Download Bioconjugation Protocols - Strategies and Methods [Methods in Molec Bio 283] - C. Niemeyer (Humana, 2004) WW PDF

TitleBioconjugation Protocols - Strategies and Methods [Methods in Molec Bio 283] - C. Niemeyer (Humana, 2004) WW
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Page 1

Edited by

Christof M. Niemeyer


Strategies and Methods

Volume 283



Strategies and Methods

Edited by

Christof M. Niemeyer

Page 2

Bioconjugation Protocols

Page 171



158 Pljevaljcic et al.

serves to release the product duplex 10·12 from the complex and the product
duplex 10·12 elutes after 19.6 min (Fig. 3B, trace c). A coinjection of starting
duplex 10·11 and product duplex 10·12 is also shown in Fig. 3B (trace d). This
anion exchange HPLC method can also be used to isolate the labeled duplex and
further modification of duplex 10·12 with amine-reactive probes should be
possible (see Note 9).

3.3.2. Labeling With Biotin

Although the product duplex 10·12 with a primary amino group could be
further modified with succinimidyl biotin to yield biotinylated DNA, it
appears more convenient to first attach the biotin group to the cofactor and
then use the aziridine cofactor 9 for labeling. The labeling reaction with cofac-
tor 9 (Fig. 4A) is performed as described for the labeling reaction with cofac-
tor 8 (see Subheading 3.3.1.).

1. Prepare a solution of aziridine cofactor 9 (80 µM, predissolved in DMSO), duplex
10·11 (10 µM), and M·TaqI (11 µM) in Tris acetate buffer (20 mM, pH 6.0) con-
taining magnesium acetate (10 mM), potassium acetate (50 mM), and Triton X-
100 (0.01%) and incubate at 37°C for 22 h. The progress of the reaction can be
monitored by anion exchange HPLC (Fig. 4B). At the end of the reaction the
starting duplex 10·11 has disappeared and a fast eluting complex between M·TaqI
and the product duplex 10·13 is formed.

2. Release the product duplex 10·13 from the complex by treatment with Proteinase K
as described above (see Subheading 3.3.1.). The free product duplex 10·13 elutes
about 1 min earlier during anion exchange HPLC than the starting duplex 10·11.

3.4. Labeling of Plasmid DNA With Biotin

Labeling of plasmid DNA is illustrated with linearized pUC19 DNA (see
Note 10).

1. Linearize pUC19 DNA (0.25 µg/µL) by treatment with R·EcoRI (10 U per µg of
plasmid DNA) in Tris hydrochloride buffer (50 mM, pH 7.5) containing magne-
sium chloride (10 mM), sodium chloride (100 mM), Triton X-100 (0.02%), and
bovine serum albumin (0.1 mg/mL; the recommended buffer supplied by the
manufacturer) and incubate at 37°C for 1 h.

2. Prepare a solution of R·EcoRI-linerized pUC19 (0.025 µg/µL, 14.1 nM, three
recognition sequences for M·TaqI), M·TaqI (46.5 nM), and aziridine cofactor 9
(80 µM, predissolved in DMSO) in Tris acetate buffer (20 mM, pH 6.0) contain-
ing magnesium acetate (10 mM), potassium acetate (50 mM), and Triton X-100
(0.01%) and incubate at 60°C for 3 h. The progress of the labeling reaction can be
monitored in a DNA protection assay (Fig. 5).

3. Remove aliquots (8 µL, 0.2 µg linearized pUC19) after different incubation times,
add restriction endonuclease R·TaqI (1 µL, 5 U) and 10X reaction buffer (1 µL)
supplied by the manufacturer to each aliquot and incubate the mixtures at 65°C

Page 172

DNA Labeling 159

for 1 h. Afterward, add a solution (2 µL) of glycerol (30%) containing bromophe-
nol blue (0.25%) to each aliquot and analyze the samples by standard agarose gel

4. Remove M·TaqI from the plasmid DNA by adjusting the pH of the solution to 8.0,
add Proteinase K (10 µg per µg DNA) and incubate the solution at 65°C for 1 h.

5. Purify the labeled plasmid DNA with the QiagenPCR purification Kit according
to the instructions given by the manufacturer.

4. Notes
1. Caution: Aziridine is hazardous and should be handled with care in a fume hood.
2. The mesylated nucleosides 5 and 6 have a strong tendency to form cyclo-

nucleosides by nucleophilic attack of the nitrogen at the 3-position of the adenine
ring on the activated 5'-carbon. Thus, it is best to use nucleosides 5 and 6 as
quickly as possible in the next step.

