Nucleotide

Not to be confused with nucleoside or nucleobase.

Nucleotides are organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules within all life-forms on Earth. Nucleotides are obtained in the diet and are also synthesized from common nutrients by the liver.^[1]

This nucleotide contains the five-carbon sugar deoxyribose (at center), a nucleobase called adenine (upper right), and one phosphate group (left). The deoxyribose sugar joined only to the nitrogenous base forms a Deoxyribonucleoside called deoxyadenosine, whereas the whole structure along with the phosphate group is a nucleotide, a constituent of DNA with the name deoxyadenosine monophosphate.

Nucleotides are composed of three subunit molecules: a nucleobase, a five-carbon sugar (ribose or deoxyribose), and a phosphate group consisting of one to three phosphates. The four nucleobases in DNA are guanine, adenine, cytosine and thymine; in RNA, uracil is used in place of thymine.

Nucleotides also play a central role in metabolism at a fundamental, cellular level. They provide chemical energy—in the form of the nucleoside triphosphates, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP)—throughout the cell for the many cellular functions that demand energy, including: amino acid, protein and cell membrane synthesis, moving the cell and cell parts (both internally and intercellularly), cell division, etc.^[2] In addition, nucleotides participate in cell signaling (cyclic guanosine monophosphate or cGMP and cyclic adenosine monophosphate or cAMP), and are incorporated into important cofactors of enzymatic reactions (e.g. coenzyme A, FAD, FMN, NAD, and NADP⁺).

In experimental biochemistry, nucleotides can be radiolabeled using radionuclides to yield radionucleotides.

5-nucleotides are also used in flavour enhancers as food additive to enhance the umami taste, often in the form of a yeast extract.^[3]

Structure

 

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 the 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 nucleic acid, shown at upper left.

A nucleotide is composed of three distinctive chemical sub-units: a five-carbon sugar molecule, a nucleobase (the two of which 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.

In nucleic acids, nucleotides contain either a purine or a pyrimidine base—i.e., the nucleobase molecule, also known as a nitrogenous base—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.

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.

In addition to being building blocks for the construction of nucleic acid polymers, singular nucleotides play roles in cellular energy storage and provision, cellular signaling, as a source of phosphate groups used to modulate the activity of proteins and other signaling molecules, and as enzymatic cofactors, often carrying out redox reactions. Signaling cyclic nucleotides are formed by binding the phosphate group twice to the same sugar molecule, bridging the 5'- and 3'- hydroxyl groups of the sugar.^[2] Some signaling nucleotides differ from the standard single-phosphate group configuration, in having multiple phosphate groups attached to different positions on the sugar.^[4] Nucleotide cofactors include a wider range of chemical groups attached to the sugar via the glycosidic bond, including nicotinamide and flavin, and in the latter case, the ribose sugar is linear rather than forming the ring seen in other nucleotides.

 

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).

Examples of non-nucleic acid nucleotides

•  

  cAMP, a cyclic nucleotide signaling molecule with a single phosphate linked to both 5- and 3-positions.

•  

  pppGpp, a nucleotide signaling molecule with both 5'- and 3'-phosphates.

•  

  NADP, a dinucleotide enzymatic cofactor.

•  

  FAD, a dinucleotide enzymatic cofactor in which one of the ribose sugars adopts a linear configuration rather than a ring.

Synthesis

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.^[1] 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. Recently it has been also demonstrated that cellular bicarbonate metabolism can be regulated by mTORC1 signaling.^[5] 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

Main article: Pyrimidine metabolism

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^[6] 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 from 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 the 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.^[7][8]

Purine ribonucleotide synthesis

Main article: Purine metabolism

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 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 addition 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 is 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.

Pyrimidine and purine degradation

In humans, pyrimidine rings (C, T, U) can be degraded completely to CO₂ and NH₃ (urea excretion). That having been said, purine rings (G, A) cannot. Instead, they are degraded to the metabolically inert uric acid which is then excreted from the body. Uric acid is formed when GMP is split into the base guanine and ribose. Guanine is deaminated to xanthine which in turn is oxidized to uric acid. This last reaction is irreversible. Similarly, uric acid can be formed when AMP is deaminated to IMP from which the ribose unit is removed to form hypoxanthine. Hypoxanthine is oxidized to xanthine and finally to uric acid. Instead of uric acid secretion, guanine and IMP can be used for recycling purposes and nucleic acid synthesis in the presence of PRPP and aspartate (NH₃ donor).

