What is dTTP?

2′-deoxythymidine 5′-triphosphate (or dTTP) is one of the nucleotide bases among three other dNTPs (deoxynucleoside triphosphate)—dATP, dGTP, and dCTP—that are involved in the synthesis of nucleic acids. dTTP is also known as thymidine, deoxythymidine, thymine deoxyriboside, thymidine triphosphate, and deoxy ribothymidine. It is composed of three main components: deoxyribose, thymidine base, and phosphate groups. Its molecular weight is 482.168 g/mol and is chemically formulated as C₁₀H₁₇N₂O₁₄P₃.

One unique feature of dTTP is that it doesn’t have any use in RNA. Because of this, the “deoxy-” is sometimes dropped and they are often referred to as thymidine. The corresponding ribonucleotide triphosphate of the compound, used in RNA synthesis, is uridine triphosphate (UTP).

In double-stranded DNA, thymidine forms two hydrogen bonds with the dATP (deoxyadenosine triphosphate), unlike dCTP and dGTP that form three hydrogen bonds and contribute more to the strength of the structure. Commercially, they are available in a crystalline powder form and are used in a spectrum of lab workflows by making a 10 mM aqueous solution with NaOH (pH in the range of 3-7.5).

This article presents an overview of deoxythymidine triphosphate (dTTP) and covers what it is, its structural characteristics, and its functions and applications.

What Does dTTP Do?

dTTPs are utilized in both labs and living organisms for the formation of the DNA. Some of their use cases are given below:

• They are used in molecular biology labs for several in vitro PCR assays, including real-time PCR, standard PCR amplification, high fidelity PCR, LAMP-PCR, and reverse transcription (RT-PCR).

The PCR workflows are performed by involving a variety of other reagents including Tris buffer, other dNTPs (dNTP mix), Taq DNA polymerase enzymes, primers, template DNA, EDTA, HCl, and some others.

• They are used in cDNA synthesis, DNA labeling, and DNA sequencing.
• In organisms, dTTP acts as a building block in the process of DNA replication.
• dTTP is also involved in the structural formation of tRNA (transfer RNA involved in protein synthesis), specifically in the T-loop.
• dTTP, containing three phosphates, can also be turned into its two other derivatives by losing one or two phosphates: deoxythymidine monophosphate (dTMP, which is thymidine with a single phosphate), and deoxythymidine diphosphate (dTDP, which is thymidine with two phosphates).
• The excess or deficiency of thymidine results in an increase in mutations in the organisms.
• Synchronizing cells at the G1/early S phase is one of thymidine’s functions in cell biology.
• dTTP also has a role as a metabolite in Escherichia coli and mice.

Apart from its involvement in labs and metabolic processes in organisms, there are some modified analogues of thymidine with other applications. These include:

• Tritiated thymidine (3H-TdR) is a radiolabeled thymidine that has application in cell proliferation assays.
• Bromodeoxyuridine (analog of thymidine) is often used to detect proliferating cells in living tissues.
• Edoxudine works as an antiviral drug.
• Azidothymidine (AZT) hinders the process of reverse transcription, thus, has application in HIV treatment.
• 5-Ethynyl-2´-deoxyuridine (EdU) is used to access DNA synthesis in living tissues or cell culture.

In the case of commercially available dTTP, it’s recommended to go through the safety data sheet (SDS) or material safety data sheet (MSDS) for detailed information and quality of the nucleotide. Additionally, ensure that it highlights three points:

• The purity of the dTTP is equal to or more than 99% and tested by HPLC.
• The nucleotide product is free of humans and E.coli DNA.
• The nucleotide product is highly stable.

How Does dTTP Work?

dTTP is an integral structural part of organisms’ genomes. It is involved in the synthesis of DNA and contributes to the functional aspects of the genetic material.

It is incorporated into DNA structure in the process of DNA replication. The process occurs in the 5′-3’ direction and is facilitated by the enzyme DNA polymerase.

The incorporation of dTTP to DNA is similar to other deoxynucleotides. In the replication process, the phosphodiester bond present in dNTPs is cleaved off, which leads to the formation of nucleotide monophosphates.

Then, the nucleotide monophosphate is attached at the 3’ end of the polymerizing DNA strand.

Figure: Structural representation of the formation of hydrogen bonds between dATP & dTTP, and dCTP & dGTP in double stranded DNA.

Synthesis of dTTP

dTTP is synthesized in a variety of ways by different organisms. For example, in Bacillus subtilis, it is synthesized via two pathways:

1. De novo synthesis:

• Thymine synthesis occurs by the formation of an intermediate dUMP (deoxyuridine monophosphate).
• dUMP forms either by deamination of dCTP (deoxycytidine triphosphate) or through phosphorolysis of dUMP, which is the result of dUTP (deoxyuridine triphosphate).
• An enzyme thymidylate synthase causes catalysis of dUMP to synthesize dTMP, which then leads to the formation of dTTP.

2. By converting thymine to thymidine:

• Thymine can be directly converted to thymidine by utilizing deoxyribose-1-phosphate. It is not commonly present in the cell and is only generated in some specific metabolic processes.
• Thymidine is then converted to deoxythymidine monophosphate in the presence of thymidine kinase.

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Figure: An illustration of the pathways of thymine synthesis.

dTTP Structure

Deoxythymidine contains a deoxyribose sugar (pentose sugar) attached to thymine, a pyrimidine (or nitrogenous) base. It possesses three phosphate groups, but can also have one or two phosphates that form deoxythymidine monophosphates and deoxythymidine diphosphates.

Figure: The structure of deoxythymidine triphosphate (dTTP).

It has a phosphate group attached at the 5’ position and is thus also referred to as Thymidine-5′-triphosphate. The chemical name given by IUPAC to the compound based on its chemical composition and structural composition is:

O1-{[(2R,3S,5R)-5-(2,4-Dioxo-5-methyl-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy oxolan-2-yl]methyl} tetrahydrogen triphosphate.

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Deoxythymidine triphosphate is a nucleotide involved in the synthesis of DNA. Structurally, the thymidine molecule consists of a nitrogenous base (thymine), a pentose sugar, and three phosphate groups. It has an array of applications in lab workflows and organisms. Apart from contributing to the structure of DNA, they also act as a metabolite in some living forms.

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