Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Chemistry of Nucleic Acids

Today it is hard to name a branch of natural science that is not concerned with the Structure AND Functions of Nucleic Acids. Despite the immense progress achieved in recent decades in studying their Chemical Composition and architecture, many challenges remain in elucidating the relationship between their structure and biological role. There is no doubt that further research in this direction will yield discoveries of profound significance for biology, medicine, and the life sciences as a whole. The epoch-making Discovery of the complementary principle in nucleic acids has provided insight not only into the Fine Structure of these Biopolymers, but also into the mechanisms governing the synthesis and Replication of biological macromolecules. Nucleic acids perform A number of vital biological functions not shared by other polymers. Specifically, they ensure the storage and transmission of hereditary information and take a direct part in executing this information by programming the synthesis of all cellular Proteins. Furthermore, Structural components of nucleic acids act as Cofactors (such as coenzyme A and uridine diphosphate glucose) and allosteric effectors, and form parts of Coenzymes (including nicotinamide adenine dinucleotide and flavin adenine dinucleotide), thereby directly participating in METABOLISM as well as in the accumulation, transfer, and transformation of energy. They also serve as precursors to second messengers—cyclic mononucleotides (such as cAMP and cGMP)—which play a critical role in intracellular signal Transduction. The primary functions of nucleic acids are discussed in detail in Chapter 14.

Methods for nucleic acid isolation. When studying The chemical composition and Structure of Nucleic Acids, researchers invariably face the task of isolating them from biological sources. As noted in Chapter 2, nucleic acids are integral components of complex proteins known as Nucleoproteins, which are found in all Cells of animals, Bacteria, Viruses, and plants. Nucleic acids exhibit strongly pronounced acidic properties—owing to the orthophosphoric acid residues in their structure—and carry a net negative charge at physiological pH values. This accounts for one of their key properties: The ability to interact via ionic bonds with basic proteins (Histones), Metal Ions (predominantly Mg²⁺), and Polyamines such as spermine, spermidine, and putrescine. Consequently, to isolate nucleic acids from Protein Complexes, it is first necessary to disrupt the numerous strong electrostatic bonds between the positively charged protein molecules and the negatively charged nucleic acid molecules. To achieve this, homogenized biological material is treated with concentrated salt solutions (such as a 10% sodium chloride solution), followed by the precipitation of nucleic acids with ethanol. Currently, to isolate nucleic acids in their Native State, researchers prefer the milder phenol method, based on treating a neutral, buffered nucleoprotein solution with phenol. This Procedure is typically carried out in the presence of protein-Denaturing Agents, such as sodium dodecyl sulfate (SDS) or sodium salicylate, after which the mixture is centrifuged. The denatured protein partitions into the phenolic phase, while the nucleic acids remain in the aqueous layer and are subsequently precipitated in the cold by adding 2–3 volumes of ethanol. This method yields sufficiently purified nucleic acid preparations.

Currently, a number of advanced methods are employed to fractionate nucleic acids from the total preparation obtained via the aforementioned procedure. These primarily include calcium phosphate Gel chromatography, Ion-exchange chromatography (using DEAE-Cellulose, DEAE-Sephadex, and other adsorbents), sucrose density gradient ultracentrifugation, Affinity Chromatography on protein Supports, Gel filtration through agarose and Sepharose gels, and gel Electrophoresis, among others.

Once obtained in pure form, nucleic acids are subjected to Hydrolysis to determine their chemical composition. Both enzymatic methods (using exo- and endonucleases) and purely chemical hydrolysis techniques—such as heating nucleic acids with perchloric acid—are employed for these purposes.