Fig. 5. M·TaqI-catalyzed labeling of plasmid DNA with biotin. The progress of the
labeling reaction with the aziridine cofactor 9 is analyzed in a DNA protection assay.
At the beginning of the labeling reaction (t = 0 h) fragmentation of R·EcoRI-linearized
pUC19 DNA (L-pUC19) with the restriction endonuclease R·TaqI (one of the four
recognition sequences of R·TaqI in pUC19 overlaps with the R·EcoRI recognition
sequence) leads to three major bands (1444, 734, and 476 bp) on an agarose gel (an
additional 32-bp fragment is too small to be observed on the gel). With increasing
reaction times (t = 0.5 and 1 h), these bands disappear and bands corresponding to
longer intermediates or the full-length linearized plasmid (2686 bp) appear. After 3 h
the DNA is almost completely protected against fragmentation by R·TaqI, indicating
that the three recognition sequences of R·TaqI are blocked by covalent modification.
No DNA protection against cleavage by R·TaqI is observed in the absence of either
M·TaqI or the aziridine cofactor 9 (not shown).

Page 342

Index 329

deoxyadenosine, 153, 160

adenosine, 149, 151

deoxyadenosine, 152,
153, 160

adenosine, 151

mesyladenosine adenosine,
151, 152, 159

5'-deoxyadenosine, 153, 154

aziridine, 148, 149, 159

adenosine, 149
biotin labeling of plasmid DNA,

158, 159
materials, 147, 148
principles, 145–147
recombinant Thermus aquaticus

DNA methyltransferase
expression and purification,
154, 155, 160

short duplex

biotin labeling, 158
methyltransferase recognition

sequence, 155, 160
primary amino group labeling,

155, 157, 158, 160
Single-walled carbon nanotubes, see

Atomic force microscopy
Smart polymer–streptavidin

molecular gate concept, 37, 38
preparation of temperature-

sensitive molecular gate,
conjugation conditions, 41
immobilization of conjugate,

41, 42
materials, 38
overview, 38, 39
polymer synthesis, 39, 40, 42
purification of conjugate, 41, 42
streptavidin site-directed

mutagenesis, 40, 41
SMILing, see Sequence-specific


Streptavidin-biotin system,
affinity, 4, 181
antibody–enzyme conjugates, see


atomic force microscopy probe
tip functionalization, see
Atomic force microscopy

Carathor’s equation, 5, 12
DNA sequence-specific

labeling, see Sequence-
specific methyltransferase-
induced labeling

DNA–streptavidin coupling, see

Page 343

330 Index

scheme of protein conjugation, 4, 5
stability of streptavidin, 181, 182
transglutaminase biotinylation of

antibodies, see

Streptavidin–smart polymer
conjugates, see Smart

Subtilisin-catalyzed glycopeptide

amino acid immobilization onto
Rink resin through PAM
linkers, 271, 272, 277

condensation reaction, 277, 278
glycopeptide amide synthesis for

ligation, 274, 276, 277
materials, 269, 270
principles, 267–269
solid-phase peptide synthesis and

cleavage conditions, 272–274


hydrazide (TPCH),
synthesis, 92–94

mercaptoproprionic acid
hydrazide (TPMPH),
synthesis, 94

Tissue plasminogen activator–
antibody conjugates,

antithrombotic therapy, 22
biodistribution of radiolabeled

conjugates after intravenous
administration in rats,

injection route effects, 31
pulmonary targeting, 31
targeting specificity, 31, 34
technique, 25, 26

targeted drug delivery, 21

TNBS, see Trinitrobenzenesulfonic

TPCH, see S-(2-Thiopyridyl)-L-
cysteine hydrazide

TPMPH, see S-(2-Thiopyridyl)-3-
mercaptoproprionic acid

biotechnology applications,

110, 111
biotinylation of antibodies,

biotinylation reaction, 112,
113, 120

enzyme-linked immunosorbent
control of biotinylation,

113, 114
kinetic analysis, 114, 120, 121

kinetics, 114
materials, 111, 112

catalytic reaction, 109, 110
functions, 110
hapten–protein conjugate

synthesis for enzyme-linked
immunosorbent assay,

aminofunctionalization of
herbicide, 116

enzyme-linked immunosorbent
assay, 119, 120

herbicide–casein conjugate
batch procedure, 117, 118
in situ procedures, 118,

121, 122
polyethylene glycol–protein

conjugate preparation, 58, 59
Trinitrobenzenesulfonic acid

(TNBS), polyethylene
glycol–protein conjugate
characterization, 48, 56

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