Protocol

AUMsilence V+ sdASO protocol

In vitro protocol for viral RNA targeting with self-delivering antisense oligonucleotides

AUMsilence V+ sdASOs are specifically designed to target viral genomes or transcripts with high precision, enabling efficient inhibition of virus replication without the need for transfection reagents.

Protocol overview

AUMsilence V+ sdASO delivery is a three-step process:

01Plate cells at optimal density and infect with virus (if cells are not already infected)
02Add AUMsilence V+ sdASO directly to culture medium
03Incubate and analyze viral inhibition (typically 24-72 hours after treatment)

This protocol can be adapted for different cell types, viral systems, 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)
Viral-specific design: Optimized to target conserved regions of viral genomes
Dual mechanism: Acts through RNase H1-mediated degradation and/or steric blocking of viral RNA function
Works in infected cells: Maintains efficacy even in virus-infected cells where other methods may be compromised

Materials and reagents

Required items

AUMsilence V+ sdASO (lyophilized or stock solution)
Appropriate cell culture medium
Culture plates or vessels
Mammalian cells susceptible to virus of interest
Viral stock (if conducting infection during the experiment)
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
Appropriate viral detection reagents (depending on your readout system):
- qRT-PCR primers for viral RNA
- Antibodies for viral proteins (Western blot or immunofluorescence)
- Viral titer assay materials (e.g., plaque assay components)

Detailed protocol

01
Step 1: Cell preparation and viral infection
Plate cells in their optimum growth medium at appropriate density for your cell type and viral system.
- For pre-infection treatment: Plate cells the day before, at a seeding density that brings them to 50-70% confluency by the time they are treated. Allow cells to adhere overnight, then add AUMsilence V+ sdASO (Step 3) prior to viral infection.
- For post-infection treatment: Plate cells, allow adherence, then infect with your virus of interest according to established protocols. After the desired infection period (typically when viral replication begins), proceed to Step 3 to add AUMsilence V+ sdASO.
- For persistently infected cells: Plate chronically infected cells at 50-70% confluency and proceed directly to Step 3.
Note: The optimal cell density may vary depending on the viral system used.
02
Step 2: AUMsilence V+ sdASO stock preparation
Prepare AUMsilence V+ 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 AUMsilence V+ 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: AUMsilence V+ sdASO delivery to cells
Add AUMsilence V+ 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 AUMsilence V+ 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 AUMsilence V+ 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 viral target.
- For viral RNA targeting, you may include multiple AUMsilence V+ sdASO designed against different conserved regions of the viral genome to enhance efficacy and reduce the likelihood of viral escape.
Optimization tip: When targeting viral RNA, starting at 10 μM and moving up may be more effective, especially for viruses with high replication rates or abundant transcripts. For pre-infection treatment, lower concentrations may be sufficient.
Add the self-delivering oligonucleotide straight to the medium: no transfection reagent.
04
Step 4: Incubation and analysis
Incubate cells with AUMsilence V+ sdASO and analyze viral inhibition at appropriate time points.
- Return cells to the incubator and maintain under standard culture conditions appropriate for your viral system.
- Analyze AUMsilence V+ sdASO-treated cells after the desired time point, typically 24-72 hours post-treatment. The optimal timepoint will depend on the viral replication cycle.
- Viral inhibition can be assessed by various methods:
  - Measuring viral RNA levels (qRT-PCR)
  - Detecting viral protein levels (Western blot, ELISA, immunofluorescence)
  - Quantifying infectious virus production (plaque assay, TCID50, etc.)
  - Observing virus-induced cytopathic effects (CPE)
Note: For viruses with rapid replication cycles, earlier timepoints (12-24 hours) may be appropriate. For slower-replicating viruses, later timepoints (48-96 hours) may be needed to observe significant inhibition effects. Monitoring multiple timepoints is recommended for initial optimization.
Return the plate to the humidified incubator under standard culture conditions for your viral system, 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. AUMsilence V+ 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 AUMsilence V+ 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 for viral targeting

Optimization tips and best practices

Viral target selection

Target conserved regions of the viral genome that are essential for replication. These include viral polymerase genes, protease regions, or structural genes that cannot tolerate mutations. Targeting multiple regions simultaneously can enhance antiviral effects and reduce escape mutation probability.

Timing considerations

For pre-infection treatment, add AUMsilence V+ sdASO 4-24 hours before viral infection. For post-infection treatment, add AUMsilence V+ sdASO as early as possible after infection is established. The timing will depend on the specific viral replication cycle.

