Structural Biochemistry - Study Guide - E. A. Bessolitsyna 2015

NUCLEOTIDES participate in a multitude of biochemical processes and also serve as the monomers of Nucleic Acids. Nucleic acids orchestrate all genetic processes. Each nucleotide consists of Three types of chemical building blocks:

✵ a nitrogenous base;

✵ a monosaccharide;

✵ 1 to 3 phosphoric acid residues.

Unlike Monosaccharides, nucleotides are complex monomeric molecules composed of structures belonging to different classes of chemical compounds; therefore, it is necessary to examine the properties and Structure of each component individually.

Nitrogenous bases belong to heterocyclic compounds. In addition to carbon atoms, the heterocycle incorporates nitrogen atoms. All nitrogenous bases found in nucleotides are classified into two major groups: Purines and Pyrimidines. Purine bases are derivatives of purine—a heterocycle consisting of two rings, one five-membered and the other six-membered, with numbering shown in the figure. Pyrimidine bases are derivatives of pyrimidine and consist of a single six-membered ring, with the numbering also indicated in the figure (Figure 31). The principal pyrimidine bases in both PROKARYOTES AND EUKARYOTES are cytosine, thymine, and uracil. Among the purine bases, adenine and guanine are the most common. Two others—xanthine and hypoxanthine—serve as intermediates in their metabolic pathways. In humans, the end product of purine Catabolism is an oxidized purine base known as uric acid. In addition to the five main bases mentioned above, less widespread minor bases are also known. Some of these occur exclusively in bacterial and viral nucleic acids, but many have also been found in pro- and eukaryotic DNA as well as transfer and Ribosomal RNAs. For instance, both bacterial and human DNA contain significant amounts of 5-methylcytosine, while 5-hydroxymethylcytosine has been discovered in Bacteriophages. Unusual bases identified in Messenger RNA include N⁶-methyladenine, N⁶, N⁶-dimethyladenine, and N⁷-methylguanine. A modified uracil with an (α-amino, α-carboxy)propyl group attached at the N₃ position has also been found in Bacteria. The Functions of these substituted purines and pyrimidines are not yet fully understood, although they may form non-canonical Base Pairs (discussed below), thereby facilitating the Formation of secondary and tertiary structures of nucleic acids.

Figure 31. Structure of nitrogenous bases

A series of purine bases with methyl substituents has been identified in plant Cells, many of which are pharmacologically active. Examples include coffee beans containing caffeine (1,3,7-trimethylxanthine), tea leaves containing theophylline (1,3-dimethylxanthine), and cocoa beans, which contain theobromine (3,7-dimethylxanthine).

The nitrogenous base molecule forms a system of alternating single and double bonds (a conjugated double bond system). This structural Organization creates a rigid molecule incapable of conformational transitions. Consequently, conformational changes do not occur in nitrogenous bases.

Nitrogenous bases exhibit only a single type of isomerism: keto-enol transition, or Tautomerism.

Tautomerism

Due to keto-enol tautomerism, nucleotides can exist in either lactim or lactam forms, with the lactam form predominating under physiological conditions for guanine and thymine (Figure 32). The Significance of this phenomenon will become apparent when discussing base-pairing processes.

Figure 32. Nucleotide tautomerism

Solubility

At neutral pH, guanine exhibits the lowest solubility, followed by xanthine. Uric acid, in the form of urates, is relatively soluble at neutral pH but shows extremely poor solubility in more acidic fluids such as urine. Guanine is normally absent from human urine, whereas xanthine and uric acid are its regular constituents. The latter two purines frequently form Urinary Tract stones.

Light absorption

Due to The system of conjugated double bonds, all nitrogenous bases absorb light in the ultraviolet region of the spectrum. The absorption spectrum is a plot of optical density as a function of wavelength. Each nitrogenous base has a characteristic absorption spectrum, which allows for the differentiation of solutions containing various nitrogenous bases or compounds incorporating them (nucleotides); however, all share a common absorption maximum at a wavelength of 260 nm. This enables rapid and straightforward Determination of the concentration of nitrogenous bases, nucleotides, and nucleic acids alike. The absorption spectrum also depends on the pH of the solution (Figure 33).

