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Antisense Oligo - A Precision Tools for Gene
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Custom Antisense Oligonucleotide (ASO) Synthesis

Potent, stable ASOs for RNase H–mediated knockdown or steric‑block applications.

Overview Advantages Applications Synthesis Options Modifications QC How to Order FAQ Contact

Overview

Antisense oligonucleotides (ASOs) modulate gene expression by sequence‑specific binding to RNA. Designs include RNase H–competent gapmers that induce mRNA cleavage and steric‑block chemistries that alter splicing or translation without cleavage.

Bio‑Synthesis offers custom ASO synthesis across PS DNA, 2′‑OMe, 2′‑MOE, 2′‑F, and LNA mixmer/gapmer architectures, plus advanced GalNAc, peptide, and lipid conjugations. Projects are delivered with comprehensive QC and optional functional testing.

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At-a-Glance

  • Chemistries: PS DNA, 2′‑OMe, 2′‑MOE, 2′‑F, LNA (mixmer/gapmer), steric‑block (backbone‑modified)
  • Typical length: 12–30 nt (gapmers 14–20 nt; steric‑block 18–25 nt)
  • Purification: RP‑HPLC or IEX‑HPLC; PAGE optional
  • Conjugations: GalNAc clusters, cholesterol/lipids, CPPs/peptides, PEGs
  • QC: MS (ESI/MALDI), analytical HPLC, OD260; optional Tm and functional assays
  • Scales: 1 mg to gram quantities (RUO → GLP/cGMP on request)

Key Advantages

Proven Potency

Gapmer designs recruit RNase H for efficient target knockdown with optimized wings/gap layout.

Enhanced Stability

Phosphorothioate (PS) backbones and 2′‑substitutions boost nuclease resistance and PK profile.

Versatile Conjugations

GalNAc, peptides, and lipids tailor biodistribution and cellular uptake for your model system.

Applications

  • mRNA knockdown (RNase H–dependent)
  • Splice modulation (exon inclusion/skipping)
  • Translation start‑site blocking
  • Functional genomics and target validation
  • Allele/SNP‑selective designs
  • In vitro, ex vivo, and preclinical studies
  • Fluorescently labeled uptake/trafficking studies
  • Controls: scrambled or mismatch ASOs
  • Diagnostic probe development

Custom Synthesis Options

Parameter Options
Scale mg to 1000 gram/batch
Length 12–30 nt standard (gapmers 14–20 nt; steric-block 18–25 nt)
Backbone Full or partial PS, PO, PN segments where specified
Sugar Mods 2′-OMe, 2′-MOE, 2′-F, cEt, LNA/BNA/UNA mixmer, morpholino; DNA in central gap for RNase H
Gapmer Layout Typical 8–10 nt DNA gap with 2–5 nt modified wings
Termini 5′ phosphate, 5′/3′ inverted dT, 3′-propanol, 3′-cholesterol (via linker)
Purification RP-HPLC, IEX-HPLC; desalting/UF; PAGE on request
Delivery Format Lyophilized sodium salt by default; buffer or counter-ion exchange on request
Controls Scrambled, 3–5 mismatch, and non-targeting controls available
QC Package MS (ESI/MALDI), analytical HPLC, OD260; optional Tm, nuclease-stability, endotoxin (LAL)

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Popular RNA Modifications

2′-O-Methyl (2′-OMe)

Improves nuclease resistance and reduces innate immune signaling.

2′-Fluoro (2′-F)

Enhances stability; often combined with 2′-OMe for siRNA.

Pseudouridine (ψ) / m1ψ

Base-level modification for reduced immunogenicity and improved translation.

Phosphorothioate (PS)

Terminal or patterned PS for exonuclease protection.

LNA / cEt Locks

Boosts Tm and affinity; strategic placements in guides/aptamers.

Conjugates

GalNAc, cholesterol, tocopherol, PEG, peptides; biotin/dyes/quencher sets.

