rNTPs and dNTPs - Using Stable Isotope Labeled Nucleotides in RNA and DNA Synthesis

Stable isotope labeling is the incorporation of stable (non-radioactive) isotopes into molecules, a technique used in molecular biology and biochemistry. By replacing specific atoms in a nucleotide or nucleoside with corresponding stable isotopes, scientists can analyze the structure of a molecule and trace the path of a molecule and its interactions within a biological system. Nucleotide labeling acts as molecular “tags” and provides invaluable information about biochemical processes such as metabolism, DNA replication, RNA transcribing, and the interaction of proteins with nucleic acids.

Isotope-Labeled rNTPs and dNTPs for RNA and DNA

Stable isotope-labeled ribo-nucleoside triphosphates (rNTPs) and deoxyribo-nucleoside triphosphates (dNTPs) are essential components of various research applications, enabling accurate NMR measurements, quantification by mass spectrometry and metabolic tracking. These labeled nucleotides play a critical role in studies involving the synthesis of nucleotides, the replication of DNA, and the transcription of RNA.

Structural studies on large DNA and RNA molecules by nuclear magnetic resonance (NMR) require labeling of these molecules with stable isotopes for detection. Some of the stable isotopes that are commonly used in molecular biology are typically ²H, ¹³C, ¹⁵N, ¹⁸O and ¹⁹F. These isotopes have the same chemical properties as their more abundant analogs, but can be readily distinguished by their atomic mass using techniques such as NMR and mass spectrometry (MS).

Silantes Stable Isotope Labeled Nucleotides

Silantes offers a wide range of stable isotope-labeled ribo- and deoxyribo-nucleoside triphosphates in many combinations of the stable isotopes. On our web site you will find a selection of the most requested products in any combination of the stable isotopes ²H, ¹³C, ¹⁵N, ¹⁸O and ¹⁹F for the enzymatic synthesis of oligonucleotides.

The isotopic purity of all products is >98 atom%. Site specifically labeled nucleotides and modifications are available on request. Please contact our customer service to see the complete product list or for special requests.

Selected products in this field (complete product range at bottom of page):

Silantes Technology for Stable Isotope Labeled rNTPs and dNTPs

Silantes stable isotope-labelled nucleotides are prepared from bacterial DNA and RNA. The strain is a chemolithoautotrophic organism which grows on H₂, O₂ and CO₂. The extracted DNA or RNA is enzymatically hydrolysed. The isolated 5’-NMPs are enzymatically phosphorylated to 5’-NDPs and 5’-NTPs and purified by IC- and RP-HPLC.

This biotechnological process results in the following stable isotope-labeled products, among others:

• Ribo-nucleosides (rNs) – Products and Prices
• Deoxyribo-nucleosides (dNs) – Products and Prices
• Ribo-nucleoside monophosphates (rNMPs) – Products and Prices
• Modified ribo-nucleoside monophosphates (rNMPs) – Products and Prices
• Deoxyribo-nucleoside monophosphates (dNMPs) – Products and Prices
• Ribo-nucleoside diphosphates (rNDPs) – Products and Prices
• Deoxyribo-nucleoside diphosphates (dNDPs) – Products and Prices
• Ribo-nucleoside triphosphates (rNTPs)
• Fluoro-ribo-nucleoside triphosphates (rNTPs) – Products and Prices
• Ribo-nucleoside triphosphates (rNTPs) with special labeling patterns – and
• Deoxyribo-nucleoside triphosphates (dNTPs)
• Nucleoside building blocks – Products and Prices

In vivo enrichment technology of biomass with stable isotopes results in:

• efficient incorporation of the stable isotopes and thus cost-effectively labelled rNTPs and dNTPs.
• high and homogeneous isotope labelling (> 98 atom %) of the nucleotides by using a closed system.

High Quality Commitment

We guarantee an isotopic enrichment of >98 atom % with a chemical purity of >95 % determined by HPLC. This figure shows an example of the HPLC elution profile of rCTP.

In addition, the biological competence, i.e., the suitability of our rNTPs and dNTPs in enzymatic oligosyntheses, is validated by in vivo RNA and DNA syntheses, respectively.

FAQs

What techniques are used for the detection of stable isotope-labeled nucleic acids?

