What are the common RNA and DNA modification reagents?
Chemical modification of RNA and DNA is widely used in biological research, drug development and diagnostic technology. The following are the common modifiers and their applications:
DNA modifying reagents
1. Methylating reagents
5-Azacytidine: DNA methyltransferase inhibitor for demethylation studies.
- SAM (S-adenosylmethionine): Natural methyl donor for methylation reactions.
2. Fluorescence labeling
- FAM (6-carboxyfluorescein), Cy3/Cy5: for fluorescent probe labeling.
Biotin-dUTP: Biotin-labeled for affinity purification or detection.
3. Crosslinking agent
Formaldehyde: used for DNA-protein cross-linking (ChIP assay).
- Psoralen: UV-activated crosslinking agent for DNA double-stranded crosslinking.
4. Nucleotide analogs
- BrdU (5-bromo-2 '-deoxyuridine): for cell proliferation assay.
- EdU (5-ethynyl-2 '-deoxyuridine): Click on the chemical label (more efficient than BrdU).
5. Other modifications
- DMS (dimethyl sulfate): for DNA methylation footprint analysis.
RNA modification reagents
1. Methylation-related
- N6-methyladenosine (m6A) reagents: such as SAM (methyl donor), METTL3/METTL14 (methyltransferase complex).
2. Fluorescence labeling
- FITC-UTP: incorporation of fluorescently labeled RNA during in vitro transcription.
- Alexa Fluor-labeled nucleotides: Highly luminous markers.
3. Biotin labeling
- Biotin-16-UTP: for RNA pull-down experiments.
4. End modifications
- T4 RNA ligase: for RNA 3 '-end ligation aptamers or fluorophores.
- 5 '-terminal phosphorylation reagents: such as T4 polynucleotide kinase (T4 PNK).
5. Crosslinking agent
- Formalaldehyde: RNA-protein cross-linking (e.g. CLIP-seq).
- UV irradiation: direct induction of RNA-protein crosslinking.
6. Nucleotide analogs
- 4SU (4-thiouridine): Photoactivated cross-linking for PAR-CLIP technology.
- Pseudouridine : Enhances mRNA stability and translation efficiency (for mRNA vaccine design).
III. General modification technology
1. Click Chemistry
DBCO-azide: used to label nucleic acids containing alkynyl or azide groups (e.g. EdU-modified DNA/RNA).
2. Enzymatic modification
APOBEC: Cytosine deaminase for C-to-U editing.
- TET enzyme: Oxidation of 5mC generates 5hmC/5fC/5caC.
IV. Application scenarios
- Epigenetic studies: DNA/RNA methylation reagents.
Sequencing library construction: end-modified or linker-linked reagents.
Live cell imaging: fluorescently labeled nucleotides (e.g. BrdU, FITC-UTP).
Therapeutic mRNA: pseudouridine, 5 'cap analogs (e.g. CleanCap).
Precautions
Stability: Some modified nucleotides (e.g. 5-azacytidine) need to be protected from light.
Toxicity: Cross-linking agents (e.g. formaldehyde, DMS) need to be handled in a fume hood.
Compatibility: Modifications may affect downstream applications (e.g. PCR efficiency).
Select appropriate modification strategies and reagents according to experimental requirements, and optimize reaction conditions to ensure efficiency.