Figure 33. Absorption spectra of various nitrogenous bases

Nitrogenous bases practically never occur in a free state, with the exception of certain Alkaloids and uric acid.

Nitrogenous bases perform the following functions:

They are components of nucleotides;

Some alkaloids are nitrogenous bases, such as caffeine in coffee or theophylline in tea;

They serve as intermediates in the METABOLISM of nitrogenous bases and nucleotides;

Uric acid is the underlying cause of urolithiasis;

In some organisms, nitrogen is excreted in the form of uric acid.

Nucleoside molecules are composed of a purine or pyrimidine base attached via a β-linkage to a carbohydrate (typically D-ribose or 2-deoxyribose) at the N₉ or N₁ position, respectively. Thus, the adenine ribonucleoside (adenosine) consists of adenine and D-ribose attached at the N₉ position; guanosine consists of guanine and D-ribose at the N₉ position; cytidine consists of cytosine and ribose at the N₁ position; and uridine consists of uracil and ribose at the N₁ position. Accordingly, in purine nucleosides (nucleotides), the nitrogenous base and the sugar are linked by a 1–9 β-glycosidic bond, whereas in pyrimidines, they are linked by a 1–1 β-glycosidic bond.

2′-Deoxyribonucleosides contain purine or pyrimidine bases and 2′-deoxyribose attached to the same N₁ and N₉ atoms. The attachment of ribose or 2′-deoxyribose to the ring STRUCTURE OF THE base occurs via a relatively acid-labile N-glycosidic bond (Figure 34).

Nucleotides are derivatives of nucleosides that are phosphorylated at one or more hydroxyl groups of the ribose (or deoxyribose) moiety. For example, adenosine monophosphate (AMP or adenylate) is built from adenine, ribose, and phosphate. 2′-Deoxyadenosine monophosphate (dAMP or deoxyadenylate) is a molecule consisting of adenine, 2′-deoxyribose, and phosphate. Typically, ribose is attached to uracil, and 2′-deoxyribose is attached to thymine. Therefore, thymidylic acid (TMP) consists of thymine, 2′-deoxyribose, and phosphate. In addition to the aforementioned forms, nucleotides with unusual structures have also been discovered. For instance, tRNA molecules contain a nucleotide in which ribose is attached to uracil at the fifth position—that is, via a carbon-carbon bond rather than a nitrogen-carbon bond. The product of this unusual linkage is named pseudouridine (ψ). tRNA molecules also contain another unusual nucleotide structure: thymine linked to ribose monophosphate. This nucleotide is formed post-transcriptionally in tRNA via the methylation of the UMP residue by S-adenosylmethionine. Pseudouridylic acid (ψMP) is similarly formed through the rearrangement of UMP following tRNA synthesis.

Figure 34. Structure of Purine and pyrimidine Nucleosides and Nucleotides

THE POSITION OF the phosphate group in a nucleotide molecule is indicated by a number. For example, adenosine with a phosphate group attached to the 3rd carbon of ribose is designated as 3′-monophosphate. The prime symbol after the number is used to distinguish the carbon numbering in the purine or pyrimidine base from the position of the atom in the deoxyribose residue. When numbering the carbon atoms of the base, a prime is not used. The nucleotide 2′-deoxyadenosine with a phosphate residue at carbon-5 of the sugar molecule is designated as 2′-deoxyadenosine-5′-monophosphate. Nucleosides containing adenine, guanine, cytosine, thymine, and uracil are conventionally denoted by the letters A, G, C, T, and U, respectively. The presence of the letter d before the abbreviation indicates that the carbohydrate component of the nucleoside is 2′-deoxyribose. Guanosine containing 2′-deoxyribose can be denoted as dG (deoxyguanosine), and its corresponding monophosphate with a phosphate group attached to the third carbon atom of deoxyribose as dG-3′-MP. As a rule, when the phosphate is attached to carbon-5 of ribose or deoxyribose, the 5′- symbol is omitted. Thus, guanosine 5′-monophosphate is commonly designated as GMP, and 2′-deoxyguanosine 5′-monophosphate is abbreviated as dGMP. If 2 or 3 phosphoric acid residues are attached to the carbohydrate moiety of the nucleoside, the Abbreviations DP (diphosphate) and TP (triphosphate) are used. Consequently, adenosine + triphosphate with three phosphate groups at the 5′-position of the carbohydrate will be designated as ATP. Since the phosphates in nucleotide molecules exist as phosphoric acid anhydrides—that is, in a low-Entropy state—they are referred to as high-energy compounds (possessing a large store of potential energy). The Hydrolysis of 1 mole of ATP to ADP releases 7.3 kcal of potential energy.

