In our previous post, we discussed an overview of what stable isotope-labeled nucleic acids are, but in order to successfully study nucleic acids in a wide range of experimental needs, synthesizing stable isotope-labeled nucleic acids becomes the next challenge.
In this article, we take a look at the different methods in which stable isotope-labeled nucleic acids can be synthesized, and how we do it here at Silantes.
Metabolic Labeling
The process of synthesizing stable isotope-labeled RNA typically involves in vitro transcription reactions, wherein labeled rNTPs are used as substrates by RNA polymerases to generate the desired RNA molecules.
This method allows for the introduction of isotopic labels at specific positions or uniformly throughout the RNA molecule, depending on the choice of labeled rNTPs. Similarly, the synthesis of stable isotope-labeled DNA can be achieved through primer extension or PCR amplification using labeled dNTPs. The incorporation of labeled dNTPs results in the generation of DNA molecules with defined isotopic signatures.
This is how we convert isotope-labeled molecules into complex organic molecules using our custom technology here at Silantes.
Chemical Synthesis
An alternative approach to synthesizing labeled nucleic acids is through solid-phase chemical synthesis using phosphoramidite chemistry for the controlled means of creating custom-labeled oligonucleotides with specific sequences and modifications.
Site Specific Labeling
At Silantes, we offer site-specific labeled phosphoramidites.
The site-specific labeling patterns are chosen to create independent spin systems either intramolecularly within the nucleotide, or intermolecularly to other molecules, providing high quality NMR information.
This method is particularly useful for the synthesis of short oligonucleotides with specific sequences and modifications, as well as for the incorporation of the fluorine atom, which is significant in the context of 19F NMR spectroscopy studies.
Enzymatic Incorporation
Enzymatic incorporation offers a highly precise way to introduce labeled nucleotides into nucleic acids in vitro.
Enzymes, such as polymerases, are used to incorporate stable isotope-labeled nucleotides during primer extension, in vitro transcription, or DNA replication assays. The high fidelity and processivity of enzymatic incorporation make it ideal for generating long labeled nucleic acids for various applications.
Uniform Labeling
Our uniformly stable isotope-labeled phosphoramidites are synthesized using a combination of biotechnological and chemical steps. In the first step, we obtain nucleosides from 2H, 13C and/or 15N-labeled bacterial biomass. In a second step, these nucleosides are chemically protected to obtain the phosphoramidites.
The enzymatic approach stands apart from traditional solid-phase synthesis, offering a more effective way to convert isotope-labeled dNTPs into oligonucleotides.
Introducing SILAC
SILAC (Stable Isotope Labeling by Amino acids in Cell culture) is a widely used technique in molecular biology that involves the incorporation of stable isotopes into proteins. Although SILAC primarily focuses on protein labeling, it indirectly contributes to the synthesis of stable isotope-labeled nucleic acids.
In SILAC, cells are cultured in a growth medium containing amino acids labeled with stable isotopes such as 13C and 15N. These isotopically labeled amino acids are metabolically incorporated into the newly synthesized proteins of the cells. The incorporation of stable isotopes allows for the discrimination between labeled and unlabeled proteins during subsequent analyses.
SILAC has significant implications for studying nucleic acids because proteins play critical roles in nucleic acid metabolism, DNA replication, transcription, and translation. By accurately labeling proteins, SILAC enables the precise tracking and analysis of protein-nucleic acid interactions. This, in turn, provides valuable insights into the function, regulation, and dynamics of nucleic acids within the cellular environment.
SILAC can be coupled with various downstream techniques to investigate specific aspects of nucleic acids. For example, by combining SILAC with chromatin immunoprecipitation (ChIP), researchers can identify protein binding sites on chromatin and determine their influence on gene expression. This approach helps unravel the complex network of interactions between proteins and nucleic acids in the context of chromatin structure and epigenetic regulation.
Furthermore, SILAC-based quantitative proteomics allows for the identification and characterization of nucleic acid-associated proteins, including transcription factors, polymerases, and chromatin modifiers. By comparing protein expression levels between different experimental conditions, researchers can uncover key players involved in nucleic acid-related processes and dissect their roles in cellular pathways.
But regardless of the synthetic method employed, the resulting stable isotope-labeled nucleic acids serve as invaluable tools for molecular biology research. They offer researchers the ability to study nucleic acid structure, dynamics, and interactions with other biomolecules in unparalleled detail.
Through the use of isotope labeling, scientists can gain deeper insights into the complex molecular mechanisms that govern gene expression, regulation, and the functional roles of nucleic acids within living organisms.
How Do You Synthesize Stable Isotope-Labeled Nucleic Acids?
Synthesizing stable isotope-labeled nucleic acids involves using stable isotopes like carbon-13 (13C) or nitrogen-15 (15N) in labeled precursors (e.g., glucose or ammonium salts). Microorganisms metabolize these precursors, incorporating the isotopes into their nucleotides. These labeled nucleotides are extracted, purified, and used for the enzymatic synthesis of labeled DNA or RNA, effectively producing stable isotope-labeled nucleic acids
Why is Isotope Labeling Important?
Isotope labeling is vital as it allows scientists to trace the pathways and interactions of molecules within biological systems. This method uses isotopes as ‘molecular tags’ to track how a molecule behaves, its location, and its role in biological processes such as metabolism, gene expression, or DNA replication. The non-radioactive nature of stable isotopes allows these studies to be conducted in living organisms, providing real-time observations of these processes in their natural context.
About Silantes
At Silantes, we are experts in producing a wide range of stable isotope labeled molecules.
We are excited to offer you a product line that is truly unrivaled in the industry. Whether you need isotope-labeled RNA and DNA building blocks (NTPs and phosphoramidites) or top-notch services for oligonucleotide synthesis.
For RNA and DNA technology products and services, our existing customers choose us because:
- we are uniquely equipped to produce a range of labeling that ensures high purity, and lower costs than our competitors
- of our extensive knowledge of the special requirements for handling stable isotope-labeled reagents in oligonucleotide synthesis
- of our ability to provide optimisation of oligonucleotide synthesis services with respect to minimal use of stable isotopes and small sample sizes.
For researchers and industry professionals interested in harnessing the power of custom RNA/DNA synthesis and stable isotope labeling, we invite you to take the next steps in accessing these services.
Whether you have a specific research project in mind or are simply curious about the possibilities, we encourage you to reach out for more information and explore the services available to you.
Want to learn more about stable isotope-labeled nucleic acids? Read our follow up articles:
- 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
Alternatively, you can request the pdf version of all of these articles to read at your convenience:
