Nuclease Resistance & Stabilizer Bases for Oligonucleotides

Engineer oligonucleotides for nuclease resistance and hybridization stability using stabilizer bases, 2′‑sugar chemistries (2′‑OMe, 2′‑F, MOE, LNA), and backbone controls (PS, end‑caps, spacers). From screening to RUO → GMP‑like, Bio‑Synthesis provides HPLC/PAGE, optional LC‑MS, ISO‑aligned docs, and tech transfer.

Overview & Services

Lewisville, TX ASO • siRNA • Aptamer 2′-OMe • 2′-F • LNA • PS RUO → GMP-like

Bio-Synthesis Inc. engineers oligonucleotides for nuclease resistance and hybridization stability using a toolkit of stabilizer bases, 2′-sugar chemistries (2′-OMe, 2′-F, MOE, LNA) and backbone controls (phosphorothioate, terminal caps, spacers). We help you balance potency and protection—tuning Tm, enzyme compatibility, and off-target risk—from screening to RUO → GMP-like manufacturing with HPLC/PAGE and optional LC-MS, ISO-aligned documentation, and tech transfer support.

Service At-a-Glance
  • RUO → GMP-like manufacturing with ISO-aligned SOPs (ISO 9001/13485-ready docs).
  • Purification: Desalt, PAGE, HPLC; optional LC-MS verification.
  • Plates & kitting, barcoding, custom buffer/format, cleanroom aliquoting.
  • Method development & validation (HPLC, LC-MS, UV) and protocol transfer.
  • Release testing: ID, purity, loading, endotoxin (where applicable).
  • Stability studies (real-time/accelerated) with interim reporting.
  • OEM / private-label programs and long-term supply agreements.
  • IND/IDE support packages (method summaries, CoA templates, change control).
  • Tech transfer, stability studies, and release testing with certificate packages.
Formats
Tubes • Plates • Kitting
Scale
µmol → multi-gram
QC
HPLC/PAGE • LC-MS
Supply
RUO → GMP-like
Additional Services
  • Method development & validation (HPLC, LC-MS, UV) and protocol transfer.
  • Release testing: ID, purity, loading, endotoxin (where applicable).
  • Stability studies (real-time/accelerated) with interim reporting.
  • Custom packaging & kitting: plates, vials, barcodes, cold-chain logistics.
  • OEM / private-label programs and long-term supply agreements.
  • Scale-up: pilot to multi-gram/kilo with dedicated synthesizers.

Sugar Modifications

Modification Structure (schematic) Mechanism & Notes Stability Impact Typical Use
2′‑O‑Methyl (2′‑OMe) O‑Me 2′‑O substitution reduces RNase access; maintains A‑form geometry and improves duplex stability. Moderate nuclease resistance; +Tm. ASO wings, probes
2′‑Fluoro (2′‑F) F Electronegative F biases C3′‑endo; tightens duplex; strongly reduces RNase susceptibility. High resistance; +Tm. siRNA, ASO
2′‑MOE MOE Bulky ethoxy group blocks nucleases; widely used in clinical ASOs (gapmers). Strong resistance; +Tm. ASO gapmer wings
LNA / BNA / cEt Bridge between 2′‑O and 4′‑C preorganizes sugar; exceptional affinity and exonuclease resistance. Very high resistance; large +Tm. ASO, probes
ENA / AmNA bridge Alternative bridged sugars; balance affinity, toxicity, and stability for newer ASO designs. High resistance; +Tm. ASO
UNA (Unlocked) Acyclic sugar increases flexibility; often reduces Tm; used to modulate structure. Resistant but lower binding. Structure tuning
GNA / TNA / HNA XNA Artificial sugars resist biological enzymes; enable specialized duplexing and high biostability. Excellent resistance; variable Tm. Diagnostics, research
Technical notes
2′-OMe vs 2′-F vs 2′-MOE
  • 2′-OMe: moderate resistance; great for probes & ASO wings.
  • 2′-F: strong resistance; raises rigidity (A-form bias).
  • 2′-MOE: clinical workhorse for ASOs (gapmer wings).
LNA/BNA/cEt Dosing
  • High Tm boost; excellent nuclease resistance.
  • Limit %LNA in long wings to control off-target binding.
  • Consider alternating LNA/2′-OMe mixmers for balanced affinity.
XNA (GNA/TNA/HNA)
  • Exceptional biostability; enzyme-orthogonal.
  • Use as segments; validate duplexing to DNA/RNA targets.

