Birgitta R. Knudsen

RESEARCH

Topoisomerase I activity: Biological role and technological applications

DNA topoisomerase enzymes are the cellular targets of one of the most widely used classes of anti-cancer chemotherapy, the camptothecins. Theses DNA modifying enzymes are ubiquitously expressed in all organisms and maintain essential functions in vital cellular processes such as transcription, replication and recombination. This makes the DNA topoisomerases ideal drug targets, and their structure and function have attracted intense investigations.

The catalytic mechanism of topoisomerase I includes several unique features such as formation of a covalent protein-DNA complex and a DNA ligation step. This makes it possible to unambiguously identify the activity of the enzyme even in cell extracts.

It is our aim to apply our detailed knowledge about the biological, structural and chemical mechanism of Topoisomerase action in the development of novel nano-technologies for the diagnosis, prognosis and treatment of human and animal diseases.

To pursue this aim, our research group maintains several lines of research in collaboration with scientific collaborators as well as with medical doctors and engineers:

1. Biochemical characterization of Topoisomerase I:

We continue our investigation of the function and mechanism of topoisomerases from different species. We clone, express and purify recombinant topoisomerase proteins and perform functional analysis by the characterization of mutant forms of the proteins. Our expertise from such studies is an excellent platform for the medical and technological applications described in the following.

2. Single molecule and single-cell analysis of topoisomerase I activity:

We spend much effort on technology development and have developed a series of assays for the measurement of enzyme activities at the single molecule level.

In the so-called REEAD assay, we use specially designed DNA substrates (DNA sensors) to monitor the activity of topoisomerase I. Using the sensor, topoisomerase I activity cause formation of a circular reaction product. This product is amplified 1000 fold by Rolling Circle Amplification, allowing us to visualize single catalytic events by hybridization of fluorescent probes (see figure 1).

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Using droplet microfluidics, we are now capable of performing single-cell investigations of enzyme activity. In the microfluidic device, the cell suspension, the DNA sensor and the necessary lysisbuffer are provided in inlet channels. By a flow of oil, single cells and are separated in individual drops. The entire REEAD reaction can take place within the drop, and the reaction products are visualized by distributing the drops in the drop-trap (figure 2).

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3. Topoisomerase I as a marker for infectious diseases:

By development of DNA sensors specific for topoisomerase I from different pathogenic organisms such as Plasmodium (the malaria parasite) our preliminary results suggest that the REEAD assay can be used to monitor several types of infectious diseases.

As opposed to current diagnosis methods, REEAD can be performed in low technological settings. We therefore expect that REEAD based detection of infectious disease can be used for developing fast and easy-to-use diagnostic kits that will perform well even in the poor areas of the world.

4. Topoisomerase I in cancer development and therapy:

Cancer cell populations are characterized by an unusual high cell-to-cell variation (Figure 3). It is largely unknown how such variations, possibly involving topoisomerase activities, affect cancer development as well as overall chemo response of the entire cancer cell population. The single cell sensitivity of REEAD in combination with droplet microfluidics allows us to answer such questions. In longer terms we hope to develop a cancer predictive assay that can help clinicians to foresee which patients will benefit from a given treatment and who will only suffer the side effects of such treatment.

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5. Design, Construction and Characterization of 3D DNA nanostructures

The inherent properties of DNA as a stable polymer with unique affinity for partner molecules makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA interacting enzymes. More recently, strategies for synthesis of more complex 2D and 3D nano-structures build from DNA have emerged. We are focusing on design, construction, characterization and functionalization of 3D DNA nanostructures.

Main project lines:

6. Construction and characterization of a truncated octahedral DNA cage-structure:

We have designed a DNA nano-structure with the overall shape of a truncated octahedron defined by double-stranded DNA helices that assemble from eight oligonucleotides . We are currently characterizing the properties of the DNA octahedron cage further and we are investigating the possibilities of using other designs for efficient construction of rigid DNA nanostructures.

7. Functionalization of the DNA cage:

Some of the potential applications of the DNA cage are, i) delivery of a specific cargo e.g. peptides, enzymes or drugs to specific cell types or tissues, ii) display of biomolecules in an ordered fashion for analytical or structural investigations, or iii) as a building block for higher order structures. These possibilities are currently under investigation.