B15: Real-time monitoring of DNA hybridization, cleavage
and repair processes by FRET on a EWOD fluidic
microprocessor
Didier Gasparutto1 and Christine Peponnet2
1Lésions
Acides Nucléiques, Service Chimie Inorganique Biologique,
DRFMC, CEA Grenoble
and 2Département des Technologies pour la Biologie et la Santé, CEA-LETI Grenoble
The combination of a microsystem dedicated to biological and chemical protocols miniaturization
based on EWOD (electrowetting on dielectric) fluidic actuation and an original biological assay
aim at monitoring DNA hybridization, DNA cleavage or DNA lesions repair processes has been
recently developed. The microsystem has a microprocessor architecture having several reservoirs
with external entries for reagents feeding, reagents storage, incubation of a reactional mixing and
a bin for droplets draining. A bus is used for moving reactions from one zone to the next (heating,
detection…) as well as for mixing. A photograph of this EWOD based microchip is given in Figure
1. The elementary fluidic operations essential for any protocol have been validated on this chip:
dispensing of volume controlled droplets, coalescence and mixing of two droplets, incubation at
controlled temperature with droplets agitation and detection of fluorescence in real time. The
volume of the droplets that can be dispensed on this chip range from 28 to 64 nl.
Figure 1: Photograph of the EWOD chip
The detection and quantification of DNA hybridization, chemical or enzymatic DNA cleavage
reactions, enzymatic DNA repair activities is based on a FRET process using fluorescent DNA
molecular beacons. The relatively close proximity of the fluorescent donor and acceptor groups
within the short oligonucleotide duplex facilitates efficient energy transfer from the donor to
acceptor, thereby reducing the fluorescent emission intensity of the donor moiety. The latter assay
has been applied to monitor DNA repair activities by DNA N-glycosylases. Thus, cleavage of the
duplex molecule that contain a lesion, induced by the specific activity of repair enzymes acting by
an excision process, leads to the separation of the donor and acceptor dyes, allowing the
recovery of the donor emission intensity to its intrinsic unquenched value (Figure 2). Thus, the
latter DNA cleavage reaction induces a fluorescent signal which is proportional to the enzymatic
excision repair activity.
Figure 2: Principle of the FRET-based DNA repair assay using
lesion (L) containing oligonucleotide probes (D=Donnor, A=Acceptor)
We have shown that these chips and the associated fluorescent detection system are perfectly
adapted to real time monitoring of several chemical and enzymatic (DNA N-glycosylases,
restriction enzymes, nucleases…) cleavage activities. An example of moving droplets during
incubation protocol is shown in Figure 3B and the corresponding fluorescence curves are
presented in Figure 3A. Finally, EWOD based actuated system is very versatile and is particularly
suited to parallel enzymatic activities quantification for drug discovery and medical diagnostic
applications.
A)
B)
Figure 3: Real-time monitoring of DNA N-glycosylase mediated DNA repair
activities (enzyme: Fpg protein; lesion: 8oxoGuanine) by fluorescence measurement on the chip.
The principles and fields of application of the FRET process in biology together with the EWOD
technology will be briefly reviewed in the introduction of the practical course. During this practical
work the whole integrated microsystem will be presented. Then, the participants will make few
DNA hybridization, DNA digestion and DNA enzymatic repair assays based on homogenous realtime FRET detection, in single tubes, in a microplate format, on a quantitative-PCR instrument
(used as reference systems) and finally on a EWOD based lab-on-chip.
References:
1- Nucleic acid biosensors for real-time monitoring of DNA repair activities by FRET.
Chollat-Namy A., Gasparutto D., Cadet J., Favier A. Chemistry of Nucleic Acid Components, 2005, 7,
397-399
2- Fouillet Y., Jary D., Brachet A. G., Boutet J., Chabrol C., Clementz P., Lauro R., Charles R., Peponnet
C. Proceeding MicroTas, 2005.
ESONN 2006, Practical Course