Dye Labels
  • Fluorophores: 6-FAM, HEX, TET, JOE, VIC, TAMRA, ROX, Cy3, Cy5, Cy5.5, Cy7, Alexa Fluor 488/555/647, Atto 488/550/647
  • Quenchers: BHQ-1/2/3, Iowa Black FQ/RQ, DABCYL, Eclipse, BBQ-650
  • Pairings: FAM–BHQ1, HEX–BHQ1, Cy5–BHQ3, TAMRA–BHQ2
  • Positions: 5′, internal (base analogs), 3′; spacers HEG/TEG/PEG available

For more dye labeling, see full list of dye labelings

Target & Delvery Enhancers

Delivery Enhancer

Modification Description
GalNAc Liver-specific delivery via asialoglycoprotein receptors.
Peptide-ASO Conjugates CPPs improve celluar uptake and tissue targetings.
PEGylation reduces renal clearance, extends circulation time.
End Capping Protects from exonuclease degradation at 5' and 3' end
Nucleotide Analogues and Precision Enhancers

Analogues

Modification Description
Bridged Nucleic Acid (BNA) Improves antisense potency, reduce toxicity and degradation resistance.
Lock Nucleic Acid (LNA) Does not activate RNase H1, Enhances RNA target affinity at the cost of increase toxicity and precision of ASO activity.
Gapmer Design Facilitates RNase H-mediated cleavage; suited for cytoplasmic and nuclear RNA.
Sugar and Backbone Modifications

Sugar and Backbone Modifications

Modification Description
Phosphorothioate (PS) One non-bridging oxygen is replaced with sulfur; increases nuclease resistance and retains RNase H compatibility.
Phosphodithioate Both non-bridging oxygens replaced with sulfur; provides even greater stability.
Methylphosphonate Replaces a non-bridging oxygen with a methyl group; neutralizes backbone charge.
Boranophosphate Oxygen replaced with a borane group (BH₃); affects redox behavior, useful in biosensors.
Phosphoramidate / Phosphonamidate Substitutes one oxygen with an amine (–NR₂); reduces negative charge and alters hybridization.
PN Backbone (Phosphoramidate Nucleic Acid) Fully phosphoramidate-linked analog with synthetic stability and unique pairing properties.
Alkyl/Aryl Phosphotriesters Adds hydrophobicity and stability by modifying the phosphate group with alkyl/aryl chains.

Alternative Linkage & Orientation Modifications

Modification Description
2′,3′-Dideoxynucleosides Lacks both 2′ and 3′ hydroxyl groups; blocks extension, used in chain termination.
2′–5′ Linked Oligonucleotide Alters the typical 3′–5′ phosphodiester linkage to a 2′–5′ one, affecting structure and enzyme interaction.
5′→3′ Synthesis Reverse direction synthesis to achieve specific orientation or strand polarity.

Mirror Image & Chirality Modifications

Modification Description
Left Hand L-DNA Mirror-image of natural D-DNA; resistant to nucleases, non-immunogenic.
Left Hand L-RNA RNA enantiomer; enhances stability and evades immune detection.

Common Sugar and Backbone Modified Bases

Modification Description
2'-O-Methoxyethyl (2'-MOE) Larger alkyl group on 2'-O position; superior binding stability for antisense applications.
Morpholino Backbone Sugar-phosphate replaced with morpholine rings and phosphorodiamidate linkages; used in FDA-approved ASOs.
Thiomorpholino Backbone Sugar-phosphate replaced with morpholine rings and sulfur aton thiomorpholino phosphoramidates.
Peptide Nucleic Acid (PNA) Peptide-like backbone (polyamide); charge-neutral and highly stable.
LNA/BNA (Bridged Nucleic Acids) Sugar modifications that also rigidify the backbone, improving hybridization and stability.
Arabino Nucleic Acid (ANA) Sugar is arabinose instead of ribose; inverted stereochemistry at 2'-position use to enhance nuclease-resistant, reduced activity
Threose Nucleic Acid (TNA) Four-carbon sugar backbone; used in xenoucleic acid research, origin-of-life studies
2'-Amino RNA (2'-NH2) 2'-OH replaced by amino group; slightly stabilized dupelx, increase resistance, ideal for RNA therapeutic
Glycol Nucleic Acid (GNA) Minimalist synthetic analog using glycol backbones; useful in structural biology.
LNA/BNA (Bridged Nucleic Acids) Sugar modifications that also rigidify the backbone, improving hybridization and stability
UNA (Unlock Nucleic Acids) Missing bond between 2' and 3' carbons; increase flexility, reduce dulex stability
Triazole Linkage Non-natural linkage formed via click chemistry; biocompatible and enzyme-resistant.

Optional Add-On Services

Custom Formulation & Packaging

Buffers (TE/PBS/custom), aliquoting, concentrations, tubes or plates, OEM labels/barcodes.

Custom Quality Control

LC-MS, CE, extended HPLC traces, endotoxin/bioburden, water content, residual chemical analysis, stability program.

