS ester at both ends (Chapter 4, Section 1.2). Reaction of the reagent in excess with

diamine-modiﬁ ed DNA probes creates an activated intermediate able to conjugate with enzyme

molecules through their available amine groups ( Figure 27.15 ) (Jablonski et al., 1986). The

coupling reaction produces stable amide linkages under mildly alkaline conditions. Although

the following protocol has been used to label oligonucleotides with success, it may be less efﬁ -

cient than the previous protocol at forming the desired conjugate due to the homobifunctional

nature of the crosslinker. During the activation step, the modiﬁ ed DNA must be puriﬁ ed away

from excess DSS. Since this is done under aqueous conditions, hydrolysis of the free NHS ester

at the other end of the crosslinker takes place at the same time. Activity losses can be severe if

the separation step is not done rapidly. In fact, the original protocol called for several hours

994 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

of gel ﬁ ltration chromatography and concentration before the conjugation reaction was done.

Farmer and Castaneda (1991) made a signiﬁ cant improvement to this procedure by including

a faster separation step using alcohol extraction after activation of the oligo with DSS. The

puriﬁ cation time decreased to minutes instead of hours. This does help to limit hydrolysis, but

cannot completely avoid it.

The following protocol is based on the methods of Farmer and Castaneda (1991), Kiyama

et al . (1992), and Ruth (1993).

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 995

Figure 27.15 The homobifunctional crosslinker DSS may be used to conjugate an enzyme to a 5  -diamine-

modiﬁ ed oligonucleotide. The NHS ester groups on DSS react with the amines to form amide bonds.

Protocol

1. Prepare an amine-modiﬁ ed oligonucleotide according to any of the protocols discussed

in Section 2.1 (this chapter). Dissolve or buffer-exchange the oligo into 0.1 M sodium

borate, 2 mM EDTA, pH 8.25, at a concentration of 9 nmol (2.0 A

260 nm

units) in 15  l.

2. Dissolve DSS in dry DMSO at a concentration of 1 mg/100  l. Prepare fresh.

3. Add 30  l of the DSS solution to the oligo. Mix well.

4. React for 15 minutes at room temperature in the dark.

5. Immediately extract excess DSS and reaction by-products by the addition of 0.5 ml of

n-butanol. Mix vigorously by vortexing and centrifuge (1 minute, 15,000 rpm) to separate

the two phases. Carefully remove the upper layer and discard. Extract two more times

with n -butanol.

6. Chill the remaining sample on dry ice and lyophilize to remove the last traces of liquid.

The drying period will only take 15–30 minutes. The dried, DSS-activated DNA is stable

under anhydrous conditions.

7. Dissolve or dialyze alkaline phosphatase into 3 M NaCl, 30 mM triethanolamine, 1 mM

MgCl2, pH 7.6, at a concentration of 20 mg/ml.

8. Add 70 l of the alkaline phosphatase to the dried, DSS-activated DNA. Mix gently to

dissolve.

9. React overnight at 4 °C in the dark.

10. Remove unconjugated oligo by using a Centricon-30 centrifugal concentrator according

to step 4 of the protocol described in the previous section. Unconjugated enzyme may

be removed by ion-exchange chromatography using a MonoQ-10 (0.5  5 cm) FPLC col-

umn (GE Healthcare) or the equivalent. Binding buffer is 20 mM Tris, pH 8. Elute using

a linear gradient of 0–100 percent 20 mM Tris, 1 M NaCl, pH 8. Free enzyme will elute

before the more negatively charged oligo–enzyme conjugate.

Enzyme Conjugation to Diamine-Modiﬁ ed DNA Using PDITC

PDITC, 1,4-phenylene diisothiocyanate, is a homobifunctional crosslinker containing two

amine-reactive isothiocyanate groups on a phenyl ring. Reaction in excess with an amine-modi-

ﬁ ed oligonucleotide results in the formation of a thiourea linkage, leaving the second isothio-

cyanate group free to couple with amine-containing enzymes or other molecules (Urdea et al .,

1988) ( Figure 27.16 ).

The following protocol is adapted from Keller and Manak (1989).

Protocol

1. Prepare an amine-modiﬁ ed oligonucleotide using any of the methods of Sections 1 or 2.1

(this chapter). Dissolve or buffer exchange 70 g of the oligo into 25 l of 0.1 M sodium

borate, pH 9.3.

2. Dissolve 10 mg of PDITC (Aldrich) in 500 l of DMF. Add this solution to the oligo pre-

pared in step 1.

3. React for 2 hours at room temperature in the dark.

4. To extract excess reactant, add to the reaction medium, 3 ml of n-butanol and 3 ml of

water. Mix well. Centrifuge the mixture to separate the two phases. Discard the upper

996 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

yellow layer. Repeat the extraction process several times, and then dry the remain-

ing solution containing activated oligo using lyophilization or a rotary evaporator. The

PDITC-activated DNA is stable in a dried state.

