Technology

Transcriptome Architect

With the help of our TranscriptomeArchitect platform, there are now thousands of experimentally validated Antisense OligoNucleotides targeting hundreds of genes. More than 2 million Antisense OligoNucleotides modulating transcript degradation and exon splicing have been designed for the entire human transcriptome. This rich resource is now stored in the TranscriptomeViewer, enabling scientists to access more than 2 millions oligos to quickly and efficiently advance their research.

spliceosome
Structure of human post-catalytic P complex spliceosome (6QDV)
viewer
TranscriptomeViewer showing transcripts from BRCA1.

Transcriptome Viewer

The TranscriptomeViewer is a scientific visualizer that allows researchers to examine specific transcripts or exon targets, and understand the possible effects of their splicing modulation. This tool is developed at the Agency of Science Technology and Research, Singapore (A*STAR) and is freely available for the research community to use. More than 2 millions NOA-Switch™ and NOA-NMD™ designed by our TranscriptomeArchitect are now available in the viewer to enable scientists to have easy access to these oligos. With the viewer, researchers can examine how specific splice modulation can affect the functional protein domains for coding transcripts and create experimentally testable hypotheses on the cell phenotypes.


Publications

Experimental Papers

Computational Papers


Guidelines

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Re-suspending the AONs

The AONs are supplied in lyophilized powder or gel form and can be re-suspended in distilled, sterile water or PBS buffer. They are very soluble in water.

Storage of the AONs

The AONs must be stored at ≤-20 ºC. When stored in powder form, the AONs are very stable for more than 1 year. Once re-suspended in water or buffer, it is recommended to aliquot them and use within 1 year. It is recommended to prepare small aliquots in order to avoid repeated freeze-thaw cycles.

Use of the AONs in vitro

in vitro

In order for the 2-O-Methyl phosphorothioate AONs to be functional in the alteration of a specific splicing event, they must be able to access the nucleus of the cell, where the splicing reaction takes place. The AONs synthesized with this chemistry are capable to self-penetrate the cell (and the nucleus) when added to the media in multiple cell types. However, we recommend using transfection reagents or electroporation that will allow to achieve the desired splicing switch with a minimal amount of AONs in in vitro experiments. A titration of the AON concentration is highly recommended for all the experiments performed.

Naked AONs

Naked AONs are simply added to culture medium once re-suspended in water or PBS buffer. It must be taken in consideration that the naked AON uptake is cell-type specific: cells with “endocytic nature” (e.g. APCs) will be able to naturally have more productive switching, while secretory cells (e.g. T cells) are going to be less prone to internalize the AONs. Usually, the amount of AON tolerated by cells in the medium spans in the range of single-digit µM to nM.

Transfection reagents

We noticed that the transfection reagents and protocols used for siRNA or plasmid’s transfection are working for AON as well, leading to an improved uptake of AON in cultured cells. We noticed a sensibly better performance for AON’s transfection in adherent cells, such as lung carcinoma A549, and prostate cancer cell lines PC-3 and LnCap, using RNAiMAX and Lipofectamine 3000 compared to Lipofectamine 2000 (see Protocols section). With Lipofectamine 3000 as transfection reagent, the IC50 of an effective AONs can range from 5 nM to 250 nM depending on the cell lines and target genes.

Electroporation of AONs

For cells cultured in suspension, electroporation systems have been shown to suit the AON intra-nuclear delivery maintaining the same parameters used for siRNA and plasmids.

Use of the AONs in vivo

Our previous study (Lin J et al., 2017) with mouse xenograph models showed that intraperitoneal injection of the naked AONs has successfully induced target exon skipping and inhibited tumor growth.

Detection of the splicing switch

Splicing is a constant and dynamic process. The switches in the desired splicing event can be observed as early as within a few hours from the transfection of the AON. The factors influencing the kinetic and efficacy of the switch are multiple: transcript abundance, transcription turnover, stability, etc. The switch can last for numerous days, depending on the target transcript, cell type, and replication rate.

Non-NMD-inducing AONs

If the AON is not meant to induce the degradation of the target transcript, the detection of the splicing switch is usually straightforward. A simple RT-PCR with primers designed on the flanking exons will be sufficient to visualize on an agarose gel the two different bands (full length isoform and “skipped” isoform). This method allows a rapid and clear quantification of the AON efficiency. A TaqMan PCR, specific for the two isoforms, can be designed for improved quantification sensitivity and reproducibility (Dewaele M et al., 2016).

RT PCR

However, it might happen that the target exon doesn’t have flanking exons that are constitutively included in the transcript. We suggest to carefully look at the alternative splicing occurring in the surroundings of the desired splicing modulation. In case the alternative splicing pattern is particularly complex, we suggest to design the primers on constitutive exons and use capillary electrophoresis to detect and quantify all the possible isoforms (Tabaglio T et al.).