Regulatory & OEM

RUO→GLP→cGMP pathways, document packages, tech transfer, sequence masking.

Need something not listed?

We routinely implement bespoke chemistries and workflows.

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Our Benefits

40+ Years of Proven Expertise

Decades of experience delivering precision, reliability and innovation.

Comprehensive Chemistry Portfolio

Extensive modification options tailored to your research demands.

High-Throughput Efficiency

Hundreds of sequences per order - consistently, accurately and fast.

From Discovery to Clinical

Full-spectrum support - from concept through clinical-grade productions.

Step-by-step process from quote request to ongoing support.

Antisense Oligonucleotide (ASO) Technology & Benefits

What ASOs do: Antisense oligonucleotides are single-stranded DNA/RNA analogs that bind target RNA with high specificity to modulate gene expression. Depending on chemistry and design, ASOs (i) recruit RNase H1 to cleave the target RNA (gapmers) or (ii) act as steric blockers that alter splicing, prevent translation, or block RNA–protein interactions—often within the nucleus where siRNA is less effective.

How ASOs Work

  • RNase H1–mediated knockdown (Gapmer): Mixed-chemistry ASOs with a central DNA “gap” flanked by high-affinity wings (e.g., LNA or cEt) bind mRNA; RNase H1 cleaves the RNA strand, reducing transcript levels.
  • Steric-block mechanisms: Fully modified ASOs (e.g., 2′-MOE/2′-OMe/LNA) bind without recruiting RNase H1 to alter splicing (exon skipping/inclusion), block translation initiation, or disrupt RNA–protein binding.
  • AntimiR/miRNA inhibition: Short LNA-rich ASOs sequester mature miRNAs to de-repress target gene networks.

Design Principles

  • Length: 14–22 nt typical (gapmers often 16–20 nt).
  • Gapmer layout: Common 3-10-3 or 5-10-5 designs (LNA/cEt wings, DNA gap) on a full phosphorothioate (PS) backbone.
  • Targetability: Favor accessible RNA regions; avoid strong secondary structure and SNPs unless allele-selective design is intended.
  • Off-target minimization: Whole-transcriptome homology screens; avoid CpG motifs (TLR9) and mitigate with 2′-mods.
  • PK/PD tuning: PS content, wing composition (LNA/cEt/2′-MOE), and conjugates (e.g., GalNAc) shape exposure, distribution, and potency.

Benefits at a Glance

  • Programmable & versatile: Knockdown, splice modulation, or translational block with the same platform.
  • Nuclear activity: Effective for pre-mRNA splicing targets where siRNA is less active.
  • High potency & durability: RNase H gapmers yield sustained knockdown; steric blockers enable precise transcript remodeling.
  • Enhanced stability: 2′-MOE/2′-OMe/LNA/cEt and PS backbones resist nucleases and support systemic delivery.
  • Conjugation-ready: GalNAc (liver), peptides, lipids, and antibodies for targeted uptake.
  • Straightforward manufacturing: Single-stranded, fully synthetic; scalable from RUO to GLP/cGMP.
ASO mechanisms: RNA degradation, RNase H activity, and exon skipping.
ASO mechanisms of action: RNase H knockdown, RNA degradation, and exon-skipping steric block.

We balance potency, safety, and PK by optimizing wing chemistry (LNA/cEt/2′-MOE), PS patterning, and conjugation (e.g., GalNAc) for your target tissue.

Use Case Why ASOs Help Typical Setup
mRNA knockdown (RNase H1) Sustained transcript reduction with single-stranded agent 3-10-3 LNA gapmer, full PS; HPLC; optional GalNAc for liver
Splice modulation (exon skip/inclusion) Precise pre-mRNA editing to restore or remove exons Fully 2′-MOE/2′-OMe/LNA steric-block ASO; PS; length 18–20 nt
miRNA inhibition (antimiR) De-repress gene networks controlled by a pathogenic miRNA Short LNA-rich ASO (16–22 nt), full PS; optional cholesterol/PEG
lncRNA targeting (nuclear) Knockdown of nuclear noncoding RNAs not amenable to siRNA LNA/cEt gapmer; PS; design to accessible nuclear regions
Tissue-targeted delivery Higher exposure in desired tissue with reduced dose GalNAc-ASO for hepatocytes; peptide/lipid conjugates for others

Quality Assurance

  • Mass spectrometry (ESI/MALDI) for identity
  • Analytical HPLC for purity profile
  • OD260 and concentration report
  • Optional: Tm, nuclease-stability, endotoxin (LAL), and functional RNase H/knockdown assays
  • Documentation: RUO by default; GLP/cGMP support on request

Typical Turnaround

Standard labeled ASOs: 2–3 weeks from order confirmation. Conjugated or multi-step mixmer/gapmer architectures may require additional time.

