185
3 Methods
To facilitate hybridization-induced assembly of the enzyme- inhibitor
pair the oligonucleotide handles have to be conjugated to the individual protein domains. In particular, cysteine is an attractive target
for protein conjugation due to its low prevalence in natural proteins
and ease of insertion using site-directed mutagenesis. In contrast
to non-natural amino acids, which allow fully orthogonal protein
conjugation chemistries, the incorporation of a nonnative cysteine
typically does not result in a significant decrease in expression yield
(~0.3 mg/L culture for both TEM1-β-lactamase and BLIP).
To allow straightforward protein expression while ensuring sitespecific conjugation of the oligonucleotide handles to the proteins
(yielding homogeneous ODN-protein conjugates), thiol-maleimide
coupling was used targeting a genetically inserted cysteine on the
proteins. To install the maleimide moiety on the oligonucleotide
handles, the heterobifunctional crosslinker Sulfo- SMCC, consisting of an NHS-ester, cyclohexyl spacer, and maleimide, was coupled to a 5′- or 3′-amine-modified oligonucleotide for BLIP and
TEM1-β-lactamase, respectively. Subsequent ethanol precipitation
of the maleimide-functionalized oligonucleotides allowed the
removal of non-reacted Sulfo-SMCC. Finally, the oligonucleotides
were conjugated to the cysteine inserted in the proteins (Fig. 2).
Fig. 2 (a) Crystal structure of a complex between TEM1-β-lactamase and BLIP (PDB: 3C7V). (b) ODN-protein
conjugation strategy. Amine-modified oligonucleotides are reacted with the NHS-ester of the heterobifunctional crosslinker Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carbocylate (Sulfo-SMCC) installing a thiol-reactive maleimide moiety on the oligonucleotides. After the removal of the excess crosslinker by
ethanol precipitation, the maleimide-activated oligonucleotides are reacted with a cysteine in the proteins.
Adapted with permission from ref. 13. Copyright 2015 American Chemical Society
DNA Sensors Based on β-Lactamase
3 Methods
To facilitate hybridization-induced assembly of the enzyme- inhibitor
pair the oligonucleotide handles have to be conjugated to the individual protein domains. In particular, cysteine is an attractive target
for protein conjugation due to its low prevalence in natural proteins
and ease of insertion using site-directed mutagenesis. In contrast
to non-natural amino acids, which allow fully orthogonal protein
conjugation chemistries, the incorporation of a nonnative cysteine
typically does not result in a significant decrease in expression yield
(~0.3 mg/L culture for both TEM1-β-lactamase and BLIP).
To allow straightforward protein expression while ensuring sitespecific conjugation of the oligonucleotide handles to the proteins
(yielding homogeneous ODN-protein conjugates), thiol-maleimide
coupling was used targeting a genetically inserted cysteine on the
proteins. To install the maleimide moiety on the oligonucleotide
handles, the heterobifunctional crosslinker Sulfo- SMCC, consisting of an NHS-ester, cyclohexyl spacer, and maleimide, was coupled to a 5′- or 3′-amine-modified oligonucleotide for BLIP and
TEM1-β-lactamase, respectively. Subsequent ethanol precipitation
of the maleimide-functionalized oligonucleotides allowed the
removal of non-reacted Sulfo-SMCC. Finally, the oligonucleotides
were conjugated to the cysteine inserted in the proteins (Fig. 2).
Fig. 2 (a) Crystal structure of a complex between TEM1-β-lactamase and BLIP (PDB: 3C7V). (b) ODN-protein
conjugation strategy. Amine-modified oligonucleotides are reacted with the NHS-ester of the heterobifunctional crosslinker Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carbocylate (Sulfo-SMCC) installing a thiol-reactive maleimide moiety on the oligonucleotides. After the removal of the excess crosslinker by
ethanol precipitation, the maleimide-activated oligonucleotides are reacted with a cysteine in the proteins.
Adapted with permission from ref. 13. Copyright 2015 American Chemical Society
DNA Sensors Based on β-Lactamase