Structural Biochemistry/Nucleic Acid/DNA/Franklin's DNA X-ray Crystallography

Although the discovery of the structure of DNA was attributed to Watson and Crick, a keynote player in helping them discover this structure was a scientist by the name of Rosalind Franklin. Rosalind Franklin, along with Francis Wilkins, worked on DNA applying X-ray crystallography to find out its structural properties. X-ray crystallography required the process of exposing a crystal specimen (DNA) to X-rays to determine the locations of the atoms in the “molecules that comprises basic unit of crystal called unit cell”. The task however was not an easy one to attain.

Obtaining a clear diffraction pattern of an object required that the crystal be pure and the x-ray strong enough. However, as Franklin realized, DNA existed in two forms in equilibrium which resulted in a very unclear diffraction pattern. These forms were the A form, which is the dehydrated form of DNA, and the B form, which generated a long and fibrous structure due to humid conditions. Franklin, applying her chemistry background, then proceeded to isolate these two forms using clever laboratory techniques such as “manipulation of the critical hydration of her specimens” [2]. The A and B forms were then separated and subjected to X-ray crystallography obtaining the pictures which would be the basis of Watson and Crick’s helical DNA structure; one of them the famous photo no. 51 of the B DNA form.

Fiber Diffraction
Fiber diffraction is a method used to determine the structural information of a molecule by using scattering data from X-rays. Rosalind Franklin used this technique in discovering structural information of DNA. The experiment places a fiber in the trajectory of an X-ray beam at right angle. The diffraction pattern is obtained in the films of a detector placed few centimeters away from the fiber. The fiber diffraction pattern is a two dimension patterns showing the helical symmetry of a molecule rather than a three dimension symmetry if taken by X-ray crystallography. A good fiber diffraction patterns exhibits four quadrant symmetry, the axis aligned to the fiber is called the meridian and the axis perpendicular to the fiber is called equator. Franklin obtained a diffraction pattern using a non-crystalline DNA fiber, and from it she deduced the B-form of DNA.

EXPERIMENT

 * The diffraction pattern obtained by Franklin and Wilkins showed a X pattern which hinted of a 2 stranded helical form


 * They also observed that the patterns was consistent and inferred that the helix’s diameter must also be consistent,


 * The helical turn of DNA correlates to the horizontal lines in the picture which measures to 34 Angstroms. They also calculated that the gap between based pairs was 3.4 A as measured on the distance from the center of the X to the ends.  Simple math deduced that there are 10 nucleotides per turn

Franklin and Wilkins also showed that the sugar phosphate backbones were found to be in the outside of the helix and not inside as it was previously thought to be. They came to this conclusion because of the A and B forms of DNA. The hydrated and dry forms of DNA showed that water could easily come in and bind to DNA, a fact that could only happen if the feature showed sugar phosphate backbones being on the outside.

SUMMARY OF FINDINGS
- DNA’s helical structure was composed of two strands

- establish that DNA’s diameter was similar throughout

- calculated that 1 turn was 34 A, distance between base pairs as 3.4A, and 10 nucleotides per helical turn

- showed that sugar phosphate backbones were located outside of the structure