Figure 35. Structure of cAMP

Physicochemical Properties of Nucleotides

Because nucleotides contain nitrogenous bases, properties such as tautomerism and ultraviolet absorbance are also characteristic of nucleotides, with the absorption spectra of nitrogenous bases and their respective nucleotides being similar. The presence of sugar and phosphoric acid residues makes them more hydrophilic than nitrogenous bases. All nucleotides are acidic due to the presence of phosphoric acid residues.

Functions of Natural Nucleotides

Nucleotides serve as the monomers of nucleic acids (RNA, DNA). DNA contains deoxyribonucleotide phosphates—derivatives of adenine, thymine, guanine, and cytosine. Additionally, certain guanine and cytosine molecules within DNA are methylated, meaning they contain a methyl group. As the primary monomers, RNA contains ribonucleotide phosphates—derivatives of adenine, uracil, guanine, and cytosine. RNA also incorporates nucleotides containing various minor nitrogenous bases, such as xanthine, hypoxanthine, dihydrouridine, and others.

Nucleotides function as monomers of Coenzymes (NAD, NADP, FAD, coenzyme A, Methionine-adenosine). As part of coenzymes, they participate in enzymatic reactions. This function will be discussed in greater detail below.

Energy storage (ATP). ATP serves as the primary intracellular carrier of Free energy. The concentration of the most abundant free nucleotide in mammalian cells—ATP—is approximately 1 mmol/L.

Signaling (cGMP, cAMP) (Figure 35). Cyclic AMP (3′, 5′-adenosine monophosphate, cAMP)—a mediator of various extracellular signals in animal cells—is formed from ATP in a reaction catalyzed by adenylate cyclase. The activity of adenylate cyclase is regulated by a complex network of interactions, many of which are initiated via Hormone Receptors. The intracellular concentration of cAMP (approximately 1 µmol/L) is 3 orders of magnitude lower than that of ATP. Cyclic cGMP (3′, 5′-guanosine monophosphate, cGMP) acts as an intracellular second messenger for extracellular signals. In some instances, cGMP acts as an antagonist to cAMP. cGMP is synthesized from GTP by the action of guanylate cyclase, an enzyme that shares many features with adenylate cyclase. Like adenylate cyclase, guanylate cyclase is regulated by various effectors, including Hormones. Similar to cAMP, cGMP is hydrolyzed by phosphodiesterase to the corresponding 5′-monophosphate.

Regulatory (GTP). The activity of a group of Proteins (G proteins), which primarily perform regulatory functions, depends on the nucleotide they bind. In their inactive form, these proteins bind GDP; upon activation, GDP is exchanged for GTP. In carrying out their function, the protein hydrolyzes GTP to GDP and phosphate, and the released energy is utilized to drive protein function.

Activation in lipid and monosaccharide metabolism (UTP, CTP). Derivatives of uracil nucleotides act as activating agents in hexose metabolism and carbohydrate polymerization reactions, notably in the Biosynthesis of starch and the oligosaccharide moieties of Glycoproteins and Proteoglycans. The substrates in these reactions are uridine diphosphate sugars. For example, uridine diphosphate Glucose serves as a precursor for Glycogen. Furthermore, The conversion of glucose into galactose, glucuronic acid, or other Monosaccharide Derivatives occurs via UDP-conjugates. CTP is required for the Biosynthesis of certain phosphoglycerides in animal Tissues. Reactions involving ceramide and CDP-Choline lead to The formation of sphingomyelin and other substituted sphingosines.

