Hermanson G. Bioconjugate Techniques, Second Edition

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The formation of a phosphorimidazolide intermediate provides better reactivity toward

amine nucleophiles than the EDC phosphodiester intermediate if EDC is used without added

imidazole. The EDC phosphodiester intermediate also has a shorter half-life in aqueous condi-

tions due to hydrolysis than the phosphorimidazolide. Although EDC alone will create nucle-

otide phosphoramidate conjugates with amine-containing molecules (Shabarova, 1988), the

result of forming the secondary phosphorimidazolide-activated species is increased derivatiza-

tion yield over carbodiimide-only reactions.

The downside of EDC conjugation with oligonucleotides is the potential for reaction of the

carbodiimide at the guanosine N-1 site or with thymidine residues (von der Haar et al., 1971).

In practice, however, this cross-reactivity appears to be low enough to maintain complete bio-

logical activity and hybridization efﬁ ciency in the ﬁ nal conjugate, indicating most of the deriva-

tization occurs at the 5  phosphate group (Chu et al ., 1983).

The following protocol describes the modiﬁ cation of DNA or RNA probes at their 5  -phos-

phate ends with a bis-hydrazide compound, such as adipic acid dihydrazide or carbohydrazide.

A similar procedure for coupling the diamine compound cystamine can be found in Section 2.2

(this chapter).

Protocol

1. Weigh out 1.25 mg of the carbodiimide EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodii

mide hydrochloride; Thermo Fisher) into a microfuge tube.

2. Add to the tube 7.5 l of RNA or DNA containing a 5  phosphate group. The concentra-

tion of the oligonucleotide should be 7.5–15 nmol or total of about 57–115.5 g. Also

immediately add 5 l of 0.25 M bis-hydrazide compound dissolved in 0.1 M imidazole,

pH 6.0. Because EDC is labile in aqueous solutions, the addition of the oligo and bis-

hydrazide/imidazole solutions should be done quickly.

3. Mix by vortexing, then place the tube in a microcentrifuge and spin for 5 minutes at

maximal rpm.

4. Add an additional 20 l of 0.1 M imidazole, pH 6.0. Mix and react for 30 minutes at

room temperature.

6. Purify the hydrazide-labeled oligo by gel ﬁ ltration on desalting resin using 10 mM sodium

phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The probe now may be used to conju-

gate with an aldehyde-containing molecule.

2.2. Sulfhydryl Modiﬁ cation of DNA

Creating a sulfhydryl group on nucleic acid probes allows conjugation reactions to be done

with sulfhydryl-reactive heterobifunctional crosslinkers (Chapter 5), providing increased

control over the derivatization process. Proteins can be activated with a crosslinking agent

containing an amine-reactive and a sulfhydryl-reactive end, such as N-succinimidyl 3-(2-

pyridyldithio)propionate (SPDP) (Chapter 5, Section 1.1), leaving the sulfhydryl-reactive por-

tion free to couple with the modiﬁ ed DNA probe. Having a sulfhydryl group on the probe

directs the coupling reaction to a discrete site on the nucleotide strand, thus better preserving

hybridization ability in the ﬁ nal conjugate. In addition, heterobifunctional crosslinkers of this

type allow two- or three-step conjugation procedures to be done, which result in better yield of

the desired conjugate than when using homobifunctional reagents.

980 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

Cystamine Modiﬁ cation of 5  Phosphate Groups Using EDC

DNA or RNA may be modiﬁ ed with cystamine at the 5  phosphate group using a carbodiimide

reaction identical to that described previously (Section 2.1, this chapter). In some procedures,

the reaction is carried out in a two-step process by ﬁ rst forming a reactive phosphorimidazolide

by EDC conjugation in an imidazole buffer. Next, cystamine is reacted with the activated oligo-

nucleotide, causing the imidazole to be replaced by the amine and creating a phosphoramidate

linkage (Chu et al., 1986). An easier protocol was described by Ghosh et al. (1990) in which

the oligo, cystamine, and EDC were all reacted together in an imidazole buffer. A modiﬁ cation

of this method developed by Zanocco et al . (1993) is described below.

Reduction of the cystamine-labeled oligo using a disulﬁ de reducing agent releases 2-mer-

captoethylamine and creates a thiol group for conjugation ( Figure 27.6 ). DNA probes labeled

in this manner have been successfully coupled with SPDP-activated alkaline phosphatase

(Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP)

(Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3),

and the ﬂ uo rescent tag AMCA–HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5).

A kit designed speciﬁ cally to perform 5 -phosphate labeling on DNA probes is available

from Thermo Fisher.

