Spectroscopic studies of DNA

     Optical studies of DNA.

The research group at UCLA has in recent past explored the optical properties and conformational changes of DNA. The initial focus has been on the investigation of the electronic properties of DNA by utilizing a wide array of spectroscopic tools '. These experiments examined (conducting experiments on both on single strand and on duplex DNA) the different charge excitations of this bio-molecule. We have shown that the low energy excitations of native DNA, and also single strand and duplex DNA oligomers are due to the water molecules in the hydration layer, and not due to charge excitations, or charge migration as suggested by some dc transport studies, and we have concluded that findings on electronic transport of the helix (suggesting that DNA acts as a "molecular wire") are due to spurious effects. Our results have been featured in a New Scientist article.

     Spectroscopic investigations of the denaturation of DNA.

The opening of the dNA duplex is the first step in replication, the fundamental process of life. As such it has been studies by physicists, chemist and biologists. Our approach is to examine the simplest possible scenario: the thermally induces opening of simple DNA sequences, and to examine whether simple theoretical models can describe the state of affairs. The experiments utilize a variety of spectroscopic tools.

In addition to the exploration of the charge excitations associated with DNA a variety of spectroscopic methods have been used to examine conformational changes, and the temperature driven "melting" of the DNA, together with issues involving duplex formation.UV absorption, circular dichroism, temperature dependent fluorescence spectroscopy and also temperature dependent gel electrophoresis was used to study the details of the melting transition in various oligomers, designed to allow us to gain information on details of the separation of the two strands of the duplex. A simple example, where the different techniques lead to the same melting curve is displayed on Figure 1 together with the simplest possible theoretical description, the so-called two state model of the DNA melting. In the example shown, a two state model is appropriate and therefore the result that the various methods lead to the same melting curve are not surprising -this however is not the case in general. In order to relate the melting of DNA to simple models, we have measured the temperature driven denaturing, or melting transition in poly d(A)-ply d(T) DNA oligomers of various lengths in different buffer conditions. Our findings are in clear disagreement with two state, reaction kinetics mode, used often to describe the melting characteristics of oligomers, and we have that the so-called zipper model, where denaturing proceeds through opening of the duplex at the ends describes well the temperature dependence of the average number of open base pairs, and can be regarded as a good firs approximation of the melting process of the simple oligomers mentioned above. Analysis of the length dependence of the transition parameters however suggest that bubble formation -the partial opening of the duplex at a distance from the ends -is important and that the transition, in the thermodynamic limit, is continuous, albeit close to first order. Subsequently we have used l6 a new method to study the bubble formation during the melting transition of DNA oligonucleotides, designed so that the presence of intermediate states could be examined. The approach is to combine UV spectroscopy with a method based on separating intermediate states on a gel (using temperature dependent gel electrophoresis in order to measure both the average fraction of open base pairs and the fraction of completely open molecules throughout the transition. From these two quantities we obtain the temperature dependent relative length of sin le stranded loops in the partially open molecules. The measurements show '6 that for relatively short oligomers most molecules are in partially open intermediate states throughout the transition, and the relative length of open segments for these states increases smoothly with increasing temperatures. These observations point to a melting process that goes beyond the simple, so called zipper model description.