NMD-inducing AONs

For AONs designed to induce degradation through nonsense-mediated decay (NMD), the switch will not be easily quantifiable since the isoform subject to NMD will be degraded. A simple quantitative PCR (qPCR) to detect the decrease of the target mRNA in this case will suffice to assess the efficacy of the AON switch. In combination, a treatment of the cells for few hours with cycloheximide before cell harvest will block the NMD process, allowing a more reliable quantification of the switch.

Troubleshooting if no or low target switching could be detected

Possible reasons and solutions:

  1. The AONs cannot penetrate the cell (and/or the nucleus).
    Solution: If naked AONs was used, try to increase the concentration of AONs or use a transfection reagent such as lipofectamine. If transfection reagent was used, try to optimize the transfection conditions. For example, adjusting the ratio of AON/transfection reagent.
  2. The AONs concentration is too high.
    Solution: We have noticed that if the AONs concentration goes too high (e.g. >250 nM) in in vitro experiment with transfection reagent, their target switching effect may start to go off. Therefore, we recommend a concentration of 50 to 100 nM for each AON to start with.
  3. The transcript with the target switching has been degraded via NMD.
    Solution: If the transcript with the target switching has been degraded through nonsense-mediated decay (NMD), no or low switching could be detected. In order to protect the transcript with the target switching from NMD, cycloheximide (suggested working concentration: 10 – 100 µg/ml) can be added into the medium 5 hours after transfection.


Protocols

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AON transfection with Lipofectamine 2000/3000

Using this protocol, we have successfully transfected AONs into the following cell lines: A549, Hela, RPE, THP-1, RS4:11, SHSY5Y, SEM, LnCap, PC-3 and U87MG.

AON transfection for adherent cells (2-days protocol)

  1. 24h before transfection, seed 150,000-250,000 cells/well (in 2 ml growth medium) in 6-well plate and incubate overnight. Cells shall reach 40-60 % confluence by the time of transfection.
  2. Replace the medium with growth medium without antibiotics before transfection (1.8 ml/well).
  3. Prepare the 2 transfection mixes in 2 separate Eppendorf tubes or falcon tubes
    1. 100 µl Opti-MEM medium + 2 µl AON (from 100 µM stock, for a final concentration of 100 nM)
    2. 100 µl Opti-MEM medium + 2-5 µl Lipofectamine 2000 or lipofectamine 3000 (Different ratio of AON : Lipofectamine must be tested to determine the optimal ratio for different cell lines)
  4. Mix a. in b. gently.
  5. Leave the transfection mixture for 20 minutes at room temperature.
  6. Add dropwise the 200 µl mixture into the well with cells.
  7. (Optional) Medium can be changed 5 hours after transfection.

AON transfection for adherent cells and suspension cells (1-day protocol)

  1. Seed 150,000-300,000 cells/well (in 1.8 ml growth medium without antibiotics) in 6-well plate.
  2. Prepare the 2 diluted reagents in 2 separate Eppendorf tubes or falcon tubes
    1. 100 µl Opti-MEM medium + 2 µl AON (from 100 µM stock, for a final concentration of 100 nM)
    2. 100 µl Opti-MEM medium + 4-5 µl Lipofectamine 2000 or lipofectamine 3000 (Different ratio of AON : Lipofectamine must be tested to determine the optimal ratio for different cell lines)
  3. Mix a. in b. gently.
  4. Leave the transfection mixture for 20 minutes at room temperature.
  5. Add dropwise the 200 µl mixture into the well with cells.

AON transfection with Lipofectamine RNAiMAX

Using this protocol, we have successfully transfected AONs into LnCap and PC-3 cells with higher efficiency as compared to Lipofectamine 2000 or 3000.

  1. 24h before transfection, seed 150,000-250,000 cells/well (in 2 ml growth medium) in 6-well plate and incubate for overnight.
  2. Replace the medium with Opti-MEM medium (700 µl/well) before transfection.
  3. Prepare the 2 diluted reagents in 2 separate Eppendorf tubes or falcon tubes
    1. 150 µl Opti-MEM medium + 1 µl AON (from 100 µM stock, for a final concentration of 100 nM)
    2. 150 µl Opti-MEM medium + 2 to 4 µl of RNAiMAX (optimization must be performed)
  4. Mix a. in b. gently.
  5. Leave the transfection mixture for 10 minutes at room temperature.
  6. Add dropwise the 300 µl mixture into the well with cells.
  7. Change to normal growth medium 6 hours after transfection or the day after.