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AUMsilence V+™ ASO Protocol

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

Introduction

This protocol provides detailed instructions for using AUMsilence V+™ self-delivering antisense oligonucleotides (sdASO™) for viral RNA knockdown in mammalian cell culture. AUMsilence V+™ ASOs 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 simple, three-step process:

  1. Plate cells at optimal density and infect with virus (if cells are not already infected)
  2. Add AUMsilence V+™ sdASO™ directly to culture medium
  3. Incubate and analyze viral inhibition (typically 24-72 hours)

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: Simply add to media - no lipofection, electroporation, or viral vectors needed
  • Viral-Specific Design: Optimized to target conserved regions of viral genomes for maximum inhibition
  • Dual Mechanism: Acts through RNase H-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

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 (prophylactic approach): Plate cells the day before at 30-50% confluency. Allow cells to adhere overnight, then add AUMsilence V+™ sdASO™ (Step 3) prior to viral infection.

For post-infection treatment (therapeutic approach): 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 30-50% confluency and proceed directly to Step 3.

Note

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). The optimal cell density may vary depending on the viral system used.

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 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.

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.

3

AUMsilence V+™ sdASO™ Delivery to Cells

Add AUMsilence V+™ sdASO™ to the cells at the desired final concentration. For viral RNA targeting, the working concentration can typically range from 500 nM to 10 μM depending on the viral target and cell type.

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 2-3 working concentrations (e.g., 500 nM, 5 μM, and 10 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, higher concentrations (5-10 μM) may be more effective, especially for viruses with high replication rates or abundant transcripts. For prophylactic treatment, lower concentrations may be sufficient if administered before infection.

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.

Reference Calculations

The table below provides guidelines for AUMsilence V+™ sdASO™ amounts and cell numbers for various culture plate formats:

Cell Culture Plate96-well24-well12-well6-well
AUMsilence V+™ sdASO™ stock (μL)11 μL5 μL10 μL30 μL
AUMsilence V+™ sdASO™ used (moles)100 pmole500 pmole1 nmole3 nmole
Cell culture media (μL)100 μL500 μL1000 μL3000 μL
Cell number (per well)20.5 × 1052.5 × 1050.5 × 1061 × 106
Table Notes
  1. The amount of AUMsilence V+™ sdASO™ shown yields a final concentration of 1 μM using 100 μM stock. For viral targets, higher concentrations (5-10 μM) may be more effective.
  2. The optimal seeding cell density will vary with the cell type, cell size, viral system, and the endpoint of the assay. Adjust density based on your specific virus and cell model.

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. Ideal targets 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 prophylactic approach, add AUMsilence V+™ sdASO™ 4-24 hours before viral infection. For therapeutic approach, 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: Try higher concentrations (up to 20 μM) for efficient viral inhibition.
  • 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, try lower concentrations (500 nM - 1 μM).
  • 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.
  • AUMsilence V+™ sdASO™ can be used in combination with standard antiviral compounds for potential synergistic effects.
Research Use Only

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™ have been successfully used to target a variety of 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, effective targets often 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, effective targets often 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.

Ready to Accelerate Your Antiviral Research?

Our scientific team is available to help you design optimal AUMsilence V+™ sdASO™ for your specific viral target and application. Contact us for personalized support or to request a quote.

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