mRNA knockdown

AUMsilence sdASO

Name: AUMsilence sdASO
Brand: AUM BioTech

Self-delivering antisense oligonucleotides for mRNA knockdown, no transfection reagent

Inside a cell, a self-delivering antisense oligonucleotide, a gapmer with a pale central gap between its two wings, is paired along its length with its target messenger RNA, and RNase H1, a translucent globular enzyme, sits on the gap under a warm glow: the messenger RNA is cut at the enzyme, in two fragments with a clear gap between its capped start and its beaded tail, and the oligonucleotide stays whole as one unbroken strand. Three gapmers drift in the medium outside the cell membrane with no transfection reagent, the membrane takes one in through a pit and a vesicle, one passes through a pore into the nucleus at the top right, and a mitochondrion lies below the pair.

AUMsilence sdASOs are self-delivering antisense oligonucleotides (ASOs) designed for mRNA knockdown in cell lines and difficult-to-transfect primary cells. Built on our proprietary AUMsilence platform, they combine a phosphorothioate backbone with chemical modifications that enable self-delivery. This chemistry allows them to form stable DNA/RNA hybrids and recruit RNase H1, thereby cleaving target mRNA and silencing gene expression.

Many primary cells and disease-relevant models are difficult to transfect with conventional methods. Transfection reagents, electroporation, or viral vectors can introduce toxicity, alter the cellular response, or require additional optimization. AUMsilence sdASOs enter the cell without a transfection reagent, electroporation, or a viral vector. Add them to cell culture medium or inject them into an animal; they are taken up by endocytosis, which their chemical design makes possible. Unlike siRNAs, which operate through the RNA-induced silencing complex (RISC), AUMsilence sdASOs act via a single-stranded mechanism – simplifying the pathway and avoiding RISC-associated off-target effects.

Self-delivery in one step
No transfection reagent, electroporation, or viral vector is required to deliver AUMsilence sdASOs to cells.
Uptake in difficult-to-transfect cells and in vivo
AUMsilence sdASOs work in contexts where transfection-dependent methods can struggle.
High specificity and low off-target effects
AUMsilence sdASOs have high specificity for the intended mRNA.
No cytotoxicity at the optimized working concentration
The working concentration is determined by dose titration for each cell type and target.

Yields and purification

RPC: 10 · 25 · 50 · 100 nmol
HPLC: 25 · 50 · 100 nmol
Labeled, HPLC: 10 · 20 · 25 · 100 nmol · FAM, Cyanine 3, Cyanine 5, Cyanine 7

Prices and lead time are shown on the order page. Custom yields by quote.

At a glance

Class: Self-delivering antisense (sdASO)
Targets: mRNA knockdown
Delivery: Add to culture medium or inject in vivo.
Fluorescent label: FAM, Cyanine 3, Cyanine 5 or Cyanine 7

In the lab

The protocol in four steps

The steps below are the AUMsilence sdASO protocol's, in its own words. The full protocol carries the amounts per plate format, the tips and the troubleshooting.

Read the full protocol

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.
Step 2
AUMsilence sdASO stock preparation
Prepare AUMsilence sdASO stock solution by reconstituting lyophilized ASOs at the desired concentration. If you already have a stock solution prepared, skip to Step 3.
Step 3
AUMsilence sdASO delivery to cells
Add AUMsilence sdASO to the cells at the desired final concentration. The recommended working range is 5-20 μM, with a starting concentration of 10 μM.
Step 4
Incubation and analysis
Incubate cells with AUMsilence sdASO and analyze knockdown at appropriate time points. Knockdown is typically observed 24 to 72 hours after treatment, depending on the cell type and the target gene. Longer time points may be required in some systems. Protein knockdown can also be observed from 24 hours after treatment. Where the target protein is long-lived, existing protein must first turn over, so a later time point may be required.

Step 1, prepare cells and the oligonucleotide: a clear 24-well plate of pink medium, a microtube of pale blue solution with its lid open and a pipette tip reaching into its mouth, and three short beaded violet strands drawn in the air above the tube, the slide's motif for the oligonucleotide. — For research use only. Not for use in diagnostic or therapeutic procedures.
Sources
The pictures are the object pictures of AUM BioTech's workflow slide, rendered views of the objects on a white ground; none is a photograph of an experiment and none records a measurement.
Tile 04 is a schematic of the three readouts, not data: the two conditions are drawn with a modest difference to show what each readout is, and no magnitude is asserted.

