The Molecular Structure of DNA. Chargaff's Rules

DNA: An Astonishing Molecule

Saying today that DNA is the molecule responsible for hereditary transmission may seem trivial, but we’re often unaware of the biological greatness contained in such a statement. That is, we’re talking about life being able to persist through time thanks to a molecule with self-replicating properties—and not only that, it’s also structured in a way that allows it to pass down all the traits seen in an individual’s ontogeny, generation after generation, defining them within a specific species.

In other words, DNA makes possible one of the essential characteristics of living beings: the ability to reproduce, producing offspring similar to the parents—offspring that are also fertile.

But what is DNA from a chemical standpoint? We could say DNA is a repetitive molecule made up of three fundamental components: a sugar (deoxyribose), a nitrogenous base (which can be a purine or a pyrimidine), and orthophosphoric acid. These components link together to form a repeating structure known as a nucleotide. Nucleotides connect via a type of bond called a phosphodiester bond, formed between the phosphate group on the 5’ carbon of one nucleotide and the OH group on the 3’ carbon of the sugar of another nucleotide, releasing a water molecule in the process. This bond repeats across a series of nucleotides to form a DNA strand.

But DNA’s complexity goes even further, as in many organisms DNA is double-stranded. What does that mean? It means the DNA molecule is not made up of just one chain of nucleotides (strand) but two, joined by hydrogen bonds. These bases always pair in a specific way, forming perpendicular connections inside the molecule:

Adenine – Thymine: held together by two hydrogen bonds.
Cytosine – Guanine: held together by three hydrogen bonds.

DNA molecule with its parts

That’s why DNA strands are called complementary—but they are also antiparallel. This means that the orientation of the two polynucleotide chains is opposite. One strand has a free phosphate group at one end, known as the 5′ end, and a free OH group from the pentose sugar at the other, known as the 3′ end. The opposite strand is arranged in reverse: the 5′ end of one strand aligns with the 3′ end of the other.

In Search of the Hereditary Molecule

At the beginning of the 20th century, it was known that traits were inherited across generations, following Mendel’s laws—but little was understood about the cytological, let alone molecular, mechanisms behind heredity.

Over time, significant progress was made in cytology, and credit for the discovery of DNA goes to Swiss biologist J.F. Miescher, though he wasn’t aware of its role or significance. Miescher managed to isolate a new cellular substance from pus in surgical bandages and salmon sperm by treating cells with enzymatic pepsin and centrifugation. He called this substance “nuclein” and observed it contained phosphorus.

However, it was the experiments of O. Avery, C. McLeod, and M. McCarty that revealed DNA’s importance. They demonstrated that DNA was a transforming substance capable of changing the characteristics of a living organism—in this case, bacteria—indicating that it might be the molecule responsible for inheritance.

To prove this, they conducted a brilliant experiment using two strains of Streptococcus pneumoniae. The rough-edged strain (R) was harmless to mice, while the smooth-edged strain (S) was virulent and caused pneumonia, leading to the mice’s death. Avery and colleagues found:

Heat-killed S bacteria were harmless to mice.
When heat-killed S bacteria were mixed with live R bacteria and injected into mice, the mice became ill and died.
Live S bacteria were found in the blood of these mice.

The conclusion: the live, harmless R bacteria had taken up “something” from the dead S bacteria that transformed them into virulent S bacteria. That “something” was called the transforming principle. After isolating, purifying, and eliminating other cellular components, they identified it as DNA.

Later experiments by Hershey and Chase, which used radioactive phosphorus isotopes to track viral infections in bacteria, confirmed the findings of Avery and his team.

Chargaff’s Rules

In the early 20th century, defining DNA’s molecular structure was a matter of scientific debate. One proposed model was that of the repetitive tetranucleotide (deoxy-A, deoxy-C, deoxy-G, deoxy-T). Given the recent discoveries suggesting DNA was a transforming substance, some scientists believed a molecule so repetitive and monotonous couldn’t carry hereditary information—either DNA wasn’t the hereditary molecule, or its structure had to be different.

Erwin Chargaff provided the answer. He analyzed DNA from various living organisms, focusing on the molar composition of the nitrogenous bases. He found that nitrogenous bases did not occur in equal amounts, and even more intriguingly, the base composition varied between species but was consistent within a species.

Chargaff also found that the amount of A (adenine) always equaled T (thymine), and the amount of C (cytosine) equaled G (guanine). In other words, (A + G)/(T + C) = 1.

These conclusions were fundamental for the model of DNA’s double helix proposed by Watson, Crick, and Franklin. From these discoveries, several rules were deduced:

The A + T / G + C ratio is species-specific and thus varies between organisms. However, this ratio is the same in each complementary strand and in the complete molecule.
The ratio (A + G)/(T + C) equals one for the complete molecule. Individually, in each strand, one is the inverse of the other. That is, in one strand it would be A + G / T + C, and in the other strand, T + C / A + G.