Stable isotope-labeled nucleic acids can be detected and analyzed using various techniques, including nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). NMR is particularly useful for structural studies of large DNA and RNA molecules, while MS is employed for quantification and metabolic tracking of labeled nucleotides.

What are alternative labeling methods for nucleotides and nucleic acids?

Some alternative labeling methods for nucleotides and nucleic acids include stable isotope labeling, fluorescent labeling, biotin labeling, and hapten labeling. Each method has its advantages and applications in molecular biology and biochemistry research.

What are the differences between stable isotope labeling and fluorescent labeling?

Stable isotope labeling involves incorporating non-radioactive isotopes (e.g., 13C, 15N, 2H) into nucleotides or nucleic acids, allowing for quantitative analysis and structural studies using techniques like NMR and mass spectrometry. In contrast, fluorescent labeling involves attaching fluorescent dyes or fluorophores to nucleotides or nucleic acids, enabling visualization and detection using fluorescence-based methods.

References

Use cases of the Silantes NTPs in scientific publications:

• Mieczkowski, M., Steinmetzger, C., Bessi, I., Lenz, A., Schmiedel, A., Holzapfel, M., Lambert, C., Pena, V., & Höbartner, C. (2021). Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine. Nature Communications, 12(1). https://doi.org/10.1038/s41467-021-23932-0
• Musheev, M. U., Schomacher, L., Basu, A., Han, D., Krebs, L., Scholz, C., & Niehrs, C. (2022). Mammalian N1-adenosine PARylation is a reversible DNA modification. Nature Communications, 13(1). https://doi.org/10.1038/s41467-022-33731-w
• Xu, Y., McSally, J., Andricioaei, I., & Al-Hashimi, H. M. (2018). Modulation of Hoogsteen dynamics on DNA recognition. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-03516-1
• Li, M., Wang, Y., Wei, X., Cai, W., Wu, J., Zhu, M., Wang, Y., Liu, Y., Xiong, J., Qu, Q., Chen, Y., Tian, X., Yao, L., Xie, R., Li, X., Chen, S., Huang, X., Zhang, C., Xie, C., . . . Lin, S. (2024). AMPK targets PDZD8 to trigger carbon source shift from glucose to glutamine. Cell Research. https://doi.org/10.1038/s41422-024-00985-6
• Cromsigt, J., Schleucher, J., Gustafsson, T., Kihlberg, J., & Wijmenga, S. (2002). Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5’’ in nucleotides. Nucleic Acids Research, 30(7), 1639–1645. https://doi.org/10.1093/nar/30.7.1639
• Rangadurai, A., Szymanski, E. S., Kimsey, I., Shi, H., & Al-Hashimi, H. M. (2020). Probing conformational transitions towards mutagenic Watson–Crick-like G·T mismatches using off-resonance sugar carbon R1ρ relaxation dispersion. Journal of Biomolecular NMR, 74(8–9), 457–471. https://doi.org/10.1007/s10858-020-00337-7
• Noeske, J., Richter, C., Grundl, M. A., Nasiri, H. R., Schwalbe, H., & Wöhnert, J. (2005). An intermolecular base triple as the basis of ligand specificity and affinity in the guanine- and adenine-sensing riboswitch RNAs. Proceedings of the National Academy of Sciences, 102(5), 1372–1377. https://doi.org/10.1073/pnas.0406347102
• Ohira, T., Minowa, K., Sugiyama, K., Yamashita, S., Sakaguchi, Y., Miyauchi, K., Noguchi, R., Kaneko, A., Orita, I., Fukui, T., Tomita, K., & Suzuki, T. (2022). Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance. Nature, 605(7909), 372–379. https://doi.org/10.1038/s41586-022-04677-2
• Vögele, J., Duchardt-Ferner, E., Bains, J. K., Knezic, B., Wacker, A., Sich, C., Weigand, J. E., Šponer, J., Schwalbe, H., Krepl, M., & Wöhnert, J. (2024). Structure of an internal loop motif with three consecutive U•U mismatches from stem–loop 1 in the 3′-UTR of the SARS-CoV-2 genomic RNA. Nucleic Acids Research, 52(11), 6687–6706. https://doi.org/10.1093/nar/gkae349
• Broft, P., Rosenkranz, R. R., Schleiff, E., Hengesbach, M., & Schwalbe, H. (2022). Structural analysis of temperature-dependent alternative splicing of HsfA2 pre-mRNA from tomato plants. RNA Biology, 19(1), 266–278. https://doi.org/10.1080/15476286.2021.2024034

