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The science

Antisense-based gene silencing

How the AUMsilence platform works

The platform

The AUMsilence platform

AUMsilence sdASOs silence gene expression at the RNA level. Sequences are designed using the AUMsilence platform.

Chemistry and molecular design

sdASOs designed using the AUMsilence platform can work in cell lines, difficult-to-transfect primary cells, and in in vivo models. Antisense oligonucleotides (ASOs) are designed as short sequences of 18-22 nucleotides that are complementary to the target RNA. They feature a gapmer architecture: a central DNA-like "gap" region flanked by modified RNA analogs that increase binding affinity and protect the oligo from degradation.

The central region, once bound to the target RNA, creates an RNA:DNA duplex that is recognized by RNase H1, an endogenous enzyme present in the nucleus and cytoplasm. RNase H1 cleaves the RNA strand of the duplex, fragmenting the target RNA and preventing its translation into protein. The ASO itself is not degraded in this process and can dissociate and hybridize to further copies of the target RNA, acting in a catalytic fashion.

  • Cytoplasmic and nuclear RNA: sdASOs designed using the AUMsilence platform act on messenger RNA in the cytoplasm and on precursor and non-coding RNA in the nucleus.
  • Self-delivery: AUMsilence sdASOs are taken up by endocytosis, with no transfection reagent, no carrier and no vector.
  • Stability: Chemical modifications protect the oligonucleotide from nuclease degradation.
  • Potency: One oligonucleotide acts catalytically: RNase H1 cuts the target and the oligonucleotide moves on to the next copy.
  • Specificity: Silencing follows Watson-Crick pairing to the target sequence, with no RISC involvement.
What reaches the inside of a cell: the self-delivering AUMsilence sdASO is taken up by endocytosis with no transfection reagent, a siRNA duplex and a CRISPR complex are not, and inside the cell RNase H1 cleaves the paired target RNA in the cytoplasm and in the nucleusThe self-delivering AUMsilence sdASO, a single short gapmer drawn as beads on a ribbon with its two wings filled and its central gap open, follows a solid route to a pit in the cell membrane and is taken up by endocytosis, with no transfection reagent. A siRNA duplex, two strands and visibly larger, and a CRISPR complex, a Cas protein with its guide RNA and larger again, follow dashed routes that stop outside the membrane at a no-entry mark: neither is taken up that way. Inside the cell, the oligonucleotide sits in an endosome beside the pit, then reaches the cytoplasm, where it is paired with a messenger RNA, and the nucleus, where it is paired with a nuclear RNA; on each pair RNase H1 sits on the duplex and the target RNA is cleaved, the oligonucleotide staying whole. The cell is a rendered picture: a whole cell whose membrane cups a single strand into a pit at its upper left, a vesicle beside it, a messenger RNA carrying a paired strand under a warm glow, a nucleus at the lower right, a mitochondrion and two small organelles.OUTSIDE THE CELLINSIDE THE CELLAUMsilence™ sdASO™taken up by endocytosissiRNA duplextwo RNA strandsCRISPR complexa Cas protein with its guide RNAEndosomeCytoplasmRNase H1CleavageNucleusRNase H1 cleavagetaken up by endocytosisnot taken up this way: needs a reagent or a vector
Our AUMsilence platform uses chemical modifications that enable self-delivery.

Sequence design

Each sequence is designed using the AUMsilence platform against the target transcript and scored against the transcriptome of the selected species.

The scoring rests on the published record, data reviewed from over 500,000 published articles, and the number grows every week.

Factors considered

  • RNA folding
  • Secondary and tertiary structure
  • Protein binding
  • GC content
  • Off-target matches
  • The chemical modification pattern, chosen for each sequence

This approach ensures that each AUMsilence sdASO is customized to its specific target, resulting in 70-95% knockdown across diverse genes and experimental systems.