Involvement in the inactivation of various alcohols and phenols (UDP-glucuronic acid). Uridine diphosphate glucuronic acid functions as an "active" glucuronide in conjugation reactions, such as the formation of bilirubin glucuronide.

Coenzymes are low-molecular-weight compounds associated with Enzymes (see the "Enzymes" section) that directly participate in biochemical reactions; in other words, they act as an additional substrate that does not leave into the surrounding environment.

Coenzymes are subdivided into two groups:

proton and electron carriers, which participate in oxidation-reduction (redox) reactions;

carriers of all other groups except protons and electrons, which participate in transferase reactions.

The mechanisms of the aforementioned reactions are discussed in greater detail in the "Enzymes" chapter.

Some coenzymes contain nucleotides in their structure. They are likewise divided into these same two groups.

These coenzymes participate in redox reactions, where adenosine performs a purely structural function, while nucleotides containing Other types of bases enter the reaction. Two Types of such coenzymes are distinguished: nicotinamide and Flavin Coenzymes. They differ not only in their active group, but also in the type of reactions they mediate.

Nicotinamide Coenzymes

Figure 36. Nicotinamide coenzymes. A — structure of NAD, B — structure of NADP, C — MECHANISM OF ACTION of nicotinic acid, D — mechanism of action of nicotinamide coenzymes

Nicotinamide adenine dinucleotide (NAD⁺) is the primary electron acceptor during The oxidation of fuel molecules. The reactive part of NAD⁺ is its nicotinamide ring. Upon substrate oxidation, the nicotinamide ring of NAD⁺ accepts a hydrogen ion and two electrons, which are equivalents of a hydride ion. The reduced form of this carrier is NADH. During this dehydrogenation, one hydrogen atom from the substrate is directly transferred to NAD⁺, while the second passes into the solvent. Both electrons lost by the substrate are transferred to the nicotinamide ring. The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) serves as an electron donor in most reductive biosynthesis (anabolism) processes. NADPH differs from NAD by the presence of a phosphate group linked via an ester bond to the 2'-hydroxyl group of adenosine. The oxidized form of NADPH is designated as NADP⁺. NADPH transfers electrons in the same manner as NADH. However, NADPH is used almost exclusively in reductive biosynthetic processes, whereas NADH is utilized predominantly for ATP generation. The additional phosphate group of NADPH serves as the recognition site responsible for fulfilling the molecule's specific purpose of enzyme interaction.

Flavin Coenzymes

The first flavin coenzyme (flavin mononucleotide, FMN) was isolated from Heart Muscle by A. Szent-Györgyi in 1932; around the same time, O. Warburg and W. Christian obtained the first flavoprotein containing FMN as a coenzyme from Yeast. The second most important flavin coenzyme, flavin adenine dinucleotide (FAD), was isolated by the same researchers as the cofactor for D-Amino Acid Oxidase in 1938. Through the redox transformation of the flavin ring, flavin coenzymes mediate oxidation-reduction reactions as part of several vital enzyme systems: oxidases (specifically D- and L-amino acid oxidases, and monoamine oxidase, which regulates Blood catecholamine levels) and dehydrogenases (often involving nicotinamide adenine dinucleotide and ubiquinones).

Figure 37. Flavin coenzymes. A — structure of FAD, B — mechanism of action of nicotinic acid, C — mechanism of action of flavin coenzymes

The second major electron carrier in the oxidation of fuel molecules is flavin adenine dinucleotide. The abbreviations used to designate the oxidized and reduced forms of this carrier are FAD and FADH₂, respectively. The reactive moiety of FAD is its isoalloxazine ring. Like NAD⁺, FAD accepts two electrons; however, unlike NAD⁺, FAD accepts both hydrogen atoms lost by the substrate.