Step 2, add to the culture medium: a single-channel micropipette with a violet plunger held over a 24-well plate of pink medium, its tip in one well. — For research use only. Not for use in diagnostic or therapeutic procedures.
Sources
The pictures are the object pictures of AUM BioTech's workflow slide, rendered views of the objects on a white ground; none is a photograph of an experiment and none records a measurement.
Tile 04 is a schematic of the three readouts, not data: the two conditions are drawn with a modest difference to show what each readout is, and no magnitude is asserted.

Why

Features of AUMsilence sdASO

Self-delivery in one step
No transfection reagent, electroporation, or viral vector is required to deliver AUMsilence sdASOs to cells. Cells are treated in one step by adding AUMsilence sdASOs to the medium. In primary cells and co-culture systems, where transfection can require optimization, AUMsilence sdASOs need no transfection step.
Uptake in difficult-to-transfect cells and in vivo
AUMsilence sdASOs work in contexts where transfection-dependent methods can struggle. They are taken up by difficult-to-transfect cells and primary cultures with no transfection reagent. AUMsilence sdASOs have been used in T lymphocytes, B cells, neuronal cells and other cells that often take up siRNAs or plasmids poorly. They are also proven in animal models, including mice.
High specificity and low off-target effects
AUMsilence sdASOs have high specificity for the intended mRNA. Cleavage needs the oligonucleotide and the transcript to pair, and a transcript that pairs in part can be bound as well. That is why every candidate sequence is scored for homology across the transcriptome before it is chosen. Unlike siRNA, which can have off-targets via partial "seed" matches through RISC, AUMsilence sdASOs have no RISC-associated off-target effects.
No cytotoxicity at the optimized working concentration
The working concentration is determined by dose titration for each cell type and target. At this concentration AUMsilence sdASOs do not cause cytotoxicity. A reduction in viability reflects the knockdown rather than the chemistry, and the non-targeting control shows whether the knockdown is on target. No transfection reagent, electroporation, or viral vector is required, so the cytotoxicity associated with these delivery methods is eliminated.

Mechanism

Mechanism of action

Antisense binding and RNase H1 cleavage: Each AUMsilence sdASO is typically 18-22 nucleotides in length and is complementary to your target mRNA. Upon introduction to cells, it hybridizes to that mRNA. Its DNA-like characteristics trigger the RNase H1 enzyme to recognize the DNA/RNA duplex and cleave the RNA strand. The result is sequence-specific degradation of the mRNA, preventing translation of the target protein.

Nuclear and cytoplasmic targeting: Unlike RNA interference (RNAi) by siRNA, which mainly works in the cytoplasm, AUMsilence sdASOs reach the nucleus as well as the cytosol, where RNase H1-dependent cleavage acts on nuclear RNA in the same way. This is crucial for genes whose mRNA may predominantly reside or function in the nucleus.

Gymnotic delivery: AUMsilence sdASOs are chemically engineered to facilitate their uptake by cells without any transfection reagent. They bind proteins on the cell surface and are taken up by endocytosis over time. Uptake with no lipid reagent avoids the toxicity and stress responses that can come with lipid-based delivery.