How a sequence is designed
FigureThe transcript and the four stages
Reading the transcript.Still reading. A first read of a transcript can take a while; this page waits up to 20 seconds for it, and its scripts then bring in the figure, or a line saying what did not arrive.
  1. Stage 1: Reads the transcript. Every exon of the target transcript, as the reference annotation places it.
  2. Stage 2: Scores every candidate window. Each window along the transcript is scored for predicted activity and for specificity across the transcriptome.
  3. Stage 3: Rests on the published record. That record holds the antisense and siRNA sequences that silenced their target, and the ones that did not. Each carries the cell type or the animal model it was tried in.
  4. Stage 4: Builds them with its chemistry. Several sequences are designed against different regions of the same transcript, and the strongest knockdown is found by testing more than one, which is why ordering more than one sequence per target is recommended.
The exons of a real transcript are drawn here from the live record once the read arrives, with the accession, the assembly, the source's release and the day it was read; the candidate windows, the chosen windows and the oligonucleotides are a schematic of the method, and no score is drawn.

Two categories, three steps

Product categories

Self-delivering ASOs (sdASO)

sdASOs designed using the AUMsilence platform target mRNA, microRNA, long non-coding RNA and viral RNA. These products include:

  • AUMsilence sdASOFor mRNA knockdown
  • AUMantagomir sdASOFor miRNA inhibition
  • AUMlnc sdASOFor lncRNA knockdown
  • AUMsilence V+ sdASOFor viral RNA knockdown

AUMsilence sdASOs are self-delivering. They are added directly to cell culture or injected in vivo, with no lipid transfection reagent, electroporation, or viral vector.

Transfection-optimized ASOs (toASO)

For researchers working with standard cell lines or conducting high-throughput screens where cost-efficiency is important:

  • AUMsilence toASOFor mRNA knockdown by transfection

AUMsilence toASO is compatible with a standard lipid-based transfection reagent or electroporation. It is used for routine gene silencing where transfection is the delivery method and the budget is a consideration.

  1. Step 1: endocytosis

    Step 1: ASO entry

    AUMsilence sdASOs are chemically modified and are taken up by the cells by gymnosis. AUMsilence toASOs are compatible with a standard lipid-based transfection reagent or electroporation.

  2. Step 2: hybridization and cleavage in the cytoplasm

    Step 2: Hybridization to the target

    Once inside the cell, the ASO hybridizes to its target RNA (mRNA, microRNA, or lncRNA) with high affinity and specificity, forming an RNA:DNA hybrid duplex.

  3. Step 3: the nucleus

    Step 3: RNase H1 cleavage

    The RNA:DNA hybrid is recognized by cellular RNase H1, which cleaves the RNA strand, degrading the target RNA while leaving the ASO intact to target further copies of the RNA. This catalytic process amplifies the silencing effect.

What the cleavage produces

From mRNA to protein silencing

By cleaving messenger RNA transcripts, AUMsilence sdASOs effectively reduce or abolish the production of the target protein. Target mRNA levels drop first and protein levels follow, at a pace set by the protein's own half-life. The mode of action follows the sequence: cleavage needs the oligonucleotide and the transcript to pair, and the oligonucleotide can also hybridize to a transcript that pairs in part. Every candidate sequence is therefore scored for homology across the transcriptome before it is chosen.

Nuclear and cytoplasmic action

The antisense mechanism works in both the nucleus and the cytoplasm. siRNAs and shRNAs primarily act in the cytoplasm on processed mRNA, whereas sdASOs designed using the AUMsilence platform can also hybridize to precursor mRNAs or lncRNAs in the nucleus. This broad cellular reach allows silencing of RNAs at multiple stages (from pre-spliced nuclear RNA to mature cytoplasmic mRNA) and is particularly useful for targeting nuclear-retained RNAs.

