Nucleotide

Showing the arrangement of nucleotides within the structure of nucleic acids: At lower left, a monophosphate nucleotide; its nitrogenous base represents one side of a base-pair. At upper right, four nucleotides form two base-pairs: thymine and adenine (connected by double hydrogen bonds) and guanine and cytosine (connected by triple hydrogen bonds). The individual nucleotide monomers are chain-joined at their sugar and phosphate molecules, forming two 'backbones' (a double helix) of a nucleic acid, shown at upper left.

A nucleotide is composed of three distinctive chemical sub-units: a five-carbon sugar molecule, a nitrogenous base—which two together are called a nucleoside—and one phosphate group. With all three joined, a nucleotide is also termed a "nucleoside monophosphate", "nucleoside diphosphate" or "nucleoside triphosphate", depending on how many phosphates make up the phosphate group.

Nucleotides contain either a purine or a pyrimidine base—i.e., the nitrogenous base molecule, also known as a nucleobase—and are termed ribonucleotides if the sugar is ribose, or deoxyribonucleotides if the sugar is deoxyribose. Individual phosphate molecules repetitively connect the sugar-ring molecules in two adjacent nucleotide monomers, thereby connecting the nucleotide monomers of a nucleic acid end-to-end into a long chain. These chain-joins of sugar and phosphate molecules create a 'backbone' strand for a single- or double helix. In any one strand, the chemical orientation (directionality) of the chain-joins runs from the 5'-end to the 3'-end (read: 5 prime-end to 3 prime-end)—referring to the five carbon sites on sugar molecules in adjacent nucleotides. In a double helix, the two strands are oriented in opposite directions, which permits base pairing and complementarity between the base-pairs, all which is essential for replicating or transcribing the encoded information found in DNA.

Unlike in nucleic acid nucleotides, singular cyclic nucleotides are formed when the phosphate group is bound twice to the same sugar molecule, i.e., at the corners of the sugar hydroxyl groups.[1] These individual nucleotides function in cell metabolism rather than the nucleic acid structures of long-chain molecules. The term nucleotide can also be used to describe nucleotide compounds that have separate phosphate groups attached to different positions on the sugar.[2]

Nucleic acids then are polymeric macromolecules assembled from nucleotides, the monomer-units of nucleic acids. The purine bases adenine and guanine and pyrimidine base cytosine occur in both DNA and RNA, while the pyrimidine bases thymine (in DNA) and uracil (in RNA) occur in just one. Adenine forms a base pair with thymine with two hydrogen bonds, while guanine pairs with cytosine with three hydrogen bonds.

Structural elements of three nucleotides—where one-, two- or three-phosphates are attached to the nucleoside (in yellow, blue, green) at center: 1st, the nucleotide termed as a nucleoside monophosphate is formed by adding a phosphate (in red); 2nd, adding a second phosphate forms a nucleoside diphosphate; 3rd, adding a third phosphate results in a nucleoside triphosphate. + The nitrogenous base (nucleobase) is indicated by "Base" and "glycosidic bond" (sugar bond). All five primary, or canonical, bases—the purines and pyrimidines—are sketched at right (in blue).

Nucleotides can be synthesized by a variety of means both in vitro and in vivo.

In vitro, protecting groups may be used during laboratory production of nucleotides. A purified nucleoside is protected to create a phosphoramidite, which can then be used to obtain analogues not found in nature and/or to synthesize an oligonucleotide.

In vivo, nucleotides can be synthesized de novo or recycled through salvage pathways.[3] The components used in de novo nucleotide synthesis are derived from biosynthetic precursors of carbohydrate and amino acid metabolism, and from ammonia and carbon dioxide. The liver is the major organ of de novo synthesis of all four nucleotides. De novo synthesis of pyrimidines and purines follows two different pathways. Pyrimidines are synthesized first from aspartate and carbamoyl-phosphate in the cytoplasm to the common precursor ring structure orotic acid, onto which a phosphorylated ribosyl unit is covalently linked. Purines, however, are first synthesized from the sugar template onto which the ring synthesis occurs. For reference, the syntheses of the purine and pyrimidine nucleotides are carried out by several enzymes in the cytoplasm of the cell, not within a specific organelle. Nucleotides undergo breakdown such that useful parts can be reused in synthesis reactions to create new nucleotides.

Pyrimidine ribonucleotide synthesis

The synthesis of UMP.

