AUMblock™ ASO Protocol
Comprehensive in vitro protocol for self-delivering steric-blocking antisense oligonucleotides
Introduction
This protocol provides detailed instructions for using AUMblock™ self-delivering steric-blocking antisense oligonucleotides (sdASO™) in mammalian cell culture. AUMblock™ ASOs enable efficient modulation of RNA function without degradation by physically blocking interactions with proteins or other cellular components, making them ideal for a wide range of applications including translation inhibition, splice modulation, and RNA-protein interaction studies.
Protocol Overview
AUMblock™ sdASO™ delivery is a simple, three-step process:
- Plate cells at optimal density (30-50% confluency)
- Add AUMblock™ sdASO™ directly to culture medium
- Incubate and analyze results (typically 24-72 hours)
This protocol can be adapted for different cell types and various culture vessel formats, from 96-well plates to larger culture vessels.
- No Transfection Required: Simply add to media - no lipofection, electroporation, or viral vectors needed
- Steric Blocking Mechanism: Modulates RNA function without degradation, allowing for reversible and tunable effects
- Precise Targeting: Block specific functional domains of RNA molecules for detailed mechanistic studies
- RNA Structure Preservation: Study RNA function while maintaining its abundance and structural integrity
Materials and Reagents
Required Items
- AUMblock™ sdASO™ (lyophilized or stock solution)
- Appropriate cell culture medium
- Culture plates or vessels
- Mammalian cells of interest
- Sterile nuclease-free water or buffer (for ASO resuspension)
- Microcentrifuge tubes (for aliquoting ASO stock)
- Standard cell culture equipment:
- Sterile pipettes and tips
- Cell culture hood
- Humidified cell culture incubator
- Centrifuge
Detailed Protocol
Cell Preparation
Plate cells in their optimum growth medium at appropriate density for your cell type.
For adherent cells: Plate cells the day before treatment at 30-50% confluency (or at densities optimized for your specific cell type and assay endpoint). Allow cells to adhere overnight.
For suspension cells: Prepare cells at appropriate density shortly before treatment with AUMblock™ sdASO™.
Optimal cell density will vary with cell type, size, growth characteristics, and the endpoint of your assay. As a general guideline, aim for 30-50% confluency at the time of ASO treatment.
When using 96-well plates, approximately 0.5 × 105 cells per well is recommended for cells similar to HeLa in size. Scale accordingly for different plate formats (see reference table below).
AUMblock™ sdASO™ Stock Preparation
Prepare AUMblock™ sdASO™ stock solution by reconstituting lyophilized ASOs at the desired concentration. If you already have a stock solution prepared, skip to Step 3.
Resuspend lyophilized AUMblock™ sdASO™ using the appropriate volume of sterile nuclease-free water or buffer to achieve the desired stock concentration (typically 100 μM).
Pipette the solution up and down 3-5 times while avoiding the introduction of bubbles.
Let the vial sit at room temperature for 5-10 minutes to ensure complete resuspension.
Centrifuge for 30-45 seconds to collect the solution at the bottom of the tube.
Prepare several aliquots of the stock solution to avoid multiple freeze-thaw cycles.
To avoid degradation, minimize freeze-thaw cycles of your ASO stock. It is strongly recommended to make single-use aliquots of your stock solution and store them at -20°C.
AUMblock™ sdASO™ Delivery to Cells
Add AUMblock™ sdASO™ to the cells at the desired final concentration. The working concentration can vary from 500 nM to 10 μM depending on the cell type and RNA target.
For adherent cells: Either aspirate the growth media and overlay cells with fresh media containing AUMblock™ sdASO™, or add the ASO stock directly to the media overlaying the cells. Mix gently.
For suspension cells: Either pellet the cells by low-speed centrifugation and gently resuspend the cell pellet in media containing AUMblock™ sdASO™, or add the ASO stock directly to the media containing the cells. Mix gently.
It is highly recommended to perform a dose response using 2-3 working concentrations (e.g., 500 nM, 5 μM, and 10 μM) to determine the optimal concentration for your specific application.
For steric blocking applications, higher concentrations may be required compared to RNA degradation approaches. In some cases, concentrations up to 20 μM may be necessary for efficient blocking.
Since AUMblock™ works through stoichiometric binding (not catalytic degradation), higher concentrations are sometimes needed to effectively block a high-abundance RNA target. Start with higher concentrations (5-10 μM) and adjust based on efficacy.
Incubation and Analysis
Incubate cells with AUMblock™ sdASO™ and analyze the effect on RNA function at appropriate time points.
Return cells to the incubator and maintain under standard culture conditions.
Analyze AUMblock™ sdASO™-treated cells after the desired time point, typically 24-72 hours post-treatment.
Unlike RNA degradation, steric blocking effects should be assessed by functional readouts rather than RNA levels. These may include:
- Translation blocking: protein level changes (Western blot, ELISA, immunofluorescence) without mRNA level changes
- Splice modulation: RT-PCR to detect altered splicing patterns
- miRNA blocking: de-repression of miRNA target genes
- RNA-protein interaction: RNA immunoprecipitation (RIP) or similar assays
Since AUMblock™ does not degrade the target RNA, RNA levels should remain relatively unchanged while function is altered. This can be used as an internal control to verify steric blocking mechanism versus degradation.
