See Overview 2010

Chandra Shekhar U Murade (promotion date: 4 June 2010)

Insights into DNA Intercalation using combined optical tweezers and fluorescence microscopy

Promotion date: 4. June 2010

Promotors: Prof. dr. Vinod Subramaniam

Assistant Promotor: Dr. ir. Martin Bennink

Assistant Promotor: Dr. C. Otto

In this thesis a combined optical tweezers and line scanning fluorescence microscopy instrument is build, which is capable of probing the change in the mechanical properties of a single double-stranded DNA molecule, as it is interacting with protein molecules, having the capability to detect the number and location of the protein molecules on the DNA simultaneously. This allows us to directly correlate the effect of protein binding on the mechanical properties of the DNA, on a single molecule level.

Capability of optical tweezers is demonstrated by measuring force extension curves, using single dsDNA molecules. Combined techniques were used in experiments to study the interaction of dye molecules, YO-1 and YOYO-1, with dsDNA at various concentrations. These molecules emit fluorescence when bound to dsDNA.

YOYO-1 molecules were used as probe molecules, to investigate the structure of the dsDNA in the overstretching region. Using optical tweezers with integrated line scanning polarization-sensitive fluorescence microscopy, it could be shown and explained that dsDNA is ‘melting’ under the influence of applied force in the overstretched region.

Was there a special moment during your thesis project, that you remember very well?

Building a measuring instrument was part of the project, combining two techniques that were very different. The optical tweezers instrument, operating with light-beams of approximately two watts, was combined with a fluorescence microscope. Fluorescence signals are in the microwatt region.

The instrument was designed some ten years ago by my co-promotor Martin Bennink. However, nowadays the cameras and other optical components are really good, which allows building of sensitive instruments.

I can remember the moment that I actually could see the DNA in my system. It was a crude image only. A year later it was possible to perform serious experiments and analysing the data. I could prove two dye-molecules of the same ‘family’, interacted with DNA quite differently. Also I could describe the processes involved in some detail.

Really good were the low-concentration experiments, using YOYO-dye molecules. I could explain a phenomenon that is known for quite some time: the stretching of the DNA-molecule in a certain region where little force leads to overstretching the molecule.

Some nice publications appeared: in the Biophysical Journal and Nucleic Acids Research, for example. Two manuscripts are still under way.

Are you more of an experimentalist?

Definitely so. I can spend time in the lab, working and working, for hours and hours, whereas studying theory can make me tired within, let’s say, two hours.

Did you profit, being part of the Mesa+ community?

Yes, for example the skills of the technicians are incredible. They have in-depth knowledge of the field and the systems. I guess, 50 % of the contents of my thesis is developed talking to them. As a PhD you are learning, while these people have build up knowledge in successive years.

Furthermore, most of the research is funded by companies, in which real collaboration takes place. I think this element is missing in India. One day I hope to share this knowledge at one of the research institutes that India has. There are quite a number of them active, all with their own mix of r&d and teaching goals.

Also, I hope to contribute to changing the attitude towards research. Here, I learned, that building a team of researchers, involving professors, PhD’s, students and technical staff etcetera, is the key to success.

What are your future plans?

Now I am working as a post-doc at the Physics of Complex Fluids group. I hope to gain experience in this different field of research. Also it is a good opportunity being more responsible for the projects running.

Are you planning to go back to India one day?

Yes, I am. Also right now, I am working on little development projects in India. I am a member of the Aadhaar foundation, run by Indian students at the University of Twente. Mesa+ is one of its contributors.

The activities are targeted towards: self-employment, education, women empowerment, water management and renewable energy sources. Right now, we have a project involving small knowledge centres in rural areas. I am very enthusiastic about this initiative. A lot of talent is living in the rural areas of India. I hope a lot of young people will have the opportunity to let their talent flourish, as I was lucky to do so.