Figure 27.6 The 5  -phosphate group of oligonucleotides may be labeled with cystamine using the EDC/imid-

azole reaction. This results in the formation of an amine-terminal spacer containing an internal disulﬁ de group.

Reduction of the disulﬁ de provides a route to creating a free thiol for further derivatization.

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 981

Protocol

1. Weigh out 1.25 mg of the carbodiimide EDC (1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide hydrochloride; Thermo Fisher) into a microfuge tube.

2. Add to the tube 7.5 l of RNA or DNA containing a 5  phosphate group. The concentra-

tion of the oligonucleotide should be 7.5–15 nmol/ l or total of about 57–115.5 g. Also

immediately add 5 l of 0.25 M cystamine in 0.1 M imidazole, pH 6.0. Because EDC is

labile in aqueous solutions, the addition of the oligo and cystamine/imidazole solutions

should be done quickly.

3. Mix by vortexing, then place the tube in a microcentrifuge and spin for 5 minutes at

maximal rpm.

4. Add an additional 20 l of 0.1 M imidazole, pH 6.0. Mix and react for 30 minutes at

room temperature.

5. For reduction of the cystamine disulﬁ des, add 20 l of 1.0 M DTT and incubate at room

temperature for 15 minutes. This will release 2-mercaptoethylamine from the cystamine

modiﬁ cation site and create the free sulfhydryl on the 5  terminus of the oligonucleotide.

6. Purify the SH-labeled oligo by gel ﬁ ltration on a desalting resin using 10 mM sodium

phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The probe now may be used to con-

jugate with an activated enzyme, biotin, ﬂ uorescent tag, or other molecules containing a

sulfhydryl-reactive group.

SPDP Modiﬁ cation of Amines on Nucleotides

Oligonucleotide probes that have been modiﬁ ed with an amine-terminal spacer arm using any

of the methods discussed in Sections 1 and 2 of this chapter may be thiolated to contain a

sulfhydryl residue. Theoretically, any of the amine-reactive thiolation reagents described in

Chapter 1, Section 4.1 may be used to convert an amino group on a DNA molecule into a

thiol. One of the more common choices, both for crosslinking and for thiolation reactions, is

the heterobifunctional reagent, SPDP (Chapter 5, Section 1.1). The NHS ester end of SPDP

reacts with primary amine groups to produce stable amide bonds. The other end of the

crosslinker contains a thiol-reactive pyridyl disulﬁ de group that also can be reduced with DTT

to create a free sulfhydryl.

The reaction of a 5  -diamine-modiﬁ ed oligonucleotide probe with SPDP proceeds under

mildly alkaline conditions (optimal pH 7–9) to give the pyridyl disulﬁ de-activated intermedi-

ate ( Figure 27.7 ). This derivative has dual functionality. It can be used to couple directly with

sulfhydryl-containing detection reagents or enzymes, or it may be converted into a free sulfhy-

dryl for coupling to thiol-reactive compounds (Gaur et al., 1989; Gaur, 1991). In an alternative

approach, Chu and Orgel (1988) used 2,2  -dipyridyldisulﬁ de (Chapter 1, Section 5.2) to cre-

ate reactive pyridyl disulﬁ de groups on a reduced 5 -cystamine-labeled oligonucleotide probe.

This derivative then can be used to couple with sulfhydryl-containing molecules, forming a

disulﬁ de bond.

Reduction of the pyridyl disulﬁ de end after SPDP modiﬁ cation releases the pyridine-2-thione

leaving group and generates a terminal––SH group. This procedure allows sulfhydryl-reactive

derivatives such as maleimide-activated enzymes (Chapter 26, Section 2.3) to be conjugated

with DNA probes for use in hybridization assays (Malcolm and Nicolas, 1984).

982 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

Protocol

1. Dissolve the amine-modiﬁ ed oligonucleotide to be thiolated in 250 l of 50 mM sodium

phosphate, pH 7.5.

2. Dissolve SPDP (Thermo Fisher) at a concentration of 6.2 mg/ml in DMSO (makes a

20 mM stock solution). Alternatively, LC-SPDP may be used and dissolved at a concen-

tration of 8.5 mg/ml in DMSO (also makes a 20 mM solution). The “ LC ” form of the

crosslinker provides a longer spacer arm that often results in better probe activity after

modiﬁ cation. If the water-soluble sulfo-LC-SPDP is used, a stock solution in water may

be prepared just prior to adding an aliquot to the thiolation reaction. In this case, prepare

a 10 mM solution of sulfo-LC-SPDP by dissolving 5.2 mg/ml in water. Since an aqueous

solution of the crosslinker will degrade by hydrolysis of the sulfo-NHS ester, it should be

used quickly to prevent signiﬁ cant loss of activity.

