What are Triphosphates? – A comprehensive overview
Triphosphates short for nucleoside triphosphates are the key molecules in cellular metabolism. Nucleoside triphosphate (NTP) consists of a nitrogenous base, a five-carbon sugar, and three phosphate groups that are linked together linear. The nitrogenous base can be adenine, guanine, cytosine, thymine or uracil, while the five-carbon sugar is ribose in RNA or deoxyribose in DNA.
The synthesis of NTP involves several enzymatic reactions that utilize precursors such as adenine triphosphate (ATP) and guanine triphosphate (GTP). The enzymes involved in NTP synthesis include kinases, nucleotidyl transferases, and nucleoside diphosphate kinases. Nucleoside triphosphate serves as the primary energy currency of cells and powers many cellular processes such as muscle contraction, ion transport, and biosynthesis.
The importance of Triphosphates in biotechnology
In biotechnology, nucleoside triphosphates especially modified nucleoside triphosphates are invaluable tools in various applications, such as in Next-Generation Sequencing (NGS) assays to generate cDNA of RNA pools. Furthermore, in DNA sequencing technologies, including Sanger sequencing and Next-Generation Sequencing (NGS), they help to determine the order of nucleotides in DNA.
Modified NTPs like Pseudouridine and N1-methylpseudouridine triphosphates are important nucleotide analogues used in RNA therapy and vaccine development. These modifications improve the efficacy of mRNA drugs by increasing their stability against enzymatic degradation and reducing immunogenicity.
NTPs are also integral to Polymerase Chain Reaction (PCR) and quantitative PCR (qPCR) processes. Additionally, they are used in cell proliferation assays and in Fluorescence In Situ Hybridization (FISH) probes for molecular labeling, demonstrating their broad utility in biotechnology.
Triphosphates in DNA synthesis
Nucleoside triphosphate plays a crucial role in DNA synthesis by providing the energy required for the polymerization via DNA polymerases. Nucleoside triphosphate units consist of a nitrogenous base (cytosine, guanine, adenine or thymine), a pentose sugar (deoxyribose) and three phosphate groups. Each unit is joined by forming a covalent bond between its phosphate group and the 3`OH group of the pentose sugar of the next nucleotide, forming a sugar-phosphate backbone. DNA is a complementary, double-stranded structure because a specific base pairing (adenine and thymine, guanine and cytosine) occurs naturally when hydrogen bonds form between the nucleotide bases.
The role of Triphosphates in polymerase reaction
Triphosphates are essential for the polymerase chain reaction (PCR) process. During PCR, heat-stable DNA polymerase enzymes use NTPs as building blocks to extend a DNA strand. This process is to amplify millions of copies of a very small amount of DNA sequences. Two DNA primers, that are complementary to the 3′ end and the anti-sense strand of the DNA target, serve as the starting point of the PCR. Amplification takes place in a series of thermocycling cycles.
Advantages of Triphosphates in biochemical processes
Energy metabolism: Triphosphates, such as adenosine triphosphate (ATP), are the universal energy currency in all living organisms. They provide energy to drive and support many processes in living cells, such as muscle contraction, nerve impulse propagation, and chemical synthesis.
Nucleotides and nucleic acids: Triphosphates are an essential components of nucleotides, which are the building blocks of nucleic acids like DNA and RNA. They play a important role in our body especially in DNA synthesis and repair, as well as RNA transcription.
Click chemistry: Triphosphates are used in click chemistry, a powerful method for joining molecules quickly and efficiently. This is particularly useful for biological labeling and the development of novel nucleic acid therapeutics.
Redox and fluorescent labeling: Triphosphates are used in redox or fluorescent labeling of biomolecules to study biological structures and interactions. For example, alkyne-modified nucleotide triphosphates can be used in PCR to generate highly functionalized DNA fragments, which can then be conjugated to marker azides for use as multi-labeled primers, FISH probes, or aptamers.
Enzymatic synthesis: Triphosphates are involved in the enzymatic synthesis of nucleotides, which are essential for DNA and RNA polymerases to assemble DNA and RNA molecules.
Antiviral drugs: Triphosphates of nucleoside analogues are active as antiviral drugs. They are used in the development of antiviral therapies by inhibiting viral replication.
Types of Triphosphates and their applications
ATP (adenosine triphosphate): Often referred to as the “energy currency” of the cell. It provides energy for countless cellular reactions, including muscle contractions, the transmission of nerve impulses and chemical synthesis.
CTP (cytidine triphosphate): Plays a role in lipid synthesis and is a precursor in RNA transcription.
GTP (guanosine triphosphate): Essential for protein synthesis and signal transduction pathways.
UTP (uridine triphosphate): Important for carbohydrate metabolism and as a precursor for RNA synthesis.
TTP (thymidine triphosphate): Plays a crucial role in DNA synthesis and cellular metabolism. It is one of the four nucleoside triphosphates used in the synthesis of DNA.