Flavin coenzymes participate in the oxidation of carbon skeletons by introducing double bonds between carbon atoms (Figure 37); they also take part in redox reactions within the Electron Transport Chains of Photosynthesis and Oxidative Phosphorylation.

These coenzymes participate in The transfer of any groups other than protons and electrons; such reactions are called transferase reactions. There are numerous coenzymes of this type, of which only selected ones will be discussed here.

Coenzyme A is a central molecule in metabolism (Figure 38). In 1945, Lipmann discovered that many enzyme-catalyzed acylation processes require a heat-stable cofactor. This cofactor was named coenzyme A (CoA), where A stands for Acetylation. It was isolated, and its structure was determined a few years later. The reactive part of the CoA molecule is the terminal sulfhydryl group of pantothenic acid. Acyl groups are attached to CoA via a thioester bond, and the resulting derivative is termed acyl-CoA. The acyl group attached to CoA is frequently an acetyl group; this derivative is called acetyl-CoA. In other words, acetyl-CoA possesses a high acetyl-group transfer potential. Similar to how ATP serves as a carrier for activated phosphoryl groups, CoA acts as a carrier for activated acetyl or other acyl groups.

Figure 38. Structural formulas of coenzymes that carry other groups. A — coenzyme A, B — S-adenosylmethionine

S-Adenosylmethionine is a coenzyme involved in Methyl group transfer reactions (Figure 38). It was first described in Italy by the scientist Cantoni in 1952. S-Adenosylmethionine is synthesized from ATP and methionine by the enzyme methionine adenosyltransferase. Within The Cell, it participates in such metabolic pathways as Transmethylation, transsulfuration, and aminopropylation. Although these anabolic reactions occur in many Tissues of the Organism, the bulk of S-adenosylmethionine is produced in the Liver.

The methyl group (CH₃) attached to the sulfur atom of methionine within S-adenosylmethionine is chemically active. Consequently, this methyl group can be transferred to a substrate molecule in a transmethylation reaction. More than forty metabolic reactions require the transfer of a methyl group from S-adenosylmethionine to substrates such as nucleic acids, proteins, and Lipids. Adenosylmethionine participates in the synthesis of phosphatidylcholine, choline, adrenaline, vitamin B₁₂, and others. The Mechanism of transmethylation involves enzyme-catalyzed displacement mediated by methyltransferases specific to the methyl group acceptor. Methylation typically occurs on N and O atoms, and less frequently on C atoms.

The transfer of methyl groups to the double bonds of Unsaturated Fatty acids in microorganisms leads to the Formation of Branched-chain acids or acids containing cyclopropane rings. The methylation of certain biologically active compounds (e.g., histamine, nicotinamide) yields products that are subsequently excreted from the organism. In proteins, the amino groups of Lysine and Arginine residues can undergo methylation. The methylation of purine and pyrimidine bases as well as ribose rings is the most common modification of nucleic acids, especially Transfer RNAs. In Polysaccharides, adenosylmethionine can, for instance, methylate the O atom at position 6 of D-glucose residues.

Upon elimination of its methyl group, adenosylmethionine is converted into S-adenosyl-L-homocysteine, which is then hydrolyzed to adenosine and homocysteine. The latter can be remethylated in the organism with the participation of N₅-methyltetrahydrofolic acid to yield methionine, which can once again be incorporated into adenosylmethionine. Other pathways of S-adenosyl-L-homocysteine metabolism are also known: in mammals, Cleavage yields homoserine, adenine, and 5-methylthioribose-1-phosphate; in microorganisms, deamination of the adenosine or homocysteine moieties of the molecule occurs, along with the cleavage of adenine.

Adenosylmethionine also serves as a donor of aminopropyl groups in The biosynthesis of the Polyamines spermine and spermidine, as well as an amino group donor in biotin synthesis. Furthermore, a regulatory function of adenosylmethionine has been established for certain enzymes: the binding of adenosylmethionine to one of the enzyme's subunits (e.g., via the transfer of an adenosyl residue) alters the substrate affinity of another subunit.

