186
Conversion yields (typically 50–90%) can be estimated by SDSPAGE analysis. Subsequent Ni
2+
-affinity chromatography and
anion-exchange chromatography to remove excess oligonucleotides and unconjugated proteins, respectively, resulted in pure
ODN-protein conjugates.
Formation of the intermolecular ternary complex is performed
by mixing ODN1-TEM1-β-lactamase, ssDNA template, and
ODN2-BLIP in a 1:1.2:2 molar ratio. Using a slight excess of
ssDNA template and ODN2-BLIP ensures that no free ODN1TEM1- β-lactamase
or
ODN1-TEM1-β-lactamase-template
remains, both of which would contribute to background activity.
In order to confirm the formation of homogeneous ternary complexes upon hybridization of the individual components, the mixtures were analyzed by semi-native PAGE. Quantitative conversion
to the ternary complex was observed upon the addition of the
ssDNA template to a mixture of the ODN-enzyme and ODNinhibitor conjugates. A gel-shift to higher molecular weight after
the addition of a complementary input oligonucleotide indicates
the formation of the desired quaternary complex. Whereas seminative PAGE suggests successful formation of the ternary DNAdirected enzyme-inhibitor complex, enzyme-activity assays were
performed to test whether the enzyme-inhibitor interaction is
indeed induced upon hybridization of both ODN-protein conjugates to the ssDNA template strand. To this end, the complexes
(containing templates with target recognition sequences of 0 to 50
nucleotides) were diluted to a final concentration of 1 nM. After
the addition of the colorimetric substrate nitrocefin, substrate conversion was monitored by measuring the optical absorbance of the
formed product as a function of time. Whereas a strong inhibition
of enzyme activity was observed with all target recognition
sequence lengths, inhibition efficiencies increased as the target recognition sequence length increases. This observation suggests that
a certain degree of flexibility between the protein partners is preferable to allow more efficient intramolecular complex formation.
In contrast, full recovery of enzyme activity was only achieved
upon the addition of input sequences of >40 nucleotides, suggesting that a double-helix of at least 40 nucleotides is required to
overcome flexibility in the protein termini and SMCC-linker and
spatially separate the interacting protein partners.
Making use of an enzyme as output domain provides the
advantage over conventional fluorescent domains that substrate
molecules are catalytically converted, hence the output signal accumulates. This allows the use of much lower concentrations of the
sensor complex, hereby increasing the sensitivity of the input oligonucleotide detection. When using 100 pM of the DNA-specific
biosensor together with the more sensitive fluorescent substrate
CCF2-FA, as little as 10 pM of input oligonucleotide results in a
distinguishable increase in enzyme activity.
Wouter Engelen and Maarten Merkx
Conversion yields (typically 50–90%) can be estimated by SDSPAGE analysis. Subsequent Ni
2+
-affinity chromatography and
anion-exchange chromatography to remove excess oligonucleotides and unconjugated proteins, respectively, resulted in pure
ODN-protein conjugates.
Formation of the intermolecular ternary complex is performed
by mixing ODN1-TEM1-β-lactamase, ssDNA template, and
ODN2-BLIP in a 1:1.2:2 molar ratio. Using a slight excess of
ssDNA template and ODN2-BLIP ensures that no free ODN1TEM1- β-lactamase
or
ODN1-TEM1-β-lactamase-template
remains, both of which would contribute to background activity.
In order to confirm the formation of homogeneous ternary complexes upon hybridization of the individual components, the mixtures were analyzed by semi-native PAGE. Quantitative conversion
to the ternary complex was observed upon the addition of the
ssDNA template to a mixture of the ODN-enzyme and ODNinhibitor conjugates. A gel-shift to higher molecular weight after
the addition of a complementary input oligonucleotide indicates
the formation of the desired quaternary complex. Whereas seminative PAGE suggests successful formation of the ternary DNAdirected enzyme-inhibitor complex, enzyme-activity assays were
performed to test whether the enzyme-inhibitor interaction is
indeed induced upon hybridization of both ODN-protein conjugates to the ssDNA template strand. To this end, the complexes
(containing templates with target recognition sequences of 0 to 50
nucleotides) were diluted to a final concentration of 1 nM. After
the addition of the colorimetric substrate nitrocefin, substrate conversion was monitored by measuring the optical absorbance of the
formed product as a function of time. Whereas a strong inhibition
of enzyme activity was observed with all target recognition
sequence lengths, inhibition efficiencies increased as the target recognition sequence length increases. This observation suggests that
a certain degree of flexibility between the protein partners is preferable to allow more efficient intramolecular complex formation.
In contrast, full recovery of enzyme activity was only achieved
upon the addition of input sequences of >40 nucleotides, suggesting that a double-helix of at least 40 nucleotides is required to
overcome flexibility in the protein termini and SMCC-linker and
spatially separate the interacting protein partners.
Making use of an enzyme as output domain provides the
advantage over conventional fluorescent domains that substrate
molecules are catalytically converted, hence the output signal accumulates. This allows the use of much lower concentrations of the
sensor complex, hereby increasing the sensitivity of the input oligonucleotide detection. When using 100 pM of the DNA-specific
biosensor together with the more sensitive fluorescent substrate
CCF2-FA, as little as 10 pM of input oligonucleotide results in a
distinguishable increase in enzyme activity.
Wouter Engelen and Maarten Merkx