Uniformly Labeled RNA Synthesis Service

For synthesis of uniformly stable isotope-labeled (2H, 13C, 15N, 18O, 19F) RNA oligonucleotides, Silantes uses the T7 RNA polymerase system for in vitro transcription.

Uniformly labeled RNA oligonucleotides at a reasonable price

The synthesis costs for stable isotope-labeled RNA oligonucleotides are mainly driven by two factors: Stable isotope-labeled rNTPs raw materials and complex purification labor costs.

In order to make stable isotope-labeled RNA reagents affordable, Silantes has optimized the production process:

  • Reasonably priced raw materials: the isotope-labeled rNTPs are made in-house by the company.
  • Low purification costs: efficient synthesis using the T7 RNA polymerase system.

To save costs: The Silantes Feasibility Study

Silantes has a large and growing database of RNA sequences that have already been synthesized. Although RNA synthesis with the T7 RNA Polymerase System has been very successful, there are cases where the synthesis of the desired RNA product was challenging. To minimize the risk of synthesis projects for our customers, we offer the possibility of a prior multi-step feasibility study:

In the first step, the target RNA sequence is validated against Silantes’ internal database. Then the target RNA sequence is screened for potential 3D structures that may complicate synthesis or purification. In the third step, to estimate yields, synthesis of the target RNA sequence is performed on an analytical scale with unlabeled rNTPs.

The project is terminated if any of the above steps were unsuccessful and only the expenses for the feasibility study will be charged.

If the feasibility study is successful, the synthesis of the target RNA sequence is carried out on a preparative scale using isotope-labeled rNTPs. In this case, no costs for the feasibility study will be charged.

Silantes Method I: DNA template as the basis for transcription

T7 is a bacteriophage that produces its own RNA polymerase, which in turn uses its own promoter sequence as a start signal. This T7 promoter has the advantage that it results in very efficient RNA transcription and is not recognized by other RNA polymerases.

Silantes has optimized in vitro transcription with the T7 RNA Polymerase Sytstem and produces highly pure RNA sequences in only 3 steps which are outlined below.

In-vitro RNA synthesis using the T7 RNA Polymerase System with ds DNA template
In-vitro RNA synthesis using the T7 RNA Polymerase System with ds DNA template

Step 1: DNA template preparation

Two complementary DNA single strands are synthesised, both containing the T7 promoter and the target RNA sequence. The two DNA single strands are then annealed. The DNA template forms the basis of transcription for the T7 polymerase.

Step 2: RNA transcription

The DNA template is mixed with the T7 polyermase and Silantes’ isotope-labeled rNTPs. The T7 polyermase recognizes the promoter sequence within the DNA template and transcribes the isotope-labeled target RNA sequence.

Step 3: RNA purification

After transcription, the mixture made of isotope-labeled rNTPs, abortive transcripts, isotope-labeled target RNA sequence and run-off transcripts are fractionated by HPLC to obtain pure isotope-labeled target RNA.

Exemplary HPLC elution profile of 13C15N-labeled r25-mer
Exemplary HPLC elution profile of 13C15N-labeled r25-mer

Limitation of the DNA template as transcription basis

Longer target RNA sequences require longer DNA templates. DNA templates are usually synthesized using solid-phase synthesis. However, the efficiency and quality of solid-phase synthesis decreases significantly as the length of the product increases.

T7 polymerase sometimes adds up to five or six nucleotides at the 3′-end of RNA transcripts. The longer the target RNA sequence, the smaller the relative difference in length between target RNA sequence and run-off transcripts. Due to the small difference in length, run-off transcripts and target RNA are difficult to separate.[1] To keep the cost of purification low, at Silantes synthesizes longer RNA sequences using the T7 polymerase system with ribozyme.

Silantes Method II: Plasmid as the basis for transcription

For long RNA sequences, Silantes uses a linearised plasmid instead of the DNA double-stranded template as the basis for transcription. Unlike long chemically synthesised DNA templates, long plasmids can be amplified in bacteria in a cost-saving and efficient manner.

The linearised plasmid used by Silantes contains information the T7 promoter and target RNA sequences. When the T7 polymerase transcribes the ribozyme at the 3′ end of the plasmid, the ribozyme specifically cuts the product-ribozyme complex between the product and the ribozyme.[2]

In-vitro RNA synthesis using the T7 RNA Polymerase System with plasmid and ribozyme
In-vitro RNA synthesis using the T7 RNA Polymerase System with plasmid and ribozyme

The in vitro synthesis of long RNA sequences using the T7 polymerase system with ribozyme results in very homogeneous 3′-ends of the target RNA sequence. In contrast to the synthesis with a DNA template, no complex and cost intensive purification of run-off transcripts is required. The SDS gel in Figure 11 shows a prominent band of the target RNA sequence.

Exemplary SDS PAGE of 13C15N-labeled r69mer target RNA, plasmid, and ribozyme

[1] Gallo, S., Furler, M. and Sigel, R. K. (2005) “In vitro Transcription and Purification of RNAs of Different Size”, CHIMIA, 59(11), p. 812. doi: 10.2533/000942905777675589.

[2] Gallo, S., Furler, M. and Sigel, R. K. (2005) “In vitro Transcription and Purification of RNAs of Different Size”, CHIMIA, 59(11), p. 812. doi: 10.2533/000942905777675589.

Realize your RNA project with Silantes

If you need a stable isotope-labeled RNA sequence for your project, Silantes can take care of the synthesis work for you. For a quote we need the following information:

Sequence of the target RNA (5’-GG end is required for RNAs. This can be appended to the RNA sequence if necessary)

  • Desired isotope labeling (2H, 13C, 15N, 18O, 19F or combinations thereof) and specification of isotope-labeling (entire oligonucleotide or all positions of a specific base)
  • Desired quantity of target RNA
  • Is purification desired? Depending on the sequence, Silantes experts will decide which method of purification (gel or HPLC) is best suited for your RNA.

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

For quotation inquiries or to get more information please contact us.

References

Relevant documents:

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

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