Prebiotic synthesis of nucleotides

Theories about the origin of life require knowledge of chemical pathways that permit formation of life’s key building blocks under plausible prebiotic conditions. The RNA world hypothesis holds that in the primordial soup there existed free-floating ribonucleotides, the fundamental molecules that combine in series to form RNA. Complex molecules like RNA must have arisen from small molecules whose reactivity was governed by physico-chemical processes. RNA is composed of purine and pyrimidine nucleotides, both of which are necessary for reliable information transfer, and thus Darwinian evolution. Becker et al. showed how pyrimidine nucleosides can be synthesized from small molecules and ribose, driven solely by wet-dry cycles.^[9] Purine nucleosides can be synthesized by a similar pathway. 5’-mono- and di-phosphates also form selectively from phosphate-containing minerals, allowing concurrent formation of polyribonucleotides with both the purine and pyrimidine bases. Thus a reaction network towards the purine and pyrimidine RNA building blocks can be established starting from simple atmospheric or volcanic molecules.^[9]

Unnatural base pair (UBP)

Main article: Base pair § Unnatural base pair (UBP)

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.^[10] Examples include d5SICS and dNaM. These artificial nucleotides bearing hydrophobic nucleobases, feature two fused aromatic rings that form a (d5SICS–dNaM) complex or base pair in DNA.^[11][12] E. coli have been induced to replicate a plasmid containing UBPs through multiple generations.^[13] This is the first known example of a living organism passing along an expanded genetic code to subsequent generations.^[11][14]

Medical applications of synthetic nucleotides

Several nucleotide derivatives have been used as antivirals against hepatitis and HIV.^[15][16] Tenofovir disoproxil, Tenofovir alafenamide and Sofosbuvir are examples of NRTI used against hepatitis. Whereas certain drugs like Mericitabine, Lamivudine, Entecavir and Telbivudine for example are nucleosides, but they are metabolized into their bioactive nucleotide forms through phosphorylation.

Length unit

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.

Abbreviation codes for degenerate bases

Main article: Nucleic acid notation

The IUPAC has designated the symbols for nucleotides.^[17] 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.

Symbol^[17]	Description	Bases represented
A	adenine	A				1
C	cytosine		C
G	guanine			G
T	thymine				T
U	uracil				U
W	weak	A			T	2
S	strong		C	G
M	amino	A	C
K	keto			G	T
R	purine	A		G
Y	pyrimidine		C		T
B	not A (B comes after A)		C	G	T	3
D	not C (D comes after C)	A		G	T
H	not G (H comes after G)	A	C		T
V	not T (V comes after T and U)	A	C	G
N	any base (not a gap)	A	C	G	T	4

See also

•  Biology portal

• Biology
• Chromosome
• Gene
• Genetics
• Nucleic acid analogue – Compound analogous to naturally occurring RNA and DNA
• Nucleic acid sequence – Succession of nucleotides in a nucleic acid
• Nucleobase – Nitrogen-containing biological compounds that form nucleosides