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 983

Figure 27.7 A oligonucleotide modiﬁ ed at its 5 -phosphate with a diamine compound may be reacted with

SPDP and subsequently reduced to create a free sulfhydryl.

3. Add 50 l of the SPDP (or LC-SPDP) solution to the oligo solution. Add 100 l of the

sulfo-LC-SPDP solution, if the water-soluble crosslinker is used. Mix.

4. React for 1 hour at room temperature.

5. Remove excess reagents from the modiﬁ ed oligo by gel ﬁ ltration on a desalting resin. The

modiﬁ ed probe now may be used to conjugate with a sulfhydryl-containing molecule, or

it may be reduced to create a thiol for conjugation with sulfhydryl-reactive molecules.

6. To release the pyridine-2-thione leaving group and form the free sulfhydryl, add 20  l

of 1.0 M DTT and incubate at room temperature for 15 minutes. If present in sufﬁ cient

quantity, the release of pyridine-2-thione can be followed by its characteristic absorb-

ance at 343 nm (   8.08  10

 1

). For many oligonucleotide modiﬁ cation

applications, however, the leaving group will be present in too low a concentration to be

detectable.

7. Purify the thiolated oligonucleotide from excess DTT by dialysis or gel ﬁ ltration using

50 mM sodium phosphate, 1 mM EDTA, pH 7.2. The modiﬁ ed probe should be used

immediately in a conjugation reaction to prevent sulfhydryl oxidation and formation of

disulﬁ de crosslinks.

SATA Modiﬁ cation of Amines on Nucleotides

Oligonucleotides containing amine groups introduced by enzymatic or chemical means may be

modiﬁ ed with N-succinimidyl S-acetylthioacetate (SATA) (Chapter 1, Section 4.1), to produce

protected sulfhydryl derivatives. The NHS ester end of SATA reacts with a primary amine to

form a stable amide bond. After modiﬁ cation, the acetyl protecting group can be removed as

needed by treatment with hydroxylamine under mildly alkaline conditions ( Figure 27.8 ). The

result is terminal sulfhydryl groups that can be used for subsequent labeling with thiol-reactive

probes or activated-enzyme derivatives (Kumar and Malhotra, 1992).

The advantage of using SATA over disulﬁ de-containing thiolation reagents such as SPDP

(previous section) is that the introduction of sulfhydryl residues does not include the use of a

disulﬁ de reducing agent. Typically, the pyridyl dithiol group resulting from an SPDP thiolation

must be reduced with a sulfhydryl-containing disulﬁ de reducing compound like DTT to free

the––SH group. With SATA, the sulfhydryl is freed by hydroxylamine cleavage, thus eliminat-

ing the need for removal of sulfhydryl reductants prior to a conjugation reaction.

Protocol

1. Dissolve the amine-modiﬁ ed oligonucleotide to be thiolated in 250 l of 50 mM sodium

phosphate, pH 8.0.

2. Dissolve SATA in DMF at a concentration of 8 mg/ml.

3. Add 250  l of the SATA solution to the oligo solution. Mix.

4. React for 3 hours at 37 °C.

5. Remove excess reagents from the modiﬁ ed oligo by gel ﬁ ltration.

6. To deprotect the acetylated thiol group, add 100 l of 50 mM hydroxylamine hydrochlo-

ride, 2.5 mM EDTA, pH 7.5.

7. React for 2 hours.

8. The sulfhydryl-containing oligonucleotide may be used immediately to conjugate with

a sulfhydryl-reactive label, or it can be puriﬁ ed from excess hydroxylamine by gel

ﬁ ltration.

984 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

2.3. Biotin Labeling of DNA

Biotinylation of oligonucleotide probes provides a highly speciﬁ c biological recognition site

for detection of DNA using (strept)avidin conjugates. The preparation of biotin-labeled DNA

can be done either by enzymatic or chemical means. Enzyme-catalyzed reactions utilize bioti-

nylated nucleoside triphosphates that can be incorporated into an oligonucleotide randomly or

at the 3  terminus (Section 1, this chapter). Chemical derivatization methods make use of cer-

tain reactive biotin compounds that can couple to functionally modiﬁ ed probes or react with

DNA with the use of an activating reagent. For a description of the wide range of biotinylation

compounds available, see Chapter 11. The preparation and use of avidin and streptavidin con-

jugates is discussed in Chapter 23.

