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nonreactive toward maleimides. TCEP can also reduce native,
intramolecular disulfide-bonds. However, the BLIP and
TEM1- β- lactamase proteins did not show any (irreversible)
reduction of intramolecular disulfides.
5. To ensure efficient conjugation of maleimide-functionalized
oligonucleotides to the proteins of interest, it is crucial to perform at least three rounds of ethanol precipitation after reacting the amine-modified oligonucleotides with Sulfo-SMCC to
ensure that all excess Sulfo-SMCC is removed since this will
compete for conjugation to the protein. A single round of ethanol precipitation has shown to significantly reduce final conjugation efficiencies.
6. To avoid the formation of nonreactive disulfide-linked protein
dimers directly proceed with mixing the desalted proteins (i.e.,
after removing TCEP) with the maleimide-functionalized oligonucleotides. Various reports have published mixed results
with TCEP interfering with maleimide conjugation. In our
experience, TCEP significantly lowers conversion yields and is
therefore removed prior to the reaction with the maleimidefunctionalized oligonucleotide.
Fig. 6 Enzyme activity as a function of target oligonucleotide concentration.
ODN1-TEM1-β-lactamaseE104D, template oligonucleotide (with a target recognition sequence of 40 nucleotides), and ODN2-BLIP were incubated at low
micromolar concentrations to form the inhibited ternary complex. Subsequently,
the complex was diluted to 100 pM of ODN1-TEM1-β-lactamaseE104D in the
presence of various concentrations of target oligonucleotide. After the addition of
the fluorescent substrate CCF2-FA product formation was measured by measuring the increase in fluorescence in time (λ ex = 409 nm, λ em = 447 nm). A linear
increase in enzyme activity as a function of target concentration is observed up
to 1 equivalent of the template oligonucleotide, suggesting tight binding between
the ternary complex and target oligonucleotide. Adapted with permission from
ref. 13. Copyright 2015 American Chemical Society
DNA Sensors Based on β-Lactamase
nonreactive toward maleimides. TCEP can also reduce native,
intramolecular disulfide-bonds. However, the BLIP and
TEM1- β- lactamase proteins did not show any (irreversible)
reduction of intramolecular disulfides.
5. To ensure efficient conjugation of maleimide-functionalized
oligonucleotides to the proteins of interest, it is crucial to perform at least three rounds of ethanol precipitation after reacting the amine-modified oligonucleotides with Sulfo-SMCC to
ensure that all excess Sulfo-SMCC is removed since this will
compete for conjugation to the protein. A single round of ethanol precipitation has shown to significantly reduce final conjugation efficiencies.
6. To avoid the formation of nonreactive disulfide-linked protein
dimers directly proceed with mixing the desalted proteins (i.e.,
after removing TCEP) with the maleimide-functionalized oligonucleotides. Various reports have published mixed results
with TCEP interfering with maleimide conjugation. In our
experience, TCEP significantly lowers conversion yields and is
therefore removed prior to the reaction with the maleimidefunctionalized oligonucleotide.
Fig. 6 Enzyme activity as a function of target oligonucleotide concentration.
ODN1-TEM1-β-lactamaseE104D, template oligonucleotide (with a target recognition sequence of 40 nucleotides), and ODN2-BLIP were incubated at low
micromolar concentrations to form the inhibited ternary complex. Subsequently,
the complex was diluted to 100 pM of ODN1-TEM1-β-lactamaseE104D in the
presence of various concentrations of target oligonucleotide. After the addition of
the fluorescent substrate CCF2-FA product formation was measured by measuring the increase in fluorescence in time (λ ex = 409 nm, λ em = 447 nm). A linear
increase in enzyme activity as a function of target concentration is observed up
to 1 equivalent of the template oligonucleotide, suggesting tight binding between
the ternary complex and target oligonucleotide. Adapted with permission from
ref. 13. Copyright 2015 American Chemical Society
DNA Sensors Based on β-Lactamase