References

1. Zaharevitz DW, Anderson LW, Malinowski NM, Hyman R, Strong JM, Cysyk RL (November 1992). "Contribution of de-novo and salvage synthesis to the uracil nucleotide pool in mouse tissues and tumors in vivo". European Journal of Biochemistry. 210 (1): 293–6. doi:10.1111/j.1432-1033.1992.tb17420.x. PMID 1446677.
2. Alberts B, Johnson A, Lewis J, Raff M, Roberts K & Walter P (2002). Molecular Biology of the Cell (4th ed.). Garland Science. ISBN 0-8153-3218-1. pp. 120–121.
3. Abd El-Aleem, Fatma Sh; Taher, Mohamed S.; Lotfy, Shereen N.; El-Massry, Khaled F.; Fadel, Hoda H. M. (2017-12-18). "Influence of extracted 5-nucleotides on aroma compounds and flavour acceptability of real beef soup". International Journal of Food Properties. 20 (sup1): S1182–S1194. doi:10.1080/10942912.2017.1286506. S2CID 100497537.
4. Smith, A. D., ed. (2000). Oxford Dictionary of Biochemistry and Molecular Biology, Revised edition. Oxford: Oxford University Press. p. 460.
5. Ali E, Liponska A, O'Hara B, Amici D, Torno M, Gao P, Asara J, Yap M-N F, Mendillo M, Ben-Sahra I (June 2022). "The mTORC1-SLC4A7 axis stimulates bicarbonate import to enhance de novo nucleotide synthesis". Molecular Cell. 82 (1): 3284–3298.e7. doi:10.1016/j.molcel.2022.06.008. PMC 9444906. PMID 35772404.
6. See IUPAC nomenclature of organic chemistry for details on carbon residue numbering
7. Jones ME (1980). "Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and regulation of UMP biosynthesis". Annual Review of Biochemistry. 49 (1): 253–79. doi:10.1146/annurev.bi.49.070180.001345. PMID 6105839.
8. McMurry JE, Begley TP (2005). The organic chemistry of biological pathways. Roberts & Company. ISBN 978-0-9747077-1-6.
9. Becker, Sidney; Feldmann, Jonas; Wiedemann, Stefan; Okamura, Hidenori; Schneider, Christina; Iwan, Katharina; Crisp, Antony; Rossa, Martin; Amatov, Tynchtyk; Carell, Thomas (2019-10-04). "Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides" (PDF). Science. 366 (6461): 76–82. Bibcode:2019Sci...366...76B. doi:10.1126/science.aax2747. PMID 31604305. S2CID 203719976.
10. Malyshev DA, Dhami K, Quach HT, Lavergne T, Ordoukhanian P, Torkamani A, Romesberg FE (July 2012). "Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet". Proceedings of the National Academy of Sciences of the United States of America. 109 (30): 12005–10. Bibcode:2012PNAS..10912005M. doi:10.1073/pnas.1205176109. PMC 3409741. PMID 22773812.
11. Malyshev DA, Dhami K, Lavergne T, Chen T, Dai N, Foster JM, Corrêa IR, Romesberg FE (May 2014). "A semi-synthetic organism with an expanded genetic alphabet". Nature. 509 (7500): 385–8. Bibcode:2014Natur.509..385M. doi:10.1038/nature13314. PMC 4058825. PMID 24805238.
12. Callaway E (May 7, 2014). "Scientists Create First Living Organism With 'Artificial' DNA". Nature News. Huffington Post. Retrieved 8 May 2014.
13. Fikes BJ (May 8, 2014). "Life engineered with expanded genetic code". San Diego Union Tribune. Retrieved 8 May 2014.
14. Sample I (May 7, 2014). "First life forms to pass on artificial DNA engineered by US scientists". The Guardian. Retrieved 8 May 2014.
15. Ramesh, Deepthi; Vijayakumar, Balaji Gowrivel; Kannan, Tharanikkarasu (December 2020). "Therapeutic potential of uracil and its derivatives in countering pathogenic and physiological disorders". European Journal of Medicinal Chemistry. 207: 112801. doi:10.1016/j.ejmech.2020.112801. PMID 32927231. S2CID 221724578.
16. Ramesh, Deepthi; Vijayakumar, Balaji Gowrivel; Kannan, Tharanikkarasu (12 February 2021). . ChemMedChem. 16 (9): 1403–1419. doi:10.1002/cmdc.202000849. PMID 33427377. S2CID 231576801. Archived from the original on 14 December 2021. Retrieved 13 March 2021.
17. Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1984). "Nomenclature for Incompletely Specified Bases in Nucleic Acid Sequences". Retrieved 2008-02-04.

Further reading

• 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. Vol. 17. de Gruyter. pp. 319–402. doi:10.1515/9783110434330-011. ISBN 9783110434330. PMID 28731304.
• Freisinger, E., & Sigel, R. K. (2007). From nucleotides to ribozymes—a comparison of their metal ion binding properties. Coordination Chemistry Reviews, 251(13-14), 1834-1851.

External links

• Abbreviations and Symbols for Nucleic Acids, Polynucleotides and their Constituents (IUPAC)
• (IUPAC)