Biotin-LC-dUTP

Perhaps the most common method of DNA biotinylation is through enzymatic incorporation

with the use of a biotin-labeled deoxynucleoside triphosphate. First reported by Langer et al. in

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 985

Figure 27.8 SATA may be used to modify a 5 -amine derivative of an oligonucleotide, forming a protected sulf-

hydryl. Deprotection with hydroxylamine results in generation of a free thiol.

1981, the procedure is probably the most popular nonradioactive labeling technique reported

for oligonucleotide probes. Although biotinylated derivatives of dCTP and dATP are reported

in the literature, by far the most frequently employed derivative is biotin–dUTP prepared from

the reaction of an amine-modiﬁ ed dUTP with an amine-reactive biotinylation reagent, such as

NHS-LC-biotin (Chapter 11, Section 3.1).

Biotin–dUTP derivatives are formed by modiﬁ cation of the C-5 position of uridine. This

location is not involved in hydrogen bonding activity with complementary DNA strands, thus

hybridization efﬁ ciency is not immediately compromised. By contrast, biotin–dCTP or biotin–

dATP derivatives involve modiﬁ cation of the bases at the N-4 position of cytosine and the N-6

position of adenine, locations directly involved in hydrogen bond formation with complemen-

tary bases. Thus, DNA biotinylation through the use of modiﬁ ed deoxynucleoside triphos-

phates to be incorporated into existing DNA strands may result in better activity of the probe

if dUTP is used over dATP or dCTP.

The length of the spacer arm between the C-5 position of uridine and the biotin group is

another important parameter for activity of the resulting conjugate. The spacer affects the

incorporation efﬁ ciency into existing probes using DNA polymerases, and it also affects the

ability of an (strept)avidin conjugate to effectively bind the biotinylated probe. Designation of

spacer length is usually expressed as the number of atoms separating the nucleotide base from

the biotin component. Thus, biotin- n-dUTP would describe a biotinylated deoxynucleoside tri-

phosphate having a spacer arm n atoms long. The shorter the spacer arm, the better the deriva-

tive is able to be recognized and incorporated into DNA polymers using polymerase enzymes.

Conversely, the longer the spacer arm, the better the biotinylated probe is able to hybridize

to its target and still maintain the capacity to have a streptavidin conjugate be complexed

with it. Thus, there is an optimal trade-off in spacer length between enzymatic incorporation

efﬁ ciency and labeled-probe detectability. Studies have determined that this optimal range is

rather broad—between 7 atoms and 21 atoms in length. Perhaps the most common derivative

is biotin-11-dUTP, wherein an 11-atom spacer is employed ( Figure 27.9 ).

General protocols for the enzymatic incorporation of biotin-11-dUTP into DNA probes can be

found in Section 1 (this chapter). A particularly interesting modiﬁ cation of the typical enzymatic

986 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

Figure 27.9 Biotin-11-dUTP is perhaps the most popular nucleotide derivative used for enzymatic biotinylation

of oligonucleotides. The “11” designation refers to the number of atoms in its spacer arm.

incorporation protocol for biotin is described by Didenko (1993). The single strand template

is immobilized by adsorption onto membranes before synthesis of the biotinylated probe. After

polymerase incorporation of biotin-11-dUTP, the labeled probe is removed by brief heating to

90 ° C in water. The result is highly pure probe with no contaminating complementary DNA

strands.

Photobiotin Modiﬁ cation of DNA

The photoreactive biotin derivative, N -(4-azido-2-nitrophenyl)-aminopropyl- N  -( N -d-biotinyl-

3-aminopropyl)-N-methyl-1,3-propanediamine, simply called photoactivatable biotin or pho-

tobiotin (Forster et al., 1985) contains a 9-atom diamine spacer group on the biotin valeric

acid side chain on one end, while the other end of the spacer terminates in an phenyl azide

group. The phenyl azide group can be photolyzed with UV light (350 nm) resulting in the for-

mation of a highly reactive nitrene intermediate. In most instances, this nitrene rapidly reacts

via ring expansion to form a dehydroazepine that is reactive with nucleophiles, such as amine

groups (Chapter 5, Section 3).

When photobiotin is irradiated in the presence of DNA the reaction process nonselectively

couples a biotin label to every 100–200 base residues. The result is an oligonucleotide probe

detectable by the use of (strept)avidin conjugates. The uses of photobiotin for DNA or RNA

modiﬁ cation are summarized in Chapter 11, Section 4.

The following protocol is based on the method of Forster et al. (1985). Some optimization may

be necessary to obtain the best signal and activity for particular probes in hybridization assays.

