In this article we will discuss about the physico-chemical properties of nucleic acids.
The size of nucleic acids varies immensely. The smallest ribonucleic acids are the tRNAs which comprise about 80 nucleotides; their molecular weight is about 30 000. Ribosomal ribonucleic acids are larger; they contain several thousands of nucleotides and their molecular weight can exceed 1 million.
The RNA of the tobacco mosaic virus has about 6 500 nucleotides, which corresponds to a molecular weight greater than 2 million; the polycistronic messenger ribonucleic acids have comparable dimensions.
Deoxyribonucleic acids are much larger. A small DNA like the circular single-stranded DNA of phage ϕ X 174 already has 5.5 X 103 nucleotides and a molecular weight of 1.7 million, but that of the phage T2 comprises 2 x 105 pairs of nucleotides which corresponds to a molecular weight of 130 million.
The chromosome of E.coli is a circular double-stranded molecule comprising about 4 x 106 pairs of nucleotides; its molecular weight is therefore of the order of 2 to 3 x 109. In fact, the extraction of intact DNA molecules (from bacterial, plant or animal cells) is a rather delicate operation; cleavages occur easily and in the past, one often attributed molecular weights much lower than the real ones.
Cells of eucaryotes have a variable number of chromosomes according to the species. Each chromosome seems to consist of only one molecule of DNA (containing about 108 pairs of nucleotides), associated with basic proteins, the histones, to form chromatin, consisting of a flexible chain of repetitive units, the nucleosomes.
The nucleosome, the fundamental packing unit of the DNA, contains about 200 pairs of nucleotides coiled around a nucleus consisting of 8 molecules of histones. The nucleosomal chain is itself folded up in such a manner that finally, the length of a metaphasic chromosome is about 8000 times shorter than that of the DNA it contains. Non-histone proteins (called “acid”) are also attached to the chromatin. Some of them most probably play a role in the regulation of the genetic expression at the transcriptional level.
On the other hand, it must be noted that the deoxyribonucleic acids found in cellular organelles (mitochondria, chloroplasts) differ by their size and base sequence from the nuclear DNA present in the same cell.
The molecular weights of deoxyribonucleic acids are determined either upon the observation of molecules by electron microscopy (it is estimated that a length of 1 μ corresponds to a molecular weight of 106 for a single-stranded structure, or 2 x 106 for a double-stranded structure), or from the sedimentation constant of the DNA, obtained by comparing its rate of sedimentation in sucrose gradient (from 5 to 20%) to that of a DNA of known sedimentation constant. (Then, with the help of an empirical equation, the molecular weight is calculated from the value of S).
Very often, for preparative of analytical purposes, one uses the sedimentation equilibrium in a CsCl gradient established during the centrifugation; this gives a density gradient ranging generally from 1.65 to about 1.75 g/ml.
The DNA is concentrated in a band at the place where the density of CsCl solution is equal to its own and this density is generally determined by comparison with a DNA of known density, centrifuged in the same gradient. The density of a DNA at the sedimentation equilibrium increases with greater percentages of G-C pairs of the DNA (because G-C pairs are more dense then A-T pairs).
The DNA solutions have a very high viscosity due to the considerable length and relative rigidity of the double helix. The denaturation of DNA molecules can be followed up with the help of viscosity measurements.
On the other hand, due to the presence of purine and pyrimidine bases, they absorb in ultra-violet light (just like the RNA solutions) with a maximum around 260 nm. When a solution of native DNA is heated one observes a decrease of viscosity, and an increase of it optical density (O.D.) at 260 nm (between 30 and 40%). This phenomenon is called hyperchromia effect or hyperchromicity.
It is due to the separation of the 2 strands of the double helix (spoken of as the melting of DNA), by rupture of the interchain hydrogen bonds, and since the G ≡ C bonds are stronger then the A = T bonds, the higher the GC content of a DNA, the higher will be its melting point. The curve of O.D. as a function of temperature has a sigmoidal form.
It permits the determination of a transition point, Tm, which corresponds to the temperature at which the DNA molecules are half-denatured. A DNA whose strands are thus separated, either by heat, or under the action of other physical or chemical agents (e.g., urea) is called denatured. The DNA-passes from an ordered structure (double helix) to a disordered structure (random coil).
The DNA solution can be cooled again in 2 ways:
i. Rapidly:
The 2 strands remain separated (the DNA remains denatured);
ii. Slowly:
This permits a re-association of the 2 strands (the AT and GC bonds are formed again) and the initial helix is reconstituted. The DNA is thus renatured and recovers its biological properties (for example the transforming activity in the case of some bacterial deoxyribonucleic acids).