5. Dissolve HRP (Chapter 26) in 200 l 0.1 M sodium borate, pH 9.3, at a concentration

of 10 mg/ml. If the HRP is supplied as an ammonium sulfate suspension, all ammonium

ions must be removed by extensive dialysis prior to the conjugation reaction. Add the

HRP solution to the activated oligo.

6. React overnight at room temperature in the dark.

7. Excess enzyme may be removed through isolation of the oligo–enzyme conjugate using

electrophoresis separation under nondenaturing conditions. The reaction solution is applied

to a 7 percent polyacrylamide gel using 90 mM Tris, 90 mM Boric acid, 2.7 mM EDTA, pH

8.3, as the running buffer. The conjugate appears as a brown band in the middle of the gel.

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 997

Figure 27.16 The homobifunctional crosslinker PDITC may be used to conjugate an enzyme to a 5  -diamine-

modiﬁ ed oligonucleotide, creating isothiourea linkages.

998 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

Conjugation of SFB-Modiﬁ ed Alkaline Phosphatase to Bis-Hydrazide-Modiﬁ ed Oligonucleotides

DNA probes modiﬁ ed to contain a 5 -terminal hydrazide group (Section 2.1, this chapter)

can be conjugated to aldehyde-containing molecules, resulting in the formation of a hydra-

zone bond. The crosslinking agent succinimidyl p-formylbenzoate (SFB; Chapter 17, Section 2)

can be used to add aldehyde groups to proteins and other molecules that do not naturally

contain them (Chapter 1, Section 4.4). Reaction of SFB-modiﬁ ed alkaline phosphatase with

a hydrazide–DNA derivative can produce a conjugate having excellent sensitivity for use as a

hybridization probe ( Figure 27.17 ). Other enzymes and detection molecules modiﬁ ed to con-

tain aldehydes may be coupled to hydrazide–DNA probes using similar methods. Using an

SFB-modiﬁ ed HRP (or periodate-oxidized HRP) that contains aldehyde groups to prepare the

DNA conjugate gives about a 40-fold less sensitivity in hybridization assays when compared to

an alkaline phosphatase conjugate in this procedure (Ghosh et al., 1989). However, this result

may change if chemiluminescent detection using an HRP conjugate is done, as this method can

result in femtogram detection limits.

The following protocol assumes the prior derivatization of an oligonucleotide at the 5  end

using a bis-hydrazide compound according to the protocol of Section 2.1 (this chapter) using a

carbodiimide-mediated reaction.

Protocol

1. Dissolve alkaline phosphatase at a concentration of 10 mg/ml in 0.1 M sodium bicarbon-

ate, 3 M NaCl, pH 8.5. Dialyze against this solution if the enzyme is already dissolved in

another buffer. This protocol requires at least 0.4 ml of the enzyme solution.

2. Dissolve SFB in acetonitrile at a concentration of 50 mM (12.35 mg/ml). Make at least 100 l.

3. Add 40  l of the SFB solution to the 0.4 ml alkaline phosphatase solution with mixing.

4. React for 30 minutes at room temperature.

5. Remove excess reactants by dialysis against 50 mM MOPS, 0.1 M NaCl, pH 7.5.

6. Add the aldehyde-derivatized alkaline phosphatase to 8 nmol of a 5 -hydrazide oligonu-

cleotide preparation made according to Section 2.1 (this chapter) using a carbodiimide

coupling protocol.

7. React overnight at room temperature.

8. Remove unconjugated oligonucleotide using gel ﬁ ltration on a column of Bio-Rad P-100

(1.5  65 cm). Use 50 mM Tris, pH 8.5 as the chromatography buffer. Pool the enzyme

fractions and apply the sample to a 1  7 cm column of DEAE–cellulose equilibrated

with the same buffer. After washing the column with 0.1 M Tris, pH 8.5, a salt gradient

from 0 to 0.2 M NaCl in the same buffer is used to remove unconjugated enzyme. The

enzyme–DNA conjugate is then eluted with 0.1 M Tris, 0.5 M NaCl, pH 8.5.

2.5. Fluorescent Labeling of DNA

Oligonucleotide probes may be labeled with small ﬂ uorescent molecules for detection of

hybridization by luminescence. Fluorescent probes are widely used in assay systems involving

bio-speciﬁ c interactions (Chapter 9). Receptors for ligands may be localized in tissues or cells

by modiﬁ cation of the ligand with the appropriate ﬂ uorophore. Targeted molecules may be

quantiﬁ ed through measurement or modulation of a ﬂ uorescent signal upon binding of a tagged

ligand. The sensitivity of ﬂ uorescent assays can approach that obtained using radiolabels.

Fluorescently labeled DNA probes can be used for detection, localization, or quantiﬁ cation

of target DNA sequences. In situ hybridization mapping of genomic DNA sequences can be

Figure 27.17 SFB may be used to create aldehyde groups on enzyme molecules for subsequent conjugation to a

5 -bis-hydrazide-modiﬁ ed oligonucleotide, forming hydrazone bonds.

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 999