ATP (Adenosine Triphosphate) as the most well-known Triphosphate
ATP, found in all known forms of life, provides energy for several cellular reactions such as muscle contractions, the transmission of nerve impulses and chemical synthesis. When consumed in a metabolic process like walking, ATP converts either to adenosine diphosphate (ADP) or to adenosine monophosphate (AMP). On the other hand, other processes regenerate ATP. It is also a precursor to DNA and RNA, and is used as a coenzyme.
CTP, GTP, TTP and UTP – Other Triphosphates and their functions
Cytidine Triphosphate (CTP):
RNA Synthesis: CTP is one of the four ribonucleotides used as building blocks for RNA synthesis. It is required for the formation of phosphodiester bonds that link individual nucleotides together to create the RNA backbone
Lipid Synthesis: CTP is involved in the synthesis of phospholipids, which are essential components of cell membranes
Protein Sialylation: CTP is linked to the sialylation of proteins, a process important for cell-cell interactions and signaling
Guanosine Triphosphate (GTP):
Protein Synthesis: GTP is essential for protein synthesis as it provides energy for the binding of aminoacyl-tRNA to the ribosome during translation
Signal Transduction: GTP is involved in signal transduction pathways, particularly in the activation of G-proteins, which play a crucial role in transmitting signals from cell surface receptors to intracellular targets
Microtubule Polymerization: GTP is required for the polymerization of microtubules, which are important for cell structure, intracellular transport, and cell division
Thymidine Triphosphate (TTP):
DNA Synthesis: TTP is essential for DNA synthesis, serving as a building block for replication and repair processes. DNA polymerases incorporate TTP into a nascent DNA strand, ensuring the fidelity of genetic information transmission
Energy Source: The triphosphate group of TTP provides the energy needed for forming phosphodiester bonds during DNA polymerization
Uridine Triphosphate (UTP):
RNA Synthesis: UTP is one of the four ribonucleotides used in RNA synthesis. It is incorporated into RNA molecules during transcription
Glycogen Synthesis: UTP is involved in glycogen synthesis, where it forms UDP-glucose, a precursor for glycogen production
Signal Transduction: UTP can act as a signaling molecule, particularly in the regulation of ion channels and other cellular processes
Triphosphates and their role in baseclick products
baseclick utilizes triphosphates in various innovative products and technologies, particularly in the fields of DNA and RNA labeling, sequencing, and diagnostics.
baseclick’s ClickTech PCR Modification Kits contain all the necessary reagents for generating highly labeled PCR products through a two-step method based on click chemistry. In these kits, dTTP is partially replaced by EdUTP depending on the desired labeling rate. EdUTP can be added to the native triphosphate mixtures. This mixture is then used in standard PCR reactions with the optimized baseclick Ethynyl Polymerase. These kits include chemicals for performing multiple PCR and fluorescent labeling reactions, with different dyes available for various applications.
Additionally, baseclick offers a range of modified triphosphates for incorporation in PCR reactions and other applications. For example, C8-Alkyne-dCTP and 5-Ethynyl-dUTP are used for chemo-enzymatic labeling of long oligonucleotides.
3′-Azido-2′,3′-ddCTP is utilized in specialized enzymatic assays based on ClickSeq technologies. These modified triphosphates enable efficient and high-yield fluorescent labeling, making them valuable tools for effective sequencing techniques.
Triphosphates in Baseclick’s labeled nucleotides
BCT-42 Adenosine triphosphate (ATP)
ATP is one of the 4 ribonucleoside triphosphates used in in vitro transcription reactions and RNA polymerase-directed DNA sequencing.
BCT-43 Cytidine triphosphate (CTP)
CTP is one of the 4 ribonucleoside triphosphates used in in vitro transcription reactions and RNA polymerase-directed DNA sequencing.
BCT-44 Guansoine triphosphate (GTP)
GTP is one of the 4 ribonucleoside triphosphates used in in vitro transcription reactions and RNA polymerase-directed DNA sequencing.
BCT-45 Uridine triphosphate (UTP)
UTP is one of the 4 ribonucleoside triphosphates used in in vitro transcription reactions and RNA polymerase-directed DNA sequencing.
C8-Alkyne-dUTP can be used to introduce alkyne groups into DNA, enabling further functionalization through click chemistry. This approach allows for efficient tagging or conjugation, facilitating downstream applications like biomolecule tracking or immobilization.
Similar to C8-Alkyne-dUTP, C8-Alkyne-dCTP introduces alkyne groups into DNA for post-reaction with click chemistry, supporting applications such as DNA labeling and functionalized probe generation.
5-Ethynyl-UTP enables RNA modification, allowing for labeling with marker azides for visualization or cross-linking with biomolecules. This uridine analog, known for its immunosilent properties and prolonged expression, is also a promising candidate in mRNA-based vaccines and therapeutics.
5-Ethynyl-dUTP facilitates DNA modification by incorporating ethynyl groups, ready for click chemistry applications, useful in creating labeled or functionalized DNA fragments.
