Predicting the Crystalization of A-, B-, and Z-DNA

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9304467 Ho The first and most critical step in any crystallographic experiment is to obtain a single crystal that diffracts x-rays. The experiments in this proposal attempts to systematically develop rules for growing crystals of A-, B-, and Z-DNA for structural studies. There are two primary factors that affect the growth pattern and the ultimate structure of an oligonucleotide in a single crystal: 1) the conformational of the DNA and 2) the forces holding the molecules in the symmetric arrangement of the crystal (i.e. the crystal packing forces). Dr. Ho has previously show that the conformation of the DNA in a crystal is dependent on its conformation in the crystallization solutions. This, in turn, is affected by the solution conditions for crystallization and the sequence of the DNA being crystallized. For Z-DNA, Dr. Ho had previously predicted crystallization conditions by estimating the propensity of a particular sequence to form Z-DNA. The stability of Z-DNA is calculated as the difference in the hydration free energy for a given sequence in the B- versus Z-conformations. Dr. Ho proposes to continue this work by extending the calculations to include empirical estimates of Z-DNA stability for sequences that have steric inhibitions to Z-DNA formation. Furthermore, this same approach can be used to predict the conditions for crystallizing the A- form of DNA. To crystallize B-DNA, attention will be paid to specific sequence requirements that stabilizes only this right- handed conformation. In addition, the problem of crystal packing forces, how they affect crystallization and how they affect the structure of an oligonucleotide once in the crystal will be addressed. Experiments will be carried out to determine the enthalpic versus the entropic contributions to these energies, and to compare steric versus hydrophobic effects on the intermolecular interactions within the crystal lattice. %%% The structure of the DNA duplex was first determined forty y ears ago through x-ray diffraction studies on DNA fibers. This general structure launched a new era in the biological sciences by defining the underlying molecular principles of genetic processes in the cell. Since that time, the structure of DNA has been shown to be even more variable than originally suspected, taking new bends and twists that may act as additional signals for controlling such basic cellular functions as transcription and replication. Again x-ray diffraction has played a major role in the discovery of these new and different structures. These recent studies, however, utilize single crystal of short DNA fragments (oligonucleotides), which give us structures of specific DNA sequences at the atomic level. The primary hurdle to these studies has been the ability to grow single crystals that are suitable for structure determinations. This process is generally treated more as an art rather than as a quantitative science. This is surprising since crystallography is perceived as a very exacting science. Dr. Ho proposes studies that attempt to define the intrinsic and extrinsic conditions required to crystallize DNA sequences in the various conformations. This includes the design of the sequence for crystallization, and the types and amount of salts and alcohols that need to be added to these sequences to help stabilize a specific DNA conformation.