Commonly Asked Questions About Phosphoramidites
1. What are phosphoramidites used for?
Phosphoramidites are the building blocks for solid-phase synthesis of oligonucleotides—including DNA, RNA, and modified forms. They play a crucial role in making synthetic oligos used in research, diagnostics, and therapeutics.
2. How are phosphoramidites activated in oligonucleotide synthesis?
In oligonucleotide synthesis, phosphoramidites are activated by tetrazole derivatives (like ethylthiotetrazole). This helps them link to the growing nucleotide chain in a process known as coupling.
3. Why do phosphoramidites need protection from a DMT group?
The DMT (dimethoxytrityl) group protects the 5’-hydroxyl group of the nucleoside. This protection prevents unwanted reactions and ensures that each nucleotide is added in the correct order during synthesis.
4. What types of phosphoramidites are available?
Phosphoramidites come in several types, depending on their use:
- DNA phosphoramidites: For synthesizing DNA oligonucleotides.
- RNA phosphoramidites: With 2’-OH protection for RNA synthesis.
- Modified phosphoramidites: Functional groups like fluorophores and biotin can be added.
- Backbone-modifying phosphoramidites: Like phosphorothioate or LNA, which improve stability.
5. What are phosphorothioate phosphoramidites, and why are they important?
Phosphorothioate phosphoramidites replace one oxygen atom with sulfur, making oligonucleotides more nuclease-resistant. This modification is crucial in therapies like antisense and siRNA treatments.
6. What’s the difference between DNA and RNA phosphoramidites?
- DNA phosphoramidites: Deoxyribose sugar (no hydroxyl group at the 2’ position).
- RNA phosphoramidites: Ribose sugar, with a hydroxyl group (-OH) at the 2’ position. This requires special protection (e.g., TBDMS) during synthesis.
7. How stable are phosphoramidites?
Phosphoramidites are sensitive to moisture and can degrade when exposed to air or water. They need to be stored in dry, inert conditions (argon or nitrogen gas) and are usually dissolved in anhydrous solvents before use.
8. What challenges arise when working with phosphoramidites?
- Moisture sensitivity: Requires careful handling and storage.
- Coupling efficiency: Needs optimization to avoid incomplete synthesis.
- Purification: After synthesis, phosphoramidites often need purification to remove side products.
9. Key applications of phosphoramidites?
- Custom oligonucleotide synthesis: For PCR primers, probes, and gene-editing tools.
- Therapeutics: Such as antisense and RNAi therapies.
- Diagnostics: Used in assays like qPCR and fluorescent labeling.
- Peptide synthesis: Introducing phosphate groups for studying protein phosphorylation.
10. How long do phosphoramidites last, and how should they be stored?
Phosphoramidites can last from months to years if stored properly:
- Keep in cool, dry, inert environments (argon or nitrogen gas).
- Store in tightly sealed vials and protect from light.
- Refrigeration can extend shelf life.
11. What are non-nucleoside phosphoramidites, and how are they used?
These phosphoramidites introduce special modifications:
- Fluorophores for labeling.
- Biotin for detection and purification.
- Spacers/linkers like C3 and C6 to create space between functional groups.
12. What are common activators used with phosphoramidites?
- Tetrazole derivatives (e.g., ethylthiotetrazole) are widely used.
- Dicyanoimidazole (DCI) is sometimes employed for even better coupling efficiency.
Phosphoramidites are truly central to modern molecular biology and biotechnology, providing the backbone for custom oligonucleotide synthesis, diagnostics, and cutting-edge therapies!
Ref:
- Synthesis of Folate-Labeled siRNAs: This study explores the use of a novel folate-derivative phosphoramidite for siRNA synthesis. The folate labeling allows targeted delivery to folate receptor-expressing cancer cells, enhancing the potential of RNAi-based cancer therapies(RSC Publishing).
- Novel Phosphoramidite Building Blocks: This research highlights recent advancements in modified nucleoside phosphoramidites, which are used to create oligonucleotides with enhanced stability, nuclease resistance, and therapeutic potential. These modifications are critical for improving drug delivery and effectiveness in gene therapies(Welcome to Bentham Science Publisher).
- Green Innovations in Oligonucleotide Synthesis: This review covers the use of phosphoramidite chemistry in the context of more sustainable, green synthesis practices for oligonucleotides. It also discusses how phosphoramidites remain essential for rapid and efficient DNA synthesis(RSC Publishing).
- Chemical Synthesis of Oligonucleotide Sequences: This comprehensive review published in 2023 outlines the continued importance of phosphoramidites in DNA and RNA oligonucleotide synthesis, with applications ranging from gene editing to diagnostics(SpringerLink).