Essential to human and animal health, pharmaceutical substances consumed in large quantities are omnipresent in aquatic environments where their biological activity can have harmful effects on ecosystems. Their detection by sensitive and rapid methods is a major issue. A team from IPBS-Toulouse (CNRS / Université Toulouse III-Paul Sabatier) has shown that its microarray for high-throughput analysis of individual DNA molecules effectively detects the presence of DNA intercalating agents. This work, published in the journal Analytical Chemistry, finds applications in the monitoring of water quality as well as the screening of active molecules for the treatment of cancer.
Medicines could soon enter the list of priority substances for aquatic environments with imposed quality standards. To support this evolution and allow regular monitoring of the quality of surface water in the future, sensitive, rapid, low-cost, transportable and even on-site tools must be developed. Biosensors combining nanotechnologies, for their capacity for miniaturization, with DNA, as a material of choice for biosensing, are promising solutions.
A large number of prescribed drugs exert their activity through their interaction with DNA by intercalation between its base pairs which blocks its transcription or replication. They are thus particularly relevant targets because of the genotoxic and/or cytotoxic risk they can present. The intercalation of a compound in DNA is accompanied by structural changes in the molecule such as an increase in the contour length of the DNA and possibly a change in its rigidity. The biochip developed and used by researchers in Dr. Salomé's team was designed to access such structural modifications in basic research. It consists of nanoparticles attached to the free end of DNA molecules immobilized, by their other end, on anchoring sites isolated and organized in a regular array on a support. Using videomicroscopy and image analysis, the amplitudes of motion of several hundreds of these nanoparticles can be measured in parallel over time. Combined with this Tethered Particle Motion technique, our biochip allows the conformational dynamics of single DNA molecules to be monitored at high speed and the detection of structural changes in DNA with high resolution. In parallel with studies of DNA biophysics, the researchers are exploring the potential of the method as a biosensor. They first demonstrated the feasibility of detecting DNA intercalators by this method by observing a dose-dependent response for fluorescent intercalators whose mechanical effects are established in the literature. Then they demonstrated that the method can be applied to the measurement of the concentration in natural water of doxorubicin, a therapeutic molecule used in the treatment of cancer, over a wide range of concentrations. Thus, the method allows the detection of intercalating agents provided that their interaction with DNA does not notably reduce its rigidity. The processes necessary for its implementation being of low-tech type, environmentally friendly, cost effective and miniaturizable make this method a promising tool for environmental analyzes. Furthermore, this method also finds applications in the field of screening for active molecules interacting with DNA.
Work continues towards the development of solutions for specific detection by integrating recognition molecules into our biochip for environmental but also diagnostic applications.
Scheme of the single DNA molecule biochip: a glass slide wears an array of submicrometric sized sites of anchor protein for immobilization by one end of DNA molecules linked to a nanoparticle by the other end.
Principle of the detection of an intercalator: the increase in effective length of the DNA molecule leads to an increase in the amplitude of motion of the nanoparticle DeltaA which is measured by videomicroscopy and image analysis.
Calibration curve: The signal, i.e. the variation in the amplitude of motion, increases with the concentration of intercalating agent over several orders of magnitude.
© Laurence Salomé
Single-molecule sensing of DNA intercalating drugs in water
Serres Sandra, Tardin Catherine and Salomé Laurence. Analytical Chemistry. 2020 May 31. doi: 10.1021/acs.analchem.0c00184
- Patent « Biopuces pour l’analyse de la dynamique de molécules d’acide nucléique » Plénat T., Salomé L., Tardin C., Thibault C., Trévisiol E., Vieu C. FR 1057031 déposé le 3/9/2010 ; EP 2 611 940 B1 déposé le 1/9/2011
- Plénat T.*, Tardin C.*, Rousseau P. and Salomé L. (2012) High-throughput single-molecule analysis of DNA-protein interactions by Tethered Particle Motion Nucleic Acids Res 40(12) e89
- Guilbaud S., Salomé L., Destainville N., Manghi M., Tardin C (2019) Dependence of DNA persistence length on ionic strength and ion type. Phys Rev Lett 122, 028102
Laurence Salomé | Laurence.Salome@ipbs.fr | 05 61 17 59 39
IPBS-Toulouse, CNRS, Université Paul Sabatier
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Françoise Viala | email@example.com | 06 01 26 52 59