Frame 1 of 6, the cell and its medium — 01 **The cell and its medium.** Self-delivering AUMsilence sdASOs are added to the culture medium as they are, with no transfection reagent, no carrier and no vector; an siRNA duplex or a CRISPR ribonucleoprotein in the same medium needs a reagent, electroporation or a vector to get in. [R01] [R02] [R03] [R04] [R05] [R06] [R07] [R08] [R09]
For research use only. Not for use in diagnostic or therapeutic procedures.View at full size
References
[R01] Crooke ST, Wang S, Vickers TA, Shen W, Liang XH (2017). Cellular uptake and trafficking of antisense oligonucleotides. Nature Biotechnology 35:230-237. PMID 28244996, doi 10.1038/nbt.3779.
[R02] Wang S, Allen N, Vickers TA, Revenko AS, Sun H, Liang XH, et al. (2018). Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides. Nucleic Acids Research 46:3579-3594. PMID 29514240, doi 10.1093/nar/gky145.
[R03] Stein CA, Hansen JB, Lai J, Wu S, Voskresenskiy A, Høg A, et al. (2010). Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Research 38:e3. PMID 19854938, doi 10.1093/nar/gkp841.
[R04] Dominska M, Dykxhoorn DM (2010). Breaking down the barriers: siRNA delivery and endosome escape. Journal of Cell Science 123:1183-1189. PMID 20356929, doi 10.1242/jcs.066399.
[R05] Whitehead KA, Langer R, Anderson DG (2009). Knocking down barriers: advances in siRNA delivery. Nature Reviews Drug Discovery 8:129-138. PMID 19180106, doi 10.1038/nrd2742.
[R06] Falato L, Gestin M, Langel Ü (2021). Cell-Penetrating Peptides Delivering siRNAs: An Overview. Methods in Molecular Biology 2282:329-352. PMID 33928583, doi 10.1007/978-1-0716-1298-9_18.
[R07] Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, et al. (2015). Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. Journal of Biotechnology 208:44-53. PMID 26003884, doi 10.1016/j.jbiotec.2015.04.024.
[R08] Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, et al. (2015). Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nature Biotechnology 33:73-80. PMID 25357182, doi 10.1038/nbt.3081.
[R09] Kim S, Kim D, Cho SW, Kim J, Kim JS (2014). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research 24:1012-1019. PMID 24696461, doi 10.1101/gr.171322.113.
[R10] Beltinger C, Saragovi HU, Smith RM, LeSauteur L, Shah N, DeDionisio L, et al. (1995). Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides. The Journal of Clinical Investigation 95:1814-1823. PMID 7706488, doi 10.1172/jci117860.
[R11] Juliano RL (2018). Intracellular Trafficking and Endosomal Release of Oligonucleotides: What We Know and What We Don't. Nucleic Acid Therapeutics 28:166-177. PMID 29708838, doi 10.1089/nat.2018.0727.
[R12] Ochaba J, Powers AF, Tremble KA, Greenlee S, Post NM, Matson JE, et al. (2019). A novel and translational role for autophagy in antisense oligonucleotide trafficking and activity. Nucleic Acids Research 47:11284-11303. PMID 31612951, doi 10.1093/nar/gkz901.
[R13] Koller E, Vincent TM, Chappell A, De S, Manoharan M, Bennett CF (2011). Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes. Nucleic Acids Research 39:4795-4807. PMID 21345934, doi 10.1093/nar/gkr089.
[R14] Wang S, Sun H, Tanowitz M, Liang XH, Crooke ST (2017). Intra-endosomal trafficking mediated by lysobisphosphatidic acid contributes to intracellular release of phosphorothioate-modified antisense oligonucleotides. Nucleic Acids Research 45:5309-5322. PMID 28379543, doi 10.1093/nar/gkx231.
[R15] Cerritelli SM, Crouch RJ (2009). Ribonuclease H: the enzymes in eukaryotes. The FEBS Journal 276:1494-1505. PMID 19228196, doi 10.1111/j.1742-4658.2009.06908.x.
[R16] Wu H, Lima WF, Zhang H, Fan A, Sun H, Crooke ST (2004). Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs. The Journal of Biological Chemistry 279:17181-17189. PMID 14960586, doi 10.1074/jbc.m311683200.
[R17] Liang XH, Sun H, Nichols JG, Crooke ST (2017). RNase H1-Dependent Antisense Oligonucleotides Are Robustly Active in Directing RNA Cleavage in Both the Cytoplasm and the Nucleus. Molecular Therapy 25:2075-2092. PMID 28663102, doi 10.1016/j.ymthe.2017.06.002.
[R18] Lorenz P, Baker BF, Bennett CF, Spector DL (1998). Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies. Molecular Biology of the Cell 9:1007-1023. PMID 9571236, doi 10.1091/mbc.9.5.1007.