Multiplicity of infection (MOI)

Start with lower MOI (0.01-0.1) for initial optimization. Higher viral loads may require increased AUMsilence V+ sdASO concentrations. For persistent viral infections, higher ASO concentrations may be needed to achieve significant inhibition.

Controls

Include appropriate controls: untreated infected cells, scrambled/non-targeting AUMsilence V+ sdASO in infected cells, and positive control antiviral compounds (if available) for your viral system. Testing ASO effects on uninfected cells can help distinguish antiviral effects from potential cytotoxicity.

Troubleshooting common issues

Low viral inhibition

Increase concentration: Move up towards 20 μM, the top of the range.

Target multiple viral regions: Use a combination of AUMsilence V+ sdASO targeting different regions of the viral genome.

Check target accessibility: The target sequence might have structural constraints or be protected by viral or cellular proteins. Consider targeting more accessible regions.

Optimize timing: For rapidly replicating viruses, earlier treatment is typically more effective. Consider adding AUMsilence V+ sdASO before infection or at very early stages of viral replication.

Viral escape or resistance

Combine multiple ASOs: Target multiple conserved regions of the viral genome simultaneously to reduce escape probability.

Target essential viral regions: Focus on viral genome regions that cannot tolerate mutations without loss of fitness.

Sequence the viral target region: If resistance emerges, sequence the viral genome to identify potential escape mutations, which can inform redesign of more effective ASOs.

Cell cytotoxicity

Reduce concentration: If toxicity is observed, move down towards 5 μM, the bottom of the range.

Distinguish viral CPE from ASO toxicity: Include uninfected cells treated with the same ASO concentration to differentiate between viral cytopathic effects and potential ASO toxicity.

Check sequence specificity: Ensure your ASO does not have significant homology to host cell transcripts that could lead to off-target effects.

Inconsistent results

Standardize viral input: Ensure consistent MOI across experiments by carefully titering viral stocks.

Control cell density: Maintain consistent cell confluency at the time of infection and ASO treatment.

Multiple readouts: Use multiple methods to assess viral inhibition (e.g., viral RNA, protein, and infectious titer) for more robust conclusions.

Storage and additional information

Storage conditions

AUMsilence V+ sdASO are shipped in lyophilized form. Upon arrival, store at -20°C.
Resuspended AUMsilence V+ 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 for viral research

AUMsilence V+ sdASO are compatible with standard virus culture media, including those containing serum.
For BSL-2 or higher viral work, all appropriate biosafety procedures must be followed. AUMsilence V+ sdASO do not alter the biosafety requirements for your viral system.
When using fluorescently labeled AUMsilence V+ sdASO, consider potential spectral overlap with viral fluorescent reporters if using fluorescence-based detection methods.

Note

AUMsilence V+ sdASO are for research use only. Not for use in diagnostic or therapeutic procedures.

Common viral applications

RNA virus applications

AUMsilence V+ sdASO are designed against RNA viruses, including:

Positive-strand RNA viruses: Coronaviruses (SARS-CoV-2, MERS-CoV), flaviviruses (HCV, Zika, Dengue), picornaviruses (poliovirus, rhinovirus)
Negative-strand RNA viruses: Influenza virus, respiratory syncytial virus (RSV), vesicular stomatitis virus (VSV)
Retroviruses: HIV-1, HTLV-1

For these viruses, the regions AUM designs against include:

Viral polymerase genes
Conserved untranslated regions (UTRs)
Essential structural genes
Viral protease-encoding regions

DNA virus applications

AUMsilence V+ sdASO can also target viral transcripts from DNA viruses, especially targeting:

Herpesvirus family: HSV-1, HSV-2, CMV, EBV
Hepadnaviruses: Hepatitis B virus (HBV)
Papillomaviruses: HPV

For DNA viruses, the regions AUM designs against include:

Immediate-early gene transcripts
Essential regulatory proteins (e.g., HBx for HBV)
Viral oncogenes (e.g., E6/E7 for HPV)
Conserved regions in viral mRNAs

Application insight

For viruses with high mutation rates or genetic diversity (e.g., HIV, influenza), consider targeting highly conserved regions essential for viral function. Using a combination of ASOs targeting different viral regions can significantly reduce the probability of viral escape through mutations.

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

AUMsilence V+™ sdASO™ protocol