Protocol for Labeling DNA Probes with Photobiotin

1. In subdued lighting conditions, dissolve photobiotin in water at a concentration of

1  g/  1. Protect from light.

2. Dissolve the oligonucleotide probe in water or 0.1 mM EDTA, pH 7.0, at a concentration

of 1  g/  l.

3. Mix an equal volume of the photobiotin solution with the DNA probe solution.

4. Place the solution in an ice bath and irradiate from above (about 10 cm away) for

15 minutes using a sunlamp (such as Philips Ultrapnil MLU 300 W, General Electric

sunlamp RSM 275 W, or National Self-Ballasted BHRF 240–250 V 250 W W-P lamp).

5. Add 50 l of 0.1 M Tris, pH 9, and increase the total volume of the solution to 100 l (if

it is less than this amount).

6. To extract excess photobiotin, add 100 l of 2-butanol. Mix well and centrifuge. Discard

the upper phase. Repeat this process two more times.

7. To recover the biotinylated DNA, add 75  l of 4 M NaCl and mix.

8. Add 100 

l of ethanol and cool the sample in dry ice (CO

) for 15 minutes.

9. Centrifuge to collect the precipitated, biotinylated DNA.

Reaction of NHS-LC-Biotin with Diamine-Modiﬁ ed DNA Probes

NHS-LC-biotin is an extended spacer arm derivative of biotin containing an amine-reactive

NHS ester (Chapter 11, Section 1). The compound is a popular choice for biotinylating a wide

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 987

range of molecules containing primary amine groups, especially proteins. Oligonucleotides mod-

iﬁ ed to contain amine-terminal spacer arms also can be modiﬁ ed with NHS-LC-biotin to create

stable amide bond derivatives. Alternatively, a hydrophilic biotinylation compound containing

a PEG spacer arm can be used to form a biotin-oligo derivative without the hydrophobic

character of the alkyl chain within NHS-LC-biotin. Chapter 18, Section 3 describes the amine-

reactive NHS–PEG

–biotin compounds suitable for this purpose.

Whether an amine is incorporated into an oligo by enzymatic means or chemical deriva-

tization, an NHS-ester-containing biotinylation reagent can be used to label the derivative in

high yield. If an amine group is added to the 5  end of a DNA probe by phosphoramidate

formation (Section 2.1, this chapter), then biotinylation of such molecules directs the label to a

region totally removed from interfering in subsequent hybridization with a target DNA strand

(Figure 27.10 ).

The following protocol assumes that the amine-containing oligo has already been synthe-

sized by any of the methods discussed in Section 2.1, this chapter.

988 27. Nucleic Acid and Oligonucleotide Modiﬁ cation and Conjugation

Figure 27.10 Biotinylation of oligonucleotides may be done at the 5 -phosphate end using a diamine derivative

and reacting with NHS-LC-biotin.

Protocol

1. Prepare 10–20 g of amine-containing oligonucleotide in 200 l of water. Add to this

solution, 20  l of 1 M sodium bicarbonate, pH 9.0.

2. Dissolve NHS-LC-biotin (Thermo Fisher) in DMSO at a concentration of 10 mg/ml. Add

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

3. React for 2 hours at room temperature.

4. Isolate the biotinylated probe by ethanol/salt precipitation as described in Section 1 (this

chapter) for nick-translation modiﬁ cation of DNA probes. Alternatively, dialysis, gel ﬁ l-

tration, or n -butanol extraction may be used to remove excess reagents.

Biotin–Diazonium Modiﬁ cation of DNA

Diazonium groups are able to couple at the C-8 position of adenosine or guanosine residues,

forming diazo bonds. -Aminobenzoyl biocytin can be used in this reaction to add a biotin

handle to purine bases within oligonucleotides (Chapter 11, Section 5). This biotinylation rea-

gent contains a 4-aminobenzoic acid amide derivative off the -amino group of biocytin ’s lysine

residue (Thermo Fisher). The aromatic amine can be treated with sodium nitrite in dilute HCl

to form a highly reactive diazonium derivative, which is able to couple with active hydrogen-

containing compounds. A diazonium reacts rapidly with histidine or tyrosine residues within

proteins, forming covalent diazo bonds (Wilchek et al., 1986). It also can react with purine

residues within DNA at position 8 of the bases (Rothenberg and Wilchek, 1988; Lowe, 1979)

(Figure 27.11 ).

2. Chemical Modiﬁ cation of Nucleic Acids and Oligonucleotides 989

Figure 27.11 This diazo derivative of biocytin may be used to modify guanine bases at the C-8 position.