Use cases of the Silantes phosphoramidites in scientific publications:

• Becette, O., Olenginski, L. T., & Dayie, T. K. (2019). Solid-Phase chemical synthesis of stable Isotope-Labeled RNA to aid structure and dynamics studies by NMR spectroscopy. Molecules, 24(19), 3476. https://doi.org/10.3390/molecules24193476
• Štih, V., Amenitsch, H., Plavec, J., & Podbevšek, P. (2023). Spatial arrangement of functional domains in OxyS stress response sRNA. RNA, 29(10), 1520–1534. https://doi.org/10.1261/rna.079618.123

Use cases of the Silantes oligonucleotide synthesis service in scientific publications:

• Belfetmi, A., Zargarian, L., Tisné, C., Sleiman, D., Morellet, N., Lescop, E., Maskri, O., René, B., Mély, Y., Fosse, P., & Mauffret, O. (2016). Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein. RNA, 22(4), 506–517. https://doi.org/10.1261/rna.054445.115
• Borggräfe, J., Victor, J., Rosenbach, H., Viegas, A., Gertzen, C. G. W., Wuebben, C., … Etzkorn, M. (2021). Time-resolved structural analysis of an RNA-cleaving DNA catalyst. Nature, 601(7891), 144–149. https://doi.org/10.1038/s41586-021-04225-4
• Chernatynskaya, A. V., Deleeuw, L., Trent, J. O., Brown, T., & Lane, A. N. (2009). Structural analysis of the DNA target site and its interaction with Mbp1. Organic & Biomolecular Chemistry, 7(23), 4981. https://doi.org/10.1039/b912309a
• Van Melckebeke, H., Devany, M., Di Primo, C., Beaurain, F., Toulmé, J., Bryce, D. L., & Boisbouvier, J. (2008). Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex. Proceedings of the National Academy of Sciences, 105(27), 9210–9215. https://doi.org/10.1073/pnas.0712121105

Use cases of the Silantes 14-mer RNA Standard in scientific publications:

• Duchardt, E., & Schwalbe, H. (2005). Residue Specific Ribose and Nucleobase Dynamics of the cUUCGg RNA Tetraloop Motif by MNMR 13C Relaxation. Journal of Biomolecular NMR, 32(4), 295–308. https://doi.org/10.1007/s10858-005-0659-x
• Hartlmüller, C., Günther, J. C., Wolter, A. C., Wöhnert, J., Sattler, M., & Madl, T. (2017). RNA structure refinement using NMR solvent accessibility data. Scientific Reports, 7(1). https://doi.org/10.1038/s41598-017-05821-z
• Nozinovic, S., Fürtig, B., Jonker, H. R. A., Richter, C., & Schwalbe, H. (2009). High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA. Nucleic Acids Research, 38(2), 683–694. https://doi.org/10.1093/nar/gkp956
• Richter, C., Kovacs, H., Buck, J., Wacker, A., Fürtig, B., Bermel, W., & Schwalbe, H. (2010). 13C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides. Journal of Biomolecular NMR, 47(4), 259–269. https://doi.org/10.1007/s10858-010-9429-5
• Ferner, J., Villa, A., Duchardt, E., Widjajakusuma, E., Wöhnert, J., Stock, G., & Schwalbe, H. (2008). NMR and MD studies of the temperature-dependent dynamics of RNA YNMG-tetraloops. Nucleic Acids Research, 36(6), 1928–1940. https://doi.org/10.1093/nar/gkm1183

Relevant blog articles:

• What Are Stable-Isotope Labeled Nucleic Acids?
• Synthesizing Stable Isotope-Labeled Nucleic Acids
• The Advantages of Using Stable Isotope-Labeled Nucleic Acids
• Applications of Stable Isotope-Labeled Molecules: Exploring the Power of Isotopic Tracers
• Custom RNA & DNA Synthesis Services : Tailored Solutions for Your Nucleic Acid Needs

Relevant webinars:

• Easy 13C, 15N-Labeling of DNA through isothermal amplification: Applications to G-quadruplex, aptamer, and DNAzyme