The discovery of deoxyribonucleic acid (DNA) as the substance carrying genetic information set the stage for one of the greatest scientific quests of the mid-20th century. While scientists confirmed DNA held the blueprint for life, the exact physical organization of this complex molecule remained unknown. Determining the three-dimensional structure of DNA was the most significant unsolved puzzle in biology, as the structure held the answer to how genetic information is stored and copied. The ultimate breakthrough relied on a specialized technique capable of peering into the molecular architecture: X-ray crystallography.
The Fundamentals of X-Ray Crystallography
X-ray crystallography is an experimental method used to map the atomic and molecular structure of crystalline materials. The technique works by aiming a beam of X-rays at a highly ordered sample, such as a crystal or a fiber of repeating molecules. When the X-rays encounter the regular arrangement of atoms, the rays scatter or diffract in specific, predictable directions.
This interaction produces a diffraction pattern, typically recorded as a collection of spots. The pattern is not a direct image of the molecule but rather a translation of the molecule’s internal symmetry and atomic spacing. The angle and intensity of each diffracted spot contain information about the electron density within the molecule.
Scientists use complex mathematical analysis, based on principles like Bragg’s law, to translate this abstract diffraction pattern back into a three-dimensional representation of the molecule’s structure. The quality of the final structural map is dependent on the purity and orderliness of the crystal used. For fibrous materials like DNA, the technique is known as X-ray fiber diffraction, which provides an angularly averaged pattern that reveals overall shape, symmetry, and repeating distances.
Capturing the Image: The DNA Diffraction Data
Applying the X-ray technique to the DNA molecule required careful sample preparation. Researchers in the early 1950s, particularly Rosalind Franklin and Maurice Wilkins at King’s College London, worked to obtain the clearest diffraction patterns possible. They found that the structure of DNA changed depending on the humidity of the surrounding environment.
Franklin defined two distinct molecular conformations: the “A-form,” which occurred under low humidity conditions, and the “B-form,” found when the DNA fibers were kept highly hydrated. She concentrated on obtaining high-quality images of both forms, using finely drawn fibers of DNA. The B-form, later understood to be the biologically relevant state, yielded the most informative pattern.
In May 1952, Franklin, with her graduate student Raymond Gosling, captured an exceptionally clear image of the B-form, known as “Photo 51.” This photograph was superior to any previous DNA diffraction image, displaying a striking symmetry and clarity that hinted at a highly organized, regular structure. Photo 51 became the physical evidence needed to unlock the mystery of DNA’s architecture.
Decoding the Helix: Interpreting Photo 51
The characteristic X-shaped pattern at the center of Photo 51 provided the first and most direct geometrical clue. This specific cross-like distribution of diffraction spots is the signature of a helical structure viewed end-on. The angle of the ‘X’ arms allowed scientists to calculate the diameter and pitch angle of the helix.
By measuring the distances between the spots along the vertical axis, researchers determined the physical dimensions of the molecular repeats. The spacing between the layer lines, or horizontal rows of spots, was inversely related to the pitch, or one complete turn of the helix. This measurement established the helical pitch at approximately \(3.4\) nanometers (34 Å).
A stronger, more diffuse signal was present further out on the vertical meridian, indicating a smaller, more frequent repeating unit. This spacing corresponded to \(0.34\) nanometers (3.4 Å), representing the distance between the stacked base pairs along the axis. The image revealed that there were exactly ten base pairs per helical turn. Furthermore, the specific missing diffraction spots on the meridian indicated a structure involving two offset, intertwined helices, confirming the double-stranded nature and anti-parallel orientation of the sugar-phosphate backbone.
The Double Helix: Integrating Chemistry and Structure
The data derived from X-ray crystallography provided the geometrical framework for the DNA molecule. James Watson and Francis Crick integrated these physical measurements with existing chemical knowledge to construct the final model. One crucial piece of chemical evidence came from Erwin Chargaff’s rules, which demonstrated a consistent 1:1 ratio in DNA composition: the amount of adenine (A) always equaled the amount of thymine (T), and the amount of guanine (G) always equaled the amount of cytosine (C).
The geometrical constraints from Photo 51 suggested a uniform diameter for the helix. This width could only be maintained if a larger purine base (A or G) always paired with a smaller pyrimidine base (T or C). When this uniform width constraint was combined with Chargaff’s ratios, the deduction of complementary base pairing became necessary: A must pair with T, and G must pair with C. These pairs are held together by hydrogen bonds, two between A and T, and three between G and C.
The X-ray data implied that the sugar-phosphate components formed the external backbone of the molecule, while the base pairs were stacked horizontally inside, like rungs on a ladder. This structure, with two anti-parallel strands coiled around a central axis, satisfied all the physical requirements from the diffraction data and explained the chemical composition rules. The resulting double helix model provided an immediate explanation for how genetic information could be stored, protected, and accurately replicated.