Frame 2 of 6, adsorption at the membrane — 01 **The cell and its medium.** Self-delivering AUMsilence sdASOs are added to the culture medium as they are, with no transfection reagent, no carrier and no vector; an siRNA duplex or a CRISPR ribonucleoprotein in the same medium needs a reagent, electroporation or a vector to get in. [R01] [R02] [R03] [R04] [R05] [R06] [R07] [R08] [R09]
For research use only. Not for use in diagnostic or therapeutic procedures.View at full size
References
[R01] Crooke ST, Wang S, Vickers TA, Shen W, Liang XH (2017). Cellular uptake and trafficking of antisense oligonucleotides. Nature Biotechnology 35:230-237. PMID 28244996, doi 10.1038/nbt.3779.
[R02] Wang S, Allen N, Vickers TA, Revenko AS, Sun H, Liang XH, et al. (2018). Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides. Nucleic Acids Research 46:3579-3594. PMID 29514240, doi 10.1093/nar/gky145.
[R03] Stein CA, Hansen JB, Lai J, Wu S, Voskresenskiy A, Høg A, et al. (2010). Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Research 38:e3. PMID 19854938, doi 10.1093/nar/gkp841.
[R04] Dominska M, Dykxhoorn DM (2010). Breaking down the barriers: siRNA delivery and endosome escape. Journal of Cell Science 123:1183-1189. PMID 20356929, doi 10.1242/jcs.066399.
[R05] Whitehead KA, Langer R, Anderson DG (2009). Knocking down barriers: advances in siRNA delivery. Nature Reviews Drug Discovery 8:129-138. PMID 19180106, doi 10.1038/nrd2742.
[R06] Falato L, Gestin M, Langel Ü (2021). Cell-Penetrating Peptides Delivering siRNAs: An Overview. Methods in Molecular Biology 2282:329-352. PMID 33928583, doi 10.1007/978-1-0716-1298-9_18.
[R07] Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, et al. (2015). Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. Journal of Biotechnology 208:44-53. PMID 26003884, doi 10.1016/j.jbiotec.2015.04.024.
[R08] Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, et al. (2015). Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nature Biotechnology 33:73-80. PMID 25357182, doi 10.1038/nbt.3081.
[R09] Kim S, Kim D, Cho SW, Kim J, Kim JS (2014). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research 24:1012-1019. PMID 24696461, doi 10.1101/gr.171322.113.
[R10] Beltinger C, Saragovi HU, Smith RM, LeSauteur L, Shah N, DeDionisio L, et al. (1995). Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides. The Journal of Clinical Investigation 95:1814-1823. PMID 7706488, doi 10.1172/jci117860.
[R11] Juliano RL (2018). Intracellular Trafficking and Endosomal Release of Oligonucleotides: What We Know and What We Don't. Nucleic Acid Therapeutics 28:166-177. PMID 29708838, doi 10.1089/nat.2018.0727.
[R12] Ochaba J, Powers AF, Tremble KA, Greenlee S, Post NM, Matson JE, et al. (2019). A novel and translational role for autophagy in antisense oligonucleotide trafficking and activity. Nucleic Acids Research 47:11284-11303. PMID 31612951, doi 10.1093/nar/gkz901.
[R13] Koller E, Vincent TM, Chappell A, De S, Manoharan M, Bennett CF (2011). Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes. Nucleic Acids Research 39:4795-4807. PMID 21345934, doi 10.1093/nar/gkr089.
[R14] Wang S, Sun H, Tanowitz M, Liang XH, Crooke ST (2017). Intra-endosomal trafficking mediated by lysobisphosphatidic acid contributes to intracellular release of phosphorothioate-modified antisense oligonucleotides. Nucleic Acids Research 45:5309-5322. PMID 28379543, doi 10.1093/nar/gkx231.
[R15] Cerritelli SM, Crouch RJ (2009). Ribonuclease H: the enzymes in eukaryotes. The FEBS Journal 276:1494-1505. PMID 19228196, doi 10.1111/j.1742-4658.2009.06908.x.
[R16] Wu H, Lima WF, Zhang H, Fan A, Sun H, Crooke ST (2004). Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs. The Journal of Biological Chemistry 279:17181-17189. PMID 14960586, doi 10.1074/jbc.m311683200.
[R17] Liang XH, Sun H, Nichols JG, Crooke ST (2017). RNase H1-Dependent Antisense Oligonucleotides Are Robustly Active in Directing RNA Cleavage in Both the Cytoplasm and the Nucleus. Molecular Therapy 25:2075-2092. PMID 28663102, doi 10.1016/j.ymthe.2017.06.002.
[R18] Lorenz P, Baker BF, Bennett CF, Spector DL (1998). Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies. Molecular Biology of the Cell 9:1007-1023. PMID 9571236, doi 10.1091/mbc.9.5.1007.

Applications

Applications and use cases

Gene function studies
Assess the function of a gene by silencing its expression in cells. For example, to study a disease-related gene, treat disease model cells with AUMsilence sdASO targeting that gene. Changes in phenotype, signaling pathways, or other readouts are then measured.
Difficult-to-transfect cells
Use AUMsilence sdASO to achieve knockdown in primary cells (e.g., primary neurons, hepatocytes, stem cells), suspension cell lines (blood cancer lines, immune cells), or other cells where lipofection efficiency can be low.
In vivo gene silencing
AUMsilence sdASOs are used for in vivo studies with no nanoparticle or viral delivery. They are dissolved in a buffer such as phosphate-buffered saline (PBS) or saline and injected intravenously, intraperitoneally, intrathecally or subcutaneously, depending on your study.
High-throughput screening
AUMsilence sdASOs are used in arrayed screens to identify gene functions or candidate targets. Since no transfection reagent is added, a knockdown experiment scales to 96- or 384-well plate formats.

Every compatible cell type, 3D model, organoid and in vivo route, with the in vitro workflow.

See where it works

Experimental considerations

The working concentration is higher than that of a transfected oligonucleotide. The recommended working range for AUMsilence sdASOs is 5-20 μM. For AUMsilence toASO, delivered with a transfection reagent, the recommended range is 50-100 nM.

In the literature