RNase H1 cleaving a hybridized precursor messenger RNA in the nucleus and a mature messenger RNA in the cytoplasm.
The paired message cut in the nucleus and in the cytoplasm

Against the alternatives

Comparison with siRNA, shRNA, and CRISPR

Comparison with siRNA, shRNA, and CRISPR
FeatureSelf-delivering ASO (sdASO)siRNA (RNAi)shRNACRISPR/Cas9
Delivery methodSelf-delivering, no transfection neededRequires transfection reagents or lipid carriersRequires viral vectors or plasmid transfectionComplex delivery systems (viral vectors, electroporation)
MechanismRNase H1-mediated RNA cleavageRISC-mediated RNA cleavageProcessed to siRNA, then RISC-mediatedDNA modification/genome editing
Cell type rangeWorks in cell lines and difficult-to-transfect primary cellsChallenging in primary and difficult-to-transfect cellsLimited by viral transduction efficiencyVariable efficiency across cell types
Off-target effectsMinimal, sequence-based onlyCommon via seed region matchingSimilar to siRNA, plus insertion effectsPotential unintended genome modifications
Duration of effectTransient and dose-dependentTransientLong-term with stable integrationPermanent genetic modification
Speed to resultsFastFastSlow (weeks for stable lines)Slow (weeks for clonal selection)
Nuclear RNA targetsYes, works in nucleusLimited, primarily cytoplasmicLimited, primarily cytoplasmicIndirectly, by modifying DNA
Workflow complexityAdded to the mediumModerate (transfection optimization)Complex (cloning, viral packaging)Most complex (design, cloning, selection)

sdASO vs. siRNA (RNAi)

Small interfering RNAs (siRNAs) rely on the RISC complex to bind and cleave target mRNA. Unlike siRNAs, which typically require cationic lipid transfection, AUMsilence sdASOs are self-delivering and are taken up by endocytosis, with no transfection reagent, no carrier and no vector.

siRNAs need to unwind and integrate into RISC, which can sometimes lead to unintended gene silencing via seed-based off-target effects. AUMsilence sdASOs avoid RISC entirely, and their specificity is encoded in the direct Watson-Crick base pairing to the target RNA.

For in vivo use, delivering siRNA often requires lipid nanoparticles or viral vectors. AUMsilence sdASOs are used for in vivo studies with no special formulation, nanoparticles, conjugates or viral delivery.

sdASO vs. CRISPR/Cas9 gene editing

CRISPR systems (Cas9 protein or mRNA plus guide RNA) are large and often require delivery methods such as viral vectors or electroporation, which can be inefficient and toxic to certain cell types. AUMsilence sdASOs are much smaller, and they are taken up by endocytosis.

CRISPR achieves permanent gene knockout by causing DNA mutations. This permanence can be problematic where only a transient knockdown is wanted or where a knockout is lethal. AUMsilence sdASO-induced knockdown is transient and can be titrated.

CRISPR off-targets involve unintended DNA cuts at similar genomic sequences, potentially causing mutations in untargeted genes. AUMsilence sdASOs do not alter DNA, and their off-target effects at the RNA level are minimal due to very high sequence specificity.

Design principles

ASO design principles

Target site selection

Not all regions of an RNA are equally targetable. These are weighed for every candidate site along the transcript:

RNA structure:
Targeting accessible, single-stranded regions rather than highly structured areas
Sequence uniqueness:
Confirming that the chosen sequence has no significant homology to other transcripts
GC content:
Balancing binding strength with specificity
RNase H1 activation:
Designing the "gap" region so that its duplex with the target RNA recruits RNase H1 for cleavage

The AUMsilence platform combines these factors to select target sites for knockdown with minimal off-target effects. Knockdown is target and cell-type dependent and ranges from 70% to 95%.

Chemical modifications

The AUMsilence platform incorporates these chemical modifications:

Phosphorothioate backbone:
Replacing oxygen with sulfur in the oligonucleotide backbone raises resistance to nuclease degradation
Sugar modifications:
Chemical modifications enhance binding affinity while maintaining RNase H1 compatibility
Gapmer architecture:
The central "gap" region forms the duplex that recruits RNase H1, while modified flanking regions enhance stability and cellular uptake
Self-delivery enhancements:
Our AUMsilence platform uses chemical modifications that enable self-delivery.

These modifications are applied according to the needs of each product, for different RNA targets and experimental contexts.

