Master piece of the switchSENSE technology, the probe is
made by a single-stranded DNA bearing a fluorescent dye at one
extremity and attached on its opposite end to a gold-quenching
surface through a sulfur bond. This single-stranded DNA is
annealed to a complementary strand (RNA or DNA) that could
be used alone or conjugated coupled to a protein. Hybridization of
the complementary strand generates a rigid negatively charged
electro-switchable biosensor, also referred as nanolever (Fig. 1).
Making use of the DNA plasticity and of biochemical tools, a
wide variety of nanolevers could be adapted to the biological context of the experiment: size and type of DNA may vary, even
allowing the use of complex DNA origami constructs [12], RNA
motifs can be easily adapted [13], various types of modified extremity can be used (protein, cofactor, NTA, biotin, etc.) [2], as well as
various dyes (Cy3, Cy5, etc.).
Here we describe a specific setup adapted from Langer and
colleagues [14] for polymerase activity characterization, using an
overhang DNA as nanolever. With this kind of substrate, we show
that it is possible to follow the entire biological reaction including
DNA polymerase association, polymerization, and dissociation rate
in one set of experiment.
2 Materials
2.1 Instrument
and Accessories
1. DRX 2400 instrument (Dynamic Biosensors).
2. Nanodrop ND-1000 spectrophotometer (Thermo Fisher) or
equivalent.
3. switchCONTROL, switchBUILD, and switchANALYSER
software (Dynamic Biosensors).
4. Biochip for enzymatic studies (ENZ-80-1-Y1-S, Dynamic Biosensors) bearing an 80-mer DNA probe attached at the 3
0
extremity.
5. Passivation and regeneration solutions (Dynamic Biosensors,
SOL-PAS-1-5 and SOL-REG-12-1).
6. The P36T80 oligonucleotide, a 36-mer DNA complementary
to 3
0 end region of the 80-mer DNA probe attached to the
biochip (see Note 1).
7. Heparin sodium salt, physico-chemical grade (Sigma-Aldrich).
8. 10Â Auxilary buffer (100 mM sodium phosphate, pH 7.0,
400 mM NaCl, 0.5% Tween 20, 0.5 mM EDTA).
9. Deoxynucleotide, 100 mM solutions (dATP, dGTP, dCGT,
and dTTP).
10. Ultrapure RNase-free water.
146
Guillaume Bec and Eric Ennifar
made by a single-stranded DNA bearing a fluorescent dye at one
extremity and attached on its opposite end to a gold-quenching
surface through a sulfur bond. This single-stranded DNA is
annealed to a complementary strand (RNA or DNA) that could
be used alone or conjugated coupled to a protein. Hybridization of
the complementary strand generates a rigid negatively charged
electro-switchable biosensor, also referred as nanolever (Fig. 1).
Making use of the DNA plasticity and of biochemical tools, a
wide variety of nanolevers could be adapted to the biological context of the experiment: size and type of DNA may vary, even
allowing the use of complex DNA origami constructs [12], RNA
motifs can be easily adapted [13], various types of modified extremity can be used (protein, cofactor, NTA, biotin, etc.) [2], as well as
various dyes (Cy3, Cy5, etc.).
Here we describe a specific setup adapted from Langer and
colleagues [14] for polymerase activity characterization, using an
overhang DNA as nanolever. With this kind of substrate, we show
that it is possible to follow the entire biological reaction including
DNA polymerase association, polymerization, and dissociation rate
in one set of experiment.
2 Materials
2.1 Instrument
and Accessories
1. DRX 2400 instrument (Dynamic Biosensors).
2. Nanodrop ND-1000 spectrophotometer (Thermo Fisher) or
equivalent.
3. switchCONTROL, switchBUILD, and switchANALYSER
software (Dynamic Biosensors).
4. Biochip for enzymatic studies (ENZ-80-1-Y1-S, Dynamic Biosensors) bearing an 80-mer DNA probe attached at the 3
0
extremity.
5. Passivation and regeneration solutions (Dynamic Biosensors,
SOL-PAS-1-5 and SOL-REG-12-1).
6. The P36T80 oligonucleotide, a 36-mer DNA complementary
to 3
0 end region of the 80-mer DNA probe attached to the
biochip (see Note 1).
7. Heparin sodium salt, physico-chemical grade (Sigma-Aldrich).
8. 10Â Auxilary buffer (100 mM sodium phosphate, pH 7.0,
400 mM NaCl, 0.5% Tween 20, 0.5 mM EDTA).
9. Deoxynucleotide, 100 mM solutions (dATP, dGTP, dCGT,
and dTTP).
10. Ultrapure RNase-free water.
146
Guillaume Bec and Eric Ennifar