Protocol

AUMblock sdASO protocol

In vitro protocol for self-delivering steric-blocking antisense oligonucleotides

AUMblock are self-delivering steric-blocking antisense oligonucleotides (sdASO) for mammalian cell culture. AUMblock sdASOs modulate RNA function without RNase H mediated degradation, by physically blocking interactions with proteins or other cellular components, and are used for translation inhibition, splice modulation, and RNA-protein interaction studies.

Protocol overview

AUMblock sdASO delivery is a three-step process:

01Plate cells at optimal density (50-70% confluency at treatment)
02Add AUMblock sdASO directly to culture medium
03Incubate and analyze results (typically 24-72 hours after treatment)

This protocol can be adapted for different cell types and various culture vessel formats, from 96-well plates to larger culture vessels.

Key advantages

No transfection required: Add to media (no lipofection, electroporation, or viral vectors needed)
Steric blocking mechanism: Modulates RNA function without RNase H mediated degradation, allowing for reversible and tunable effects
Precise targeting: Block specific functional domains of the RNA for detailed mechanistic studies
No cleavage by the oligonucleotide: Study RNA function without recruiting RNase H to the target

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

01
Step 1: Cell preparation
Plate cells in their optimum growth medium at a density appropriate for the cell type. A confluency of 50-70% at the time of treatment is typical.
- For adherent cells: Plate cells the day before treatment, at a seeding density that brings them to that confluency by the time of treatment (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.
02
Step 2: 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 1 mM).
- 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.
Important: 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.
Cells seeded, here in a 24-well plate; the oligonucleotide waits in solution.
03
Step 3: AUMblock sdASO delivery to cells
Add AUMblock sdASO to the cells at the desired final concentration. The recommended working range is 5-20 μM, with a starting concentration of 10 μM. The optimal concentration varies with the target gene, the RNA class (messenger RNA, microRNA or long non-coding RNA) and the cell type, and should be determined by titration for each system.
- 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 the three concentrations of the range (5 μM, 10 μM and 20 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 the top of the range, 20 μM, is necessary for efficient blocking.
Optimization tip: Since AUMblock sdASO works through stoichiometric binding (not catalytic degradation), higher concentrations are sometimes needed to effectively block a high-abundance RNA target. Start at 10 μM and adjust based on efficacy.
Add the self-delivering oligonucleotide straight to the medium: no transfection reagent.
04
Step 4: 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)
  - 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
Note: Since AUMblock sdASO binds its target without cleaving it, transcript abundance is not the readout for a steric block. Target RNA levels can still fall without cleavage by the oligonucleotide, because a blocked or redirected transcript may be degraded by the cell, so a reduction by RT-qPCR does not establish the mechanism. Read the function that was blocked: protein level for a translation block, splice pattern for splice modulation.
Return the plate to the humidified incubator under standard culture conditions, typically 24-72 hours.

Reference calculations

Making the stock

Reconstitute the vial in nuclease-free water to 1 mM (1 μL per nmol, so 25 μL for a 25 nmol vial). For a 100 μM stock, add 10 μL per nmol.

Vial	For 1 mM	For 100 μM
10 nmol	10 μL	100 μL
25 nmol	25 μL	250 μL
50 nmol	50 μL	500 μL
100 nmol	100 μL	1 mL

Adding the stock to the well

Add the 1 mM stock to the well at 1:200 for 5 μM, 1:100 for 10 μM and 1:50 for 20 μM. At 10 μM that is 1 μL per 100 μL of medium.

Plate	Medium in the well	5 μM	10 μM	20 μM
96-well	100 μL	0.5 μL	1 μL	2 μL
48-well	250 μL	1.25 μL	2.5 μL	5 μL
24-well	500 μL	2.5 μL	5 μL	10 μL
12-well	1 mL	5 μL	10 μL	20 μL
6-well	2 mL	10 μL	20 μL	40 μL

Treatments per vial

At 10 μM, a 25 nmol vial treats 25 wells of a 96-well plate, 5 of a 24-well plate or 2 of a 12-well plate. Counts are rounded down to whole wells. AUMblock sdASO is supplied by quotation in these vials.

Plate	Medium in the well	5 μM	10 μM	20 μM
96-well	100 μL	50	25	12
48-well	250 μL	20	10	5
24-well	500 μL	10	5	2
12-well	1 mL	5	2	1
6-well	2 mL	2	1	0

Cell density at treatment

Add AUMblock sdASO when adherent cells are at 50-70% confluency. Seed suspension cells and primary cells at the density their cell type guide gives.

Tips and troubleshooting

Optimization tips and best practices

Target site selection

For 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 needs more than RNA degradation does, since the mechanism is stoichiometric rather than catalytic. Start at 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.

Reading the right level

Read function rather than transcript abundance: protein level for a translation block, splice pattern for splice modulation. A fall in target RNA level does not by itself show that AUMblock sdASO worked by degradation, since a blocked or redirected transcript may be degraded by the cell.

Troubleshooting common issues

Limited functional effect

Increase concentration: Since steric blocking is stoichiometric, ensure sufficient ASO concentration to effectively block the RNA target (the top of the range, 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 are using AUMblock sdASO rather than AUMsilence sdASO, 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: Read target RNA levels alongside the functional readout rather than in place of it. A fall in RNA level does not by itself separate the two mechanisms, since a blocked or redirected transcript may be degraded by the cell; the separation comes from the functional readout and from the knockdown control below.

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 sdASO 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. Serum proteins may reduce uptake.
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.

Note

AUMblock sdASO are for research use only. Not for use in diagnostic or therapeutic procedures.

AUMblock sdASO applications

Uses for a steric block

Translation inhibition

Block translation initiation by targeting the 5' UTR or start codon region of mRNAs. This prevents ribosome binding or scanning without RNase H mediated cleavage, allowing for the study of protein function through a non-degradative mechanism.

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.

Application note

AUMblock sdASO is particularly valuable for studying RNA function beyond simple gene silencing. For traditional mRNA knockdown applications, consider using AUMsilence sdASO which is optimized for RNase H1-mediated RNA degradation. For splice modulation applications, also consider AUMsplice sdASO, for exon skipping.

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For research use only. Not for use in diagnostic or therapeutic procedures.

AUMblock™ sdASO™ protocol