In addition, methylases containing S-adenosylmethionine as a cofactor are involved in DNA modifications that trigger heterochromatinization and alter the affinity of RNA polymerase for promoters. It is precisely these methylation and demethylation processes that influence Chromatin activity and The regulation of DNA expression. Thus, methylases play a key role in the Selection/27.html">Realization of Genetic information.

A nucleic acid (NA) molecule is a macromromolecule whose monomer is a nucleotide. Ribose and deoxyribose occur in NAs in cyclic form. Nucleotides are linked via phosphoric acid residues, thereby forming a sugar-phosphate backbone in which sugar residues alternate with phosphoric acid residues. The phosphoric acid residue connects the 3′-hydroxyl group of the preceding sugar to the 5′-hydroxyl of the next one. The molecule is asymmetric, featuring 5′ and 3′ ends; the 5′ end is considered the beginning of the molecule and the 3′ end is the terminus, since nucleic acid synthesis proceeds in the 5′ to 3′ direction. DNA and RNA differ primarily in the pentose molecules that compose them: RNA contains ribose, whereas DNA contains deoxyribose. The distinction between these pentoses lies in the presence of an OH group at the 2′ position in ribose. This group confers instability and high reactivity on RNA (enabling catalytic activity and intramolecular reactions). Uracil is formed As a result of cytosine deamination; if this base were part of DNA, The fidelity of DNA Repair would be reduced. Other differences between RNA and DΝΑ are summarized in Table 1.

Figure 39. Primary Structure of Nucleic Acids

Table 1. Differences between RNA and DNA.

The primary structure of DNA provides the foundation for the next level of organization—namely, the Secondary structure of DNA.

Figure 40. B-form of DNA. A—General structural layout of the B-form of DNA, B—hydrogen bonding pattern between base pairs in DNA

The secondary structure of DNA is a double-helical molecule. Two polynucleotide chains are wound around a common axis and run in opposite directions, meaning they are antiparallel. That is, the 3′ end of one strand interacts with the 5′ end of the complementary strand. The helices are right-handed.

The two strands are held together by Hydrogen Bonds between base pairs. Adenine always pairs with thymine, and guanine with cytosine. This is governed by THE PRINCIPLE OF complementarity—the structural and chemical correspondence between molecules. Adenosine in DNA is complementary to thymine, forming two hydrogen bonds between the bases, whereas guanine is complementary to cytosine, forming three hydrogen bonds. This is made possible by the rigid structure of the nitrogenous bases.

The purine and pyrimidine bases are located on the inside of the helix, while the phosphate and deoxyribose residues are on the outside. The planes of the bases are perpendicular to the helical axis, stacking upon one another with additional interactions forming between adjacent planes. The planes of the sugar residues lie at nearly right angles to the bases.

The diameter of the helix is 20 Å. The distance between adjacent bases along the helical axis is 3.4 Å, and they are rotated relative to each other by 36°. Thus, each turn of the helix comprises 10 nucleotides per strand, spanning a length of 34 Å.

There are no restrictions on The sequence of bases within a polynucleotide chain. A specific base sequence carries distinct Genetic information.

The secondary structure of DNA described above was elucidated by Watson and Crick; it is the most common conformation of DNA and is designated as the B-form. Several other DNA Conformations exist (Table 2).

Table 2. Conformations of DNA secondary structure

Figure 41. Structure of various DNA conformations. A — structure of the major DNA forms; B — structure of H-DNA; C — structure of non-canonical Hoogsteen interactions

