188
1. Dissolve the amine-modified oligonucleotides in PBS to a
concentration of ~1 mM.
2. Dissolve 2 mg of Sulfo-SMCC (1 vial) in 300 μL DMSO to
yield a stock concentration of ~20 mM.
3. Mix an equal volume of Sulfo-SMCC with the amine-modified
oligonucleotides yielding a 20-fold molar excess of SulfoSMCC in 1:1 PBS:DMSO.
4. Incubate the mixture at room temperature under continuous
shaking at 850 rpm for 2 h.
5. To precipitate the oligonucleotides, add 10% (v/v) 5 M NaCl
and 300% (v/v) ethanol precooled to −30 °C, and store the
mixture at −30 °C for at least 1 h.
6. Pellet the precipitated oligonucleotides by centrifugation at
19,000 × g rpm for 15 min at 4 °C.
7. Dissolve the oligonucleotides in PBS to a concentration of
~1 mM and repeat steps 5 and 6 of the ethanol precipitation
procedure twice (see Note 5).
8. Finally, dry the pelleted oligonucleotides to air overnight at
room temperature.
1. Immediately prior to oligonucleotide conjugation, desalt
the proteins to ligation buffer to remove TCEP by gel filtration using a PD-10 desalting column and dilute to ~50 μM
(see Note 6).
2. Add the appropriate volume of protein solution to the dried
oligonucleotide to yield a threefold molar excess of maleimidefunctionalized oligonucleotide.
3. Incubate the mixture at room temperature under continuous
shaking at 850 rpm for 2 h.
4. To remove excess oligonucleotides load the reaction mixture
on a 1 mL His-bind column charged with Ni
2+
ions and equilibrated with bind buffer. Wash with 10 column volumes of
bind buffer.
5. Elute the oligonucleotide-protein (ODN-protein) conjugates
in 500 μL fractions using 10 column volumes of elution
buffer.
6. Analyze the reaction efficiency and purity of the elution fractions
by 12% SDS-PAGE.
7. Combine all elution fractions that contain ODN-protein
conjugates.
8. Exchange the buffer to buffer A by gel filtration using a PD-10
desalting column.
3.2 Maleimide
Functionalization
of Amine- Modified
Oligonucleotides
3.3 Conjugation
of MaleimideFunctionalized
Oligonucleotides
to Proteins
Wouter Engelen and Maarten Merkx
1. Dissolve the amine-modified oligonucleotides in PBS to a
concentration of ~1 mM.
2. Dissolve 2 mg of Sulfo-SMCC (1 vial) in 300 μL DMSO to
yield a stock concentration of ~20 mM.
3. Mix an equal volume of Sulfo-SMCC with the amine-modified
oligonucleotides yielding a 20-fold molar excess of SulfoSMCC in 1:1 PBS:DMSO.
4. Incubate the mixture at room temperature under continuous
shaking at 850 rpm for 2 h.
5. To precipitate the oligonucleotides, add 10% (v/v) 5 M NaCl
and 300% (v/v) ethanol precooled to −30 °C, and store the
mixture at −30 °C for at least 1 h.
6. Pellet the precipitated oligonucleotides by centrifugation at
19,000 × g rpm for 15 min at 4 °C.
7. Dissolve the oligonucleotides in PBS to a concentration of
~1 mM and repeat steps 5 and 6 of the ethanol precipitation
procedure twice (see Note 5).
8. Finally, dry the pelleted oligonucleotides to air overnight at
room temperature.
1. Immediately prior to oligonucleotide conjugation, desalt
the proteins to ligation buffer to remove TCEP by gel filtration using a PD-10 desalting column and dilute to ~50 μM
(see Note 6).
2. Add the appropriate volume of protein solution to the dried
oligonucleotide to yield a threefold molar excess of maleimidefunctionalized oligonucleotide.
3. Incubate the mixture at room temperature under continuous
shaking at 850 rpm for 2 h.
4. To remove excess oligonucleotides load the reaction mixture
on a 1 mL His-bind column charged with Ni
2+
ions and equilibrated with bind buffer. Wash with 10 column volumes of
bind buffer.
5. Elute the oligonucleotide-protein (ODN-protein) conjugates
in 500 μL fractions using 10 column volumes of elution
buffer.
6. Analyze the reaction efficiency and purity of the elution fractions
by 12% SDS-PAGE.
7. Combine all elution fractions that contain ODN-protein
conjugates.
8. Exchange the buffer to buffer A by gel filtration using a PD-10
desalting column.
3.2 Maleimide
Functionalization
of Amine- Modified
Oligonucleotides
3.3 Conjugation
of MaleimideFunctionalized
Oligonucleotides
to Proteins
Wouter Engelen and Maarten Merkx