Ultrafast Molecular Dynamics

Condensed Phased Dynamics:
Exploring the unzipping of DNA on a sub-nano second time scale

Ever since the discovery of the structure of DNA, the dynamics of the DNA double helix has attracted many researchers and phenomena like energy transport, conformational dynamics and thermal fluctuations inside the double helix are still under investigation by leading scientists. One aspect of this is to predict the melting temperature of a given DNA sequence - a problem that is still unresolved. Numerous experimental and theoretical studies addressed this problem and several empirical formulas to calculate melting temperatures have been reported, but an accurate value for the melting temperature can still only be determined by time consuming measurements. Knowing the melting temperature of a specific DNA sequence is key to several molecular biology techniques like polymerase chain reaction (PCR), where specific regions of DNA are amplified for use in medical diagnostics and a number of basic research areas in biology and medicine. A detailed understanding of the un-zipping of DNA can revolutionize those techniques. The understanding of the configurational changes of nucleic acids is also a key step if one wishes to control cellular processes such as transcription or translation.

Figure 1: The left picture shows an example of non resonant CARS, formed when overlapping the three 25fs laser pulses in a microscope coverslip. The middle and right picture corresponds to blocking either one of the pump beams or the Stokes beam, respectively.

A modified azobenzene molecule can intercalate into the double helix and it has been shown that after shining UV-light onto the sample, the melting temperature can be shifted down by 10-25◦C. The azobenzene molecule is switched from a planar trans to a non-planar cis-conformation. This isomerization provides enough energy to break 4-5 hydrogen bonds, i.e. opening two base pairs. Close to the melting temperature, this is expected to induce a bubble in the double helix. Transient absorption spectroscopy can be used to monitor the dynamics of such a bubble. This can be done by using the photo-trigger itself as a probe. Alternatively, one can use fluorescence markers (F¨orster resonance energy transfer, FRET). A different Ansatz is to incorporate a different set of marker molecules that use triplet-triplet energy transfer (TTET) which has the advantage of being a better defined system: very close spacial proximity of donor and acceptor is required for quenching; also, the triplet absorption bands of donor and acceptor can be chosen to be clearly separated.

Ultimately, our goal is to extend this line of research by studying the un-zipping dynamics as a function of the base pair sequence. Every protein produced in living cells is controlled at the level of transcription by a promoter sequence that is found in the DNA before a gene sequence. Differences in this sequence, including the melting temperature and separation of the helix, control how the gene, and protein, are expressed and, ultimately, the function and health of a cell.

At the NRC, we have already successfully carried out transient absorption experiments on the modified azobenzene molecules that will be used in the experiments to un-zip the double helix. We are currently set up to perform the first series of experiments on azobenzene-containing DNA. The samples in this case will be short, six base pairs long sequences that have been shown to exhibit the largest shift in melting temperature upon trans-to-cis isomerization of the azobenzene molecule. These experiments will be able to show un-zipping dynamics on a sub-nanosecond time scale.

The general idea of this experiment is shown in Fig. 2

Figure 2. This figure shown the basic idea behind the experiments on unzipping DNA. In the trans isomer, the azobenzene fits between the basepairs, and actually stabilizes the double stranded DNA through interactions of the delocalized pi electrons (stacking), but when switched to cis by a laser pulse, it pushed neighboring basepairs away, destabilizing the helix, and initiating melting.