Lead time depends on sequence, modification density, and conjugation strategy; rush options may be available.

How to Order

  1. Share target gene/region, species, and objective (knockdown vs splice/steric-block).
  2. Select chemistry (gapmer vs steric-block), modifications, and any conjugations.
  3. Choose scale, purification, and optional QC/functional tests.
  4. Receive a same-day quote and timeline.

For padlock probes, include target sequence and intended ligase/polymerase to optimize junction design.

Design Checklist

  • Gapmer layout (wings/gap) or steric-block length
  • Backbone (PS/PO mix) and sugar mods (2′-OMe/MOE/F/LNA)
  • Conjugation type (GalNAc, lipid, peptide, antibody)
  • Controls and assay readouts

FAQ

What are antisense oligonucleotides (ASOs)?

ASOs are short, synthetic single-stranded nucleic acids designed to bind to complementary RNA sequences to modulate gene expression.

What is the typical length of an ASO?

Most ASOs are 15–22 nucleotides long, though exact length depends on target accessibility and mechanism of action.

What types of modifications are routinely used in antisense ASO experiments?

Phosphorothioate (PS) bonds are added to increase nuclease resistance but may lower melting temperature and increase non-specific protein binding.

5-Methylcytosine (5-Me-dC) is used to prevent TLR9 activation from CpG motifs. Other common modifications include 2′-MOE, LNA, and BNA to enhance affinity and stability.

References:
- Tsuyoshi Yamamoto, Mol Ther–Nucleic Acids, 2012
- Lennox KA, Behlke MA, Methods Mol Biol, 2020

Do I need HPLC purification for my ASO?

HPLC is generally not required for in vitro use. For in vivo applications, HPLC combined with Na⁺ salt exchange and SEC chromatography is recommended to reduce toxic byproducts.

How much PS should I include?

Most ASOs use a full PS backbone for stability; selective PO islands can tune properties. We’ll recommend a PS pattern appropriate for your application.

Do you offer GalNAc conjugation?

Yes. We provide triantennary GalNAc and related clusters for hepatocyte targeting via ASGPR, as well as lipid and peptide alternatives for other tissues.

How should ASOs be stored?

Store lyophilized ASOs at 4 °C (short‑term) or −20 °C (long‑term). For solutions, use nuclease‑free buffer, aliquot, and keep at −20 °C to minimize freeze‑thaw cycles.

What buffer should I use to resuspend ASOs?

Use sterile nuclease-free water or 10 mM Tris buffer (pH 7.5–8.0).

Are ASOs toxic to cells?

Toxicity depends on sequence, chemistry, delivery method, and concentration. PS-ASOs may bind non-specifically to proteins and activate immune receptors.

How much Antisense Oligo (ASO) should I use in my knockdown experiment?

Typical dose-response studies use 1–30 nM for lipid-based delivery into immortalized cell lines. Optimization is necessary per target and system.

What is the role of ASO in Gapmer design?

Gapmer ASOs contain modified flanks (e.g., 2′-MOE, LNA) and a central DNA region. This design allows RNase H to cleave the target RNA while enhancing binding and stability.

What is the best way to deliver oligos into cells in culture?

Delivery options include liposomal transfection, ligand conjugation (e.g., peptides, GalNAc, cholesterol), electroporation, or gymnotic (naked) delivery.

Choice depends on the cell type, ASO chemistry, and intended use.

Can I use ASOs in serum-containing media? What’s the expected half-life?

Yes, PS-modified ASOs can be used in serum. To reduce degradation, inactivate exonucleases by heating serum to 65°C for 30 minutes.

Half-life is highly variable and depends on the ASO sequence, chemistry, and biological context.

References:
- Lennox KA et al., Pharm Res. 2010
- Lennox KA et al., Mol Ther Nucleic Acids. 2013

Gapmer vs steric‑block—what’s the difference?

Gapmers contain a central DNA gap flanked by modified wings; they recruit RNase H to cleave the target RNA. Steric‑block ASOs use fully modified backbones to physically block splicing or translation without cleavage.

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