RNA is composed of ribonucleotides, which likewise consist of a nitrogenous base, a pentose, and a phosphoric acid residue. The differences lie in the presence of ribose and the specific set of nitrogenous bases in RNA. These differences are closely related to the distinct functions of RNA and DNA. The presence of ribose in the sugar-phosphate backbone renders it less stable (prone to strand breaks and the accumulation of errors during genetic transmission to daughter cells), yet it enables catalytic activity. Unlike DNA, RNA is single-stranded and forms complex secondary and tertiary structures, which also facilitate catalysis. The secondary Structural elements of RNA—loops and helices—allow for the formation of simple and complex hairpins. RNA helices are formed by complementary bases and resemble the DNA B-helix in architecture; the key difference is that The Double Helix is formed not by two separate strands, but by complementary and antiparallel regions of a single RNA strand. It should also be noted that both Major and minor nucleotides participate in RNA secondary structure formation, resulting in both canonical and non-canonical base pairing. The resulting helices are relatively short, spanning only a few dozen base pairs, and their length depends on the length of the complementary nucleotide sequences. RNA loops consist of unpaired bases, and unpaired bases may also bulge out from within RNA helices.

Figure 42. Secondary structure of RNA. A—structure of tRNA, B—structure of rRNA

The secondary structure of tRNA is the best understood, typically depicted as a cloverleaf. It features three main hairpins—the anticodon, pseudouridine, and dihydrouridine loops—along with The amino acid acceptor stem and a variable loop. The variable loop accounts for variations in tRNA molecule length (Figure 42). Building upon its secondary structure, RNA achieves a tertiary structure, which represents a higher level of organization. In tertiary structure, helices or stems interact with one another and/or with single-stranded regions and loops to form highly intricate architectures. Another tertiary structural motif is a pseudoknot, which may involve multiple RNA chains stabilized by both Watson-Crick complementarity and Hoogsteen interactions (similar to those found in H-DNA). The secondary structure of RNA dictates the formation of its tertiary structure as loops and hairpins fold and interact in complex ways. During this folding process, tRNA acquires an L-shaped tertiary structure: the amino acid acceptor stem and the pseudouridine hairpin interact to form one arm of the L-shape, while the anticodon and dihydrouridine hairpins associate to form the other arm. The variable loop interacts with them at the junction, forming a knot and stabilizing the overall structure.

To identify the type of nucleic acid, its constituent sugar must be identified. Colorimetric Methods are the most widely used; they can be employed for the Quantitative determination of CARBOHYDRATES as such, or, with appropriate modifications, for the detection of nucleic acids, nucleotides, and their derivatives.

Several pentose assays rely on the release of furfural upon heating with HCl. Furfural yields a red coloration with aniline acetate or a yellow coloration with π-bromophenylhydrazine. Ribose gives a characteristic color reaction when treated with orcinol under specific conditions.

When DNA is heated with diphenylamine in an acidic solution, a blue color develops. In the Feulgen reaction, deoxyribose or DNA—following Partial Acid Hydrolysis—produces a blue-violet coloration.

Nucleic acid molecules are negatively charged and therefore move toward the positive electrode in an electric field. If forced to migrate through a medium with pores—that is, a gel with a defined pore size—linear nucleic acid molecules will pass through the pores. The larger the molecule, the longer it takes to travel through the gel pores; consequently, the shorter the nucleic acid molecule, the farther it can migrate in the gel compared to a longer one. This method allows a mixture of nucleic acids to be separated by length, and in the presence of a mixture of fragments of known sizes, it also enables the determination of the length of the test fragments (Figure 43).

Figure 43. DNA agarose gel electrophoresis. M — marker (a mixture of DNA molecules of known length)

When concentrated cesium chloride solutions are centrifuged in an analytical ultracentrifuge at high speeds until equilibrium between sedimentation and diffusion is established, a stable CsCl concentration gradient is created. This corresponds to an increase in solution density in the direction of the centrifugal force. The resulting density gradient is proportional to the centrifugal force in accordance with the equation

dp/dr=aω²r,

where ρ is the density as a function of the radius r (distance from the center of rotation), ω is the angular velocity, and α is a constant depending on The Nature of the salt. If the CsCl solution contains a small amount of DNA, at equilibrium, the DNA molecules form bands in those Zones of the centrifuge cell where their density equals the density of the medium (isopycnic). The position of the DNA in the cell can be determined by ultraviolet absorbance, recorded photographically. By precisely calculating the density gradient of the solution along the cell, the density of the DNA sample can be found. This technique is called isopycnic density gradient centrifugation. The buoyant density of the DNA is empirically related to the G+C content in the molecule:

ρ (g/cm³) = 1,660 +0.100 (G + C content)

This property makes it possible to fractionate DNA molecules based on their G+C content, as well as by molecular size.