Alkyne-dATP introduces an alkyne handle into DNA, which can then be labeled through click chemistry. This is ideal for creating modified DNA for research and diagnostic applications.
BCT-23 Pseudouridine-5’-triphosphate
This modified triphosphate enhances mRNA stability and translation efficiency, reducing immunogenicity, making it valuable in mRNA-based drugs and vaccines.
3’-Azido-2’3’-ddATP supports mRNA labeling with azides, allowing modular and 3’-end site-specific modifications, beneficial in creating labeled mRNAs without complex production changes. Also used in ClickSeq’s NGS library preparation kits.
This azide-modified nucleotide enables 3’-end site-specific mRNA labeling, adding flexibility for research applications needing targeted mRNA functionalization. Also used in ClickSeq’s NGS library preparation kits.
Similarly, 3’-Azido-2’3’-ddGTP allows for azide-based labeling of mRNA at the 3’-end, ideal for chemoenzymatic labeling in mRNA studies. Also used in ClickSeq’s NGS library preparation kits.
Like other azide-modified nucleotides, 3’-Azido-2’3’-ddTTP supports mRNA labeling applications, particularly useful for mRNA research and diagnostics. Also used in ClickSeq’s NGS library preparation kits.
2-EATP is valuable for studying ATP-binding proteins and enzymes, and it can be used for polyadenylation and click chemistry-based labeling in RNA studies.
This nucleotide is incorporated at the 3’-end of RNA, allowing alkyne labeling for modular and flexible click chemistry-based RNA functionalization.
Adding 3′-(O-Propargyl)-GTP at the RNA 5’-end creates a cap-like structure for enhanced stability and compatibility with Click Chemistry, suitable for applications needing capped and labeled mRNA.
8-Azido-ATP enables copper-free RNA labeling at nucleobases through SPAAC chemistry, also useful for studying ATP-binding in proteins through UV-induced cross-linking.
2′-Azido-2′-dATP offers modular, chemoenzymatic RNA labeling, allowing flexible alkyne or DBCO-based bioconjugation at the sugar, ideal for creating labeled RNA without complex protocols.
N1-Methylpseudo-UTP significantly improves mRNA stability and reduces immunogenicity, making it an optimal choice for synthesizing stable and effective mRNA therapeutics.
The ARCA cap analog increases mRNA stability and translation efficiency, essential in producing biologically active mRNAs for research and therapeutic use.
CleanCap provides a streamlined capping method for mRNA, yielding cap-1 structures that stabilize mRNA and prevent immune activation, essential in developing efficient mRNA therapeutics.
Phosphoadenylyl- (3′ −> 5′)- guanosine (pApG) is a cap analogs and in vitro transcription (IVT) initiator, improving mRNA stability and translation efficiency. This product opens new possibilities for developing chemically modified mRNAs for various biomedical applications (e.g. circularization), including RNA-based vaccines and gene replacement therapies.
Phosphoadenylyl- (3′ −> 5′)- guanosine (pApG) is a cap analogs and in vitro transcription (IVT) initiator, improving mRNA stability and translation efficiency. This product opens new possibilities for developing chemically modified mRNAs for various biomedical applications, including RNA-based vaccines and gene replacement therapies.
Phosphoadenylyl- (3′ −> 5′)- guanosine (pApG) is a cap analogs and in vitro transcription (IVT) initiator, improving mRNA stability and translation efficiency. This product opens new possibilities for developing chemically modified mRNAs for various biomedical applications (e.g. circularization), including RNA-based vaccines and gene replacement therapies.
Why Triphosphates are essential in biotechnology
DNA and RNA synthesis: Triphosphates such as dATP, dCTP, dGTP, and dTTP are essential building blocks for DNA synthesis, while ATP, CTP, GTP, and UTP are used in RNA synthesis. These molecules provide the necessary energy and substrates for polymerases to assemble nucleic acids, ensuring the accurate replication and transcription of genetic information.
Energy metabolism: Adenosine triphosphate (ATP) is the primary energy currency in all living organisms. It powers various cellular processes, including muscle contraction, nerve impulse propagation, and chemical synthesis.
Signal transduction: Guanosine triphosphate (GTP) is involved in signal transduction pathways, particularly in the activation of G-proteins. These proteins play a critical role in transmitting signals from cell surface receptors to intracellular targets, regulating numerous physiological processes.
Fluorescent labeling: Modified triphosphates, such as alkyne-modified nucleotide triphosphates, are used in fluorescent labeling of biomolecules. This technology enables researchers to study biological structures and interactions, facilitating advancements in molecular biology, diagnostics, and therapeutic development.
Antiviral drugs: Triphosphates of nucleoside analogues are active as antiviral drugs. They inhibit viral replication by incorporating into viral DNA or RNA, making them effective in the treatment of viral infections.
Click chemistry: Triphosphates are used in click chemistry, a powerful method for joining molecules quickly and efficiently. This technique is particularly useful for biological labeling and the development of novel nucleic acid therapeutics.