Reference Calculations
The table below provides guidelines for AUMblock™ sdASO™ amounts and cell numbers for various culture plate formats:
| Cell Culture Plate | 96-well | 24-well | 12-well | 6-well |
|---|---|---|---|---|
| AUMblock™ sdASO™ stock (μL)1 | 1 μL | 5 μL | 10 μL | 30 μL |
| AUMblock™ sdASO™ used (moles) | 100 pmole | 500 pmole | 1 nmole | 3 nmole |
| Cell culture media (μL) | 100 μL | 500 μL | 1000 μL | 3000 μL |
| Cell number (per well)2 | 0.5 × 105 | 2.5 × 105 | 0.5 × 106 | 1 × 106 |
- The amount of AUMblock™ sdASO™ shown yields a final concentration of 1 μM using 100 μM stock. For steric blocking applications, consider testing higher concentrations (2-10 μM) for more effective blocking.
- The optimal seeding cell density will vary with the cell type, cell size, growth characteristics, and the endpoint of the assay. For this table, HeLa cells at 50% confluency were used at the time of AUMblock™ sdASO™ treatment.
Tips and Troubleshooting
Optimization Tips and Best Practices
Target Site Selection
For optimal steric blocking, target functionally critical regions of the RNA. For translation blocking, target the 5' UTR or start codon region. For splicing modulation, target splice junctions or splicing enhancer/silencer motifs. For miRNA blocking, target the miRNA binding site.
Concentration Optimization
Steric blocking typically requires higher concentrations than RNA degradation approaches since the mechanism is stoichiometric rather than catalytic. Start with higher concentrations (5-10 μM) and adjust based on efficacy.
Appropriate Controls
Include both scrambled ASO controls and controls targeting non-functional regions of the same RNA to distinguish between specific steric blocking effects and non-specific effects of ASO treatment.
RNA Level Verification
Confirm that target RNA levels remain unchanged while function is altered. This is a key verification that AUMblock™ is working through steric hindrance rather than degradation.
Troubleshooting Common Issues
Limited Functional Effect
- Increase concentration: Since steric blocking is stoichiometric, ensure sufficient ASO concentration to effectively block the RNA target (try up to 20 μM for high-abundance targets).
- Target site optimization: The current target site might not be functionally critical or may be inaccessible due to RNA structure or protein binding. Consider targeting alternative sites on the RNA.
- Extend incubation time: Allow sufficient time for the ASO to reach equilibrium binding with its target, particularly for nuclear targets (48-72 hours may be needed).
- Protein turnover: For translation blocking, consider the half-life of the target protein. Proteins with long half-lives may require extended treatment times to observe reduced levels.
Unexpected RNA Degradation
- Check ASO chemistry: Ensure you're using AUMblock™ rather than AUMsilence™, as the latter is designed for RNA degradation.
- Indirect effects: Consider whether the observed RNA degradation might be an indirect effect of blocking function rather than direct ASO-induced degradation.
- Dose-dependent effects: At very high concentrations, some non-specific effects may occur. Try reducing the concentration.
Difficulty Distinguishing Functional Effects
- Include RNA level measurements: Always verify that target RNA levels remain constant while functional readouts change, to confirm steric blocking mechanism.
- Use multiple readouts: Employ several different assays to measure the functional impact of blocking. For example, for translation blocking, measure both protein levels and downstream functional effects.
- Include AUMsilence™ controls: Compare with traditional knockdown approaches to distinguish steric blocking from degradation effects.
Storage and Additional Information
Storage Conditions
- AUMblock™ sdASO™ are shipped in lyophilized form. Upon arrival, store at -20°C.
- Resuspended AUMblock™ sdASO™ should be stored in aliquots at -20°C to avoid multiple freeze-thaw cycles.
- For short-term storage (up to 1 week), resuspended ASOs can be kept at 4°C.
Additional Notes
- AUMblock™ sdASO™ are compatible with standard cell culture media, including those containing serum.
- No pre-treatment or media change is required before adding AUMblock™ sdASO™ to cells.
- AUMblock™ sdASO™ are not affected by antibiotics in the culture medium.
- For translation blocking applications, consider the protein half-life when determining optimal timepoints for analysis.
- The steric blocking effect is potentially reversible upon ASO clearance, unlike degradation-based approaches.
AUMblock™ sdASO™ are for research use only. Not for use in diagnostic or therapeutic procedures.
AUMblock™ Applications
Specialized Applications for Steric Blocking ASOs
Translation Inhibition
Block translation initiation by targeting the 5' UTR or start codon region of mRNAs. This prevents ribosome binding or scanning without altering mRNA levels, allowing for the study of protein function while maintaining RNA abundance.
miRNA Target Protection
Target miRNA binding sites on mRNAs to prevent miRNA-mediated repression. This "target protector" approach allows for the specific de-repression of individual miRNA targets without altering miRNA levels or affecting other targets.
Splice Modulation
Block splice sites or regulatory elements to alter pre-mRNA splicing patterns. This can induce exon skipping, inclusion, or alternative splice site selection, enabling the study of specific transcript isoforms.
RNA-Protein Interaction Studies
Block protein binding sites on RNA to prevent specific RNA-protein interactions. This approach allows for the selective disruption of individual interactions in complex RNPs to determine their functional importance.
AUMblock™ is particularly valuable for studying RNA function beyond simple gene silencing. For traditional mRNA knockdown applications, consider using AUMsilence™ which is optimized for RNase H-mediated RNA degradation. For specialized splice modulation applications, also consider AUMskip™ which is specifically designed for exon skipping.
Ready to Advance Your RNA Modulation Research?
Our scientific team is available to help you design optimal AUMblock™ sdASO™ for your specific RNA target and application. Contact us for personalized support or to request a quote.
Related Resources
AUMsilence™ Protocol
Detailed protocol for mRNA knockdown using our self-delivering AUMsilence™ sdASO™.
View ProtocolAUMskip™ Protocol
Complete instructions for exon skipping applications using AUMskip™ sdASO™.
View Protocol