Denaturation is the disruption of secondary structure due to The breakdown of hydrogen bonds. As a result, the Double helices of DNA and RNA break down, forming random coils of single-stranded nucleic acid molecules.

Thermal denaturation is most commonly used, though chemical denaturation is also possible (using Denaturing Agents such as urea, formamide, and formaldehyde). Thermal denaturation occurs under the action of thermal energy supplied during heating; this increases the frequency and energy of vibrations, causing weak hydrogen bonds to break. During chemical denaturation, molecules of the denaturing agent compete for hydrogen bonds, causing nitrogenous bases to interact with the denaturing agents rather than with each other to form secondary structure.

The Temperature at which half of the molecules are denatured is called the melting temperature

The melting temperature depends on the length of the molecule and its G/C content, since the more hydrogen bonds that need to be broken, the more energy must be supplied to the system, and consequently, the higher the heating required. The greater the length of the molecule, the more nucleotides form secondary structure, and the more Hydrogen bonds are present within that secondary structure. When adenine pairs with thymine There are two bonds, and when guanine pairs with cytidine there are three; each G/C pair provides one additional bond, thereby increasing The amount of absorbed energy. The determination of the melting temperature should be carried out at a fixed Ionic strength and pH, as these factors significantly affect DNA stability. The value of T_m can be lowered by adding urea—a reagent that disrupts Hydrogen bonds and prevents hydrophobic interactions. For example, in 8 M urea, T_m drops to 20°C. In 95% formamide, DNA completely separates into two strands at room temperature. Similarly, in acidic solutions near pH 2–3, where Amino groups are protonated, the helix breaks down. The degree of DNA denaturation can be determined in several ways.

All nucleic acids strongly absorb light in the UV region with a maximum at 260 nm. When the Native State of DNA is disturbed, a marked hyperchromic effect is observed—an increase in absorbance. This change reflects a decrease in the number of hydrogen bonds and is noted not only for DNA, but also for RNA and synthetic polynucleotides that have a structure stabilized by hydrogen bonds.

Native DNA exhibits a strong positive optical Rotation of the plane of light polarization, which decreases noticeably upon denaturation.

Solutions of native DNA have high viscosity, which is a consequence of the relatively rigid double-helical and elongated rod-like structure of DNA. The destruction of hydrogen bonds leads to a marked decrease in viscosity.

DNA denaturation is a reversible process. If DNA is only partially denatured, for example by heating, a decrease in temperature results in the rapid renaturation of each DNA molecule, with a rate corresponding to a first-order reaction. If DNA is completely denatured, the two complementary strands will reassociate slowly (DNA "annealing"). This process involves two stages: first, complementary sequences of the two strands assemble via a second-order reaction, followed by their rapid "zipping up" via a first-order reaction. If the initial concentration of denatured DNA is C₀ (the molar concentration of DNA phosphate), The change in concentration C of single-stranded DNA over time obeys a second-order reaction equation

dc/dt — K₂C²

the integral form of which is

C/C₀ = 1/ (1 +K2C₀t)

The rate of renaturation is usually evaluated from a plot of C/C₀ versus lgC_ot. By plotting this graph, one can find C₀ t_1/2, where t_1/2 is the time at which C/C₀ = 0.5, as well as the second-order reaction rate constant K₂, equal to 1/C₀t_1/2. The constant K₂ is a characteristic parameter for a given DNA; it is inversely proportional to N—the number of DNA base pairs (assuming the DNA has no repeating sequences). C₀t_1/2 is directly proportional to N (N is referred to as DNA complexity). This method makes it possible to determine N values for bacterial and viral DNAs that are consistent with values obtained by other methods.