FigureThe gapmer
The gapmer: a modified flank at each end, a central DNA gap paired to the target RNA where RNase H1 acts, and a phosphorothioate backboneA single antisense oligonucleotide drawn as beads on a ribbon, paired base for base to a target RNA drawn beneath it. The beads at each end are filled and labeled as a modified flank; the beads between them are open and labeled as the DNA gap. RNase H1 is drawn as a translucent disc over the pair at the gap, and the RNA is marked as cut opposite the gap, with the words RNase H1 acts here beneath. Small marks between the beads mark the linkages of the backbone, labeled phosphorothioate backbone. The figure is a schematic and not to scale.MODIFIED FLANKDNA GAPMODIFIED FLANKANTISENSE OLIGONUCLEOTIDETARGET RNARNase H1 acts herePHOSPHOROTHIOATE BACKBONE
Schematic, not to scale.
Facts and references
  1. RNase H1 cleaves the RNA strand of an RNA:DNA hybrid, and the central run of DNA-like bases, the gap, is what the enzyme needs; the cut falls on the target opposite the gap. [R01] [R02] [R03]
  2. The modified flanks on either side of the gap raise the affinity of the pairing to the target and protect the oligonucleotide from nucleases, while the gap keeps the enzyme's activity. [R04] [R01]
  3. The phosphorothioate backbone, a sulfur in place of an oxygen at each linkage, raises the oligonucleotide's resistance to nucleases. [R05] [R01]
  1. [R01] Crooke ST, Liang XH, Baker BF, Crooke RM (2021). Antisense technology: A review. The Journal of Biological Chemistry 296:100416. PMID 33600796, doi 10.1016/j.jbc.2021.100416.
  2. [R02] 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.
  3. [R03] 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.
  4. [R04] Monia BP, Lesnik EA, Gonzalez C, Lima WF, McGee D, Guinosso CJ, et al. (1993). Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression. The Journal of Biological Chemistry 268:14514-14522. PMID 8390996, doi 10.1016/s0021-9258(19)85268-7.
  5. [R05] Eckstein F (2014). Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Therapeutics 24:374-387. PMID 25353652, doi 10.1089/nat.2014.0506.

In vivo

In vivo applications and experimental workflows

AUMsilence sdASOs are used for in vivo studies with no special formulation, nanoparticles, conjugates or viral delivery.

In vivo delivery (no vehicle required)

siRNA delivered in vivo often needs a lipid nanoparticle or a viral vector. sdASOs designed using the AUMsilence platform can be administered without a delivery vehicle, conjugate, or formulation, by routes such as:

  • Subcutaneous injection
  • Intraperitoneal injection
  • Intravenous injection
  • Intrathecal injection

For example, to knock down a gene in a mouse model, AUMsilence sdASO is dissolved in saline and injected subcutaneously. Testing a dose range of 3-30 mg/kg in mice is recommended.

In vivo studies on this site
A rendering of a white laboratory mouse on a white ground; a syringe of violet liquid lies along its tail and its fine needle enters the side of the tail
Intravenous dosing by the lateral tail vein (illustration)

Stability and tissue penetration

Chemical modifications provide high stability in biological fluids. AUMsilence sdASO can circulate and penetrate tissues such as liver, muscle, and tumor xenografts.

For widespread systemic targets, intravenous (IV) injection can be used. For localized action (e.g., knocking down a gene in the central nervous system), intrathecal injection can introduce the ASO directly into the cerebrospinal fluid. No special formulations or conjugates are needed to achieve tissue uptake, though formulations can be used to further enhance delivery if desired.

A capillary runs across the top of the frame as a thin translucent tube with two flat nuclei in its wall; in its plasma four antisense gapmers, strands with a pale central gap between two violet wings, drift free, and two more pass down through the lower wall into the tissue beneath, where two cells sit side by side, each with a chromatin-filled nucleus in a pored envelope and a mitochondrion, and the left cell's membrane folds into a pit around one gapmer that is entering it.
Out of a capillary, into the tissue, into a cell

Products and protocols

The same AUMsilence sdASO sequence is used in a cell-based screen and in an in vivo model. No reformulation is needed between them.

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