Uniformly Labeled DNA Synthesis Service

Silantes has developed a cost-effective technology for synthesizing stable isotope-labeled DNA oligonucleotides. The enzymatic method differs from the conventional solid-phase synthesis and allows a more efficient conversion of isotope-labeled dNTPs into oligonucleotides, either isotope-labeling the entire oligonucleotide or all positions of a particular base.

Silantes’ enzymatic DNA synthesis method consists of four steps: primer and template design, annealing, synthesis, separation and purification resulting in a high purity DNA sample at a low price.

Individual steps of enzymatic DNA oligonucleotide synthesis

Step 1: Primer and template design

A primer oligonucleotide is designed, which provides the start signal for the enzyme in a later step. Subsequently, a template oligonucleotide complementary to the primer and the product sequence is designed. This template oligonucleotide forms the transcription template for the enzyme.

Step 2: Annealing

The complementary part of the template is annealed to the primer.

Step 3: Synthesis

The enzyme and isotope-labeled dNTPs are now added to the template-primer complex. The enzyme attaches to the primer resulting in product synthesis.

The innovative step: Separation and purification

The isotope-labeled product is now bound in a product-primer-template complex. Purification of this complex presents a challenge due to the risk of destroying the product sequence when separating the complex. To overcome this potential problem, Silantes has developed an innovative approach by modifying the primer and carefully adjusting process parameters during acid hydrolysis. This allows to separate the intact product, primer and template sequence from each other.

The reaction components are then fractionated by HPLC. An exemplary HPLC elution profile is shown in Figure 16. The fraction containing the product sequence is then isolated.

Get your DNA synthesis project started with Silantes

To provide a quote for your DNA synthesis project, we need the following information:

• length of the oligonucleotide,
• sequence of the oligonucleotide,
• specification of isotopic labeling (entire oligonucleotide or all positions of a particular base)
• desired quantity

Based on this information, we prepare a quotation for the synthesis of your DNA fragment. Once an order is placed, purified DNA fragment delivery time is commonly less than 6 weeks.

References

Relevant documents:

• Stable Isotope-Labeled RNA and DNA Building Blocks & Oligonucleotide Synthesis Service at Silantes

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

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