Mechanisms of DNA Homology Recognition in Confinement

Kornyshev-Leikin theory and its extensions, some other theories, and a series of physical test-tube experiments (liquid crystalline type, as well as magnetic-bead single-molecule-experiments), suggested that homologous tracts of two different DNA molecules, or those with identical texts, may associate with each other due to the physical interactions between them alone in the absence of proteins. The next step closer to biological reality would be to investigate whether the spontaneous recognition occurs without any recognition proteins in confinement, in a simplest 'artificial' cell (large lipid vesicle or synthetic membrane), to determine the role of crowding agents, as well as the overall effect of controlled confinement.

Uncompensated negative charges on DNA and configurational entropy tend to push the molecules apart, so the existence of a recognition potential well may not necessarily indicate attraction, but rather less repulsion for homologous tracts than non-homologous ones. The confinement and crowding agents may overwhelm repulsion, allowing metastable juxtaposition of homologous tracts. The effects will be sensitive to the concentration of DNA condensing counterions, which themselves may suppress repulsion by compensating the negative charges by associating in the major or minor grooves or the DNA.

The experiments are being planned in the IC Institute of Chemical Biology (Yuval Elani, Marco Di Antonio), and so it will be important to have a theory of DNA-DNA interaction in confinement to guide them and to develop the theory in feedback with experimental results. The confinement can be formed by lipid-bilayer vesicles in physiological solutions, or a water droplet enclosed by a lipid monolayer in oil. Via marking of DNA with FRET chromophores, the proximity of homologous DNA tracts can be detected. Theoretical treatment of the system coupled with analysis of the signals in these experiments will allow us to gain insight into recognition dynamics (how much time it would take for homologous tracts to find each other depending on the degree of confinement), and distinguish between stable and metastable paired states, as well as the effects of multiple factors such as counterions, crowding, and DNA packing within the vesicle. The project will involve elements of international collaboration on theory with groups in France and Germany, as well as on the experimental front with Harvard University, and with a champion of computational microscopy Professor A. Aksimentiev, Leverhulme Visiting Professor from the University of Illinois.

The first part of the work will be focused on the interaction of homologous tracts within one molecule (as experiments with fluorescent labelling may be started in that simple configuration), and will require the development/ adjustment of statistical-mechanical theory of long of worm-like chains in confinement both with and without crowding agents. We will then proceed to the interaction of 2 DNAs in confinement. The last part of the work will deal with the same processes but with DNA associated with histones, and also involving some pertinent proteins; this part of the work will be developed in collaboration with Dr. Teif of the University of Essex.