The color scheme is as follows: enzymes, coenzymes, substrate names, inorganic molecules

The synthesis of the pyrimidines CTP and UTP occurs in the cytoplasm and starts with the formation of carbamoyl phosphate from glutamine and CO₂. Next, aspartate carbamoyltransferase catalyzes a condensation reaction between aspartate and carbamoyl phosphate to form carbamoyl aspartic acid, which is cyclized into 4,5-dihydroorotic acid by dihydroorotase. The latter is converted to orotate by dihydroorotate oxidase. The net reaction is:

(S)-Dihydroorotate + O₂ → Orotate + H₂O₂

Orotate is covalently linked with a phosphorylated ribosyl unit. The covalent linkage between the ribose and pyrimidine occurs at position C₁[4] of the ribose unit, which contains a pyrophosphate, and N₁ of the pyrimidine ring. Orotate phosphoribosyltransferase (PRPP transferase) catalyzes the net reaction yielding orotidine monophosphate (OMP):

Orotate + 5-Phospho-α-D-ribose 1-diphosphate (PRPP) → Orotidine 5'-phosphate + Pyrophosphate

Orotidine 5'-monophosphate is decarboxylated by orotidine-5'-phosphate decarboxylase to form uridine monophosphate (UMP). PRPP transferase catalyzes both the ribosylation and decarboxylation reactions, forming UMP from orotic acid in the presence of PRPP. It is from UMP that other pyrimidine nucleotides are derived. UMP is phosphorylated by two kinases to uridine triphosphate (UTP) via two sequential reactions with ATP. First the diphosphate form UDP is produced, which in turn is phosphorylated to UTP. Both steps are fueled by ATP hydrolysis:

ATP + UMP → ADP + UDP

UDP + ATP → UTP + ADP

CTP is subsequently formed by amination of UTP by the catalytic activity of CTP synthetase. Glutamine is the NH₃ donor and the reaction is fueled by ATP hydrolysis, too:

UTP + Glutamine + ATP + H₂O → CTP + ADP + P_i

Cytidine monophosphate (CMP) is derived from cytidine triphosphate (CTP) with subsequent loss of two phosphates.[5] [6]

Purine ribonucleotide synthesis

The atoms that are used to build the purine nucleotides come from a variety of sources:

The synthesis of IMP. The color scheme is as follows: enzymes, coenzymes, substrate names, metal ions, inorganic molecules

The biosynthetic origins of purine ring atoms N1 arises from the amine group of Asp C2 and C8 originate from formate N3 and N9 are contributed by the amide group of Gln C4, C5 and N7 are derived from Gly C6 comes from HCO3− (CO2)

The de novo synthesis of purine nucleotides by which these precursors are incorporated into the purine ring proceeds by a 10-step pathway to the branch-point intermediate IMP, the nucleotide of the base hypoxanthine. AMP and GMP are subsequently synthesized from this intermediate via separate, two-step pathways. Thus, purine moieties are initially formed as part of the ribonucleotides rather than as free bases.

Six enzymes take part in IMP synthesis. Three of them are multifunctional:

• GART (reactions 2, 3, and 5)
• PAICS (reactions 6, and 7)
• ATIC (reactions 9, and 10)

The pathway starts with the formation of PRPP. PRPS1 is the enzyme that activates R5P, which is formed primarily by the pentose phosphate pathway, to PRPP by reacting it with ATP. The reaction is unusual in that a pyrophosphoryl group is directly transferred from ATP to C₁ of R5P and that the product has the α configuration about C1. This reaction is also shared with the pathways for the synthesis of Trp, His, and the pyrimidine nucleotides. Being on a major metabolic crossroad and requiring much energy, this reaction is highly regulated.

In the first reaction unique to purine nucleotide biosynthesis, PPAT catalyzes the displacement of PRPP's pyrophosphate group (PP_i) by an amide nitrogen donated from either glutamine (N), glycine (N&C), aspartate (N), folic acid (C₁), or CO₂. This is the committed step in purine synthesis. The reaction occurs with the inversion of configuration about ribose C₁, thereby forming β-5-phosphorybosylamine (5-PRA) and establishing the anomeric form of the future nucleotide.

Next, a glycine is incorporated fueled by ATP hydrolysis and the carboxyl group forms an amine bond to the NH₂ previously introduced. A one-carbon unit from folic acid coenzyme N₁₀-formyl-THF is then added to the amino group of the substituted glycine followed by the closure of the imidazole ring. Next, a second NH₂ group is transferred from a glutamine to the first carbon of the glycine unit. A carboxylation of the second carbon of the glycin unit is concomitantly added. This new carbon is modified by the additional of a third NH₂ unit, this time transferred from an aspartate residue. Finally, a second one-carbon unit from formyl-THF is added to the nitrogen group and the ring covalently closed to form the common purine precursor inosine monophosphate (IMP).

Inosine monophosphate is converted to adenosine monophosphate in two steps. First, GTP hydrolysis fuels the addition of aspartate to IMP by adenylosuccinate synthase, substituting the carbonyl oxygen for a nitrogen and forming the intermediate adenylosuccinate. Fumarate is then cleaved off forming adenosine monophosphate. This step is catalyzed by adenylosuccinate lyase.

Inosine monophosphate is converted to guanosine monophosphate by the oxidation of IMP forming xanthylate, followed by the insertion of an amino group at C₂. NAD⁺ is the electron acceptor in the oxidation reaction. The amide group transfer from glutamine is fueled by ATP hydrolysis.

An unnatural base pair (UBP) is a designed subunit (or nucleobase) of DNA which is created in a laboratory and does not occur in nature. In 2012, a group of American scientists led by Floyd Romesberg, a chemical biologist at the Scripps Research Institute in San Diego, California, published that his team designed an unnatural base pair (UBP).[7] The two new artificial nucleotides or Unnatural Base Pair (UBP) were named d5SICS and dNaM. More technically, these artificial nucleotides bearing hydrophobic nucleobases, feature two fused aromatic rings that form a (d5SICS–dNaM) complex or base pair in DNA.[8][9] In 2014 the same team from the Scripps Research Institute reported that they synthesized a stretch of circular DNA known as a plasmid containing natural T-A and C-G base pairs along with the best-performing UBP Romesberg's laboratory had designed, and inserted it into cells of the common bacterium E. coli that successfully replicated the unnatural base pairs through multiple generations.[10] This is the first known example of a living organism passing along an expanded genetic code to subsequent generations.[8][11] This was in part achieved by the addition of a supportive algal gene that expresses a nucleotide triphosphate transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into E. coli bacteria.[8] Then, the natural bacterial replication pathways use them to accurately replicate the plasmid containing d5SICS–dNaM.

The successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of amino acids which can be encoded by DNA, from the existing 21 amino acids to a theoretically possible 172, thereby expanding the potential for living organisms to produce novel proteins.[10] The artificial strings of DNA do not encode for anything yet, but scientists speculate they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses.[12]

Nucleotide (abbreviated "nt") is a common unit of length for single-stranded nucleic acids, similar to how base pair is a unit of length for double-stranded nucleic acids.

A study done by the Department of Sports Science at the University of Hull in Hull, UK has shown that nucleotides have significant impact on cortisol levels in saliva. Post exercise, the experimental nucleotide group had lower cortisol levels in their blood than the control or the placebo. Additionally, post supplement values of Immunoglobulin A were significantly higher than either the placebo or the control. The study concluded, "nucleotide supplementation blunts the response of the hormones associated with physiological stress."[13]

Another study conducted in 2013 looked at the impact nucleotide supplementation had on the immune system in athletes. In the study, all athletes were male and were highly skilled in taekwondo. Out of the twenty athletes tested, half received a placebo and half received 480 mg per day of nucleotide supplement. After thirty days, the study concluded that nucleotide supplementation may counteract the impairment of the body's immune function after heavy exercise.[14]

The IUPAC has designated the symbols for nucleotides.[15] Apart from the five (A, G, C, T/U) bases, often degenerate bases are used especially for designing PCR primers. These nucleotide codes are listed here. Some primer sequences may also include the character "I", which codes for the non-standard nucleotide inosine. Inosine occurs in tRNAs, and will pair with adenine, cytosine, or thymine. This character does not appear in the following table however, because it does not represent a degeneracy. While inosine can serve a similar function as the degeneracy "D", it is an actual nucleotide, rather than a representation of a mix of nucleotides that covers each possible pairing needed.

• Sigel A, Operschall BP, Sigel H (2017). "Chapter 11. Complex Formation of Lead(II) with Nucleotides and Their Constituents". In Astrid S, Helmut S, Sigel RK (eds.). Lead: Its Effects on Environment and Health. Metal Ions in Life Sciences. 17. de Gruyter. pp. 319–402. doi:10.1515/9783110434330-011. ISBN 9783110434330. PMID 28731304.

• Abbreviations and Symbols for Nucleic Acids, Polynucleotides and their Constituents (IUPAC)
• Provisional Recommendations 2004 (IUPAC)
• Chemistry explanation of nucleotide structure