Base Analogs (Stability‑Aiding)

Modification Structure (schematic) Mechanism & Notes Impact
5‑Me‑dC / 5‑Me‑dU CH₃ Enhanced hydrophobicity and stacking; modest stabilization. Slight +Tm, limited nuclease effect.
Halogenated dU/dC (5‑Br/5‑F/5‑I) X Improved stacking; minor protection; can tune photoreactivity. Small +Tm, minor resistance.
Ψ / s²U Ψ Improves RNA stability/translation (mRNA); slightly harder to hydrolyze. Modest stability; context‑dependent.
Technical notes
Usage Note

Base analogs are best used alongside sugar/backbone changes for robust nuclease resistance.

Backbone Modifications

Modification Structure (schematic) Mechanism & Notes Stability & Use
Phosphorothioate (PS) S S substitution at non‑bridging oxygen disrupts nuclease binding; standard for ASO/siRNA stability. Moderate–strong resistance; improves PK; used in gapmers and mixmers.
Phosphorodithioate (PS₂) S S Both non‑bridging oxygens replaced with sulfur; higher hydrophobicity and resistance. Very strong resistance; advanced designs.
Boranophosphate (PB) BH₃ BH₃ substitution increases lipophilicity; reduces nuclease affinity; retains charge. Strong resistance; delivery benefits.
Phosphoramidate (PN) / Methylphosphonate (PM) N/Me Alters charge density (PN) or neutralizes (PM); lowers nuclease recognition. Excellent resistance; monitor duplex Tm.
PMO (Morpholino) neutral Morpholine rings with phosphorodiamidate linkage; neutral backbone is enzyme‑proof. Extreme resistance; FDA‑approved class.
PNA peptide-like Peptide‑like N‑(2‑aminoethyl)‑glycine backbone; no phosphodiester → no nuclease sites. Extreme resistance; diagnostics/therapeutics.
Technical notes
PS Stereochemistry
  • Rp/Sp configuration can affect protein binding and potency.
  • Stereo-defined PS offers improved consistency in some contexts.
PMO vs PNA
  • PMO: neutral, high stability; common for splice-modulating ASOs.
  • PNA: extreme stability; strong binding; consider delivery conjugates.
PB / PN / PM
  • PB: lipophilic; delivery synergy.
  • PN: charge modulation; high resistance.
  • PM: neutral; expect lower duplex Tm.

Workflow

We combine base, sugar, and backbone strategies—universal bases where appropriate—with controlled phosphorothioate (PS) patterning, 2′‑OMe/2′‑F/LNA placement, end‑caps, and spacers to improve nuclease resistance while maintaining activity.

Select Stabilizer dI • 5NI • 3NP • K‑Base 2′‑OMe, 2′‑F, LNA PS pattern & end‑caps Design Placement Limit consecutive universals Buffer T m locally Polymerase constraints Build & Purify HPLC/PAGE • LC‑MS (opt.) Format & labeling Docs & QC package Nuclease Challenge Serum/lysate tests Compare layouts Scale/transfer
Select Stabilizer

dI • 5NI • 3NP • K-Base
2′-OMe, 2′-F, LNA
PS pattern & end-caps

Design Placement

Limit consecutive universals
Buffer Tm locally
Polymerase constraints

Build & Purify

HPLC/PAGE • LC-MS (opt.)
Format & labeling
Docs & QC package

Nuclease Challenge

Serum/lysate tests
Compare layouts
Scale/transfer

Technical Notes

Gapmer & Mixmer Patterns

Typical ASO gapmer: PS backbone throughout; 2′-MOE/2′-OMe/LNA in wings; DNA core for RNase H recruitment. Mixmer probes use LNA/BNA for affinity and 2′-OMe for stability.

Toxicology & Off-Target Considerations

High LNA content raises affinity; manage off-target risk via shorter wings and sequence screens. PS stereochemistry can affect protein binding; consider stereo-defined designs.

Assay Conditions

Adjust stringency (formamide/salt) for high-affinity chemistries; validate melting profiles; protect fluorophores during stability testing.

Antisense Oligo (ASO) — Quick Recipe

Two common ASO archetypes. Start here, then tune %LNA/2′-mods and PS patterning to balance potency, off-targets, and PK.

Gapmer (RNase H)
  • Backbone: PS across full length (default); consider stereo-enrichment for consistency.
  • Layout: 2–4 nt wings with 2′-MOE/2′-OMe/LNA; DNA core for RNase H.
  • Length: 14–20 nt typical.
  • Tips: High LNA ↑ affinity & off-target risk → shorten wings or lower %LNA.
  • QC: HPLC/PAGE; LC-MS where applicable.
Steric-Blocker (no RNase H)
  • Backbone: PS at termini or patterned; optional end-caps/spacers.
  • Layout: Fully 2′-modified (2′-OMe/2′-MOE/LNA mixmers).
  • Length: 18–22 nt typical.
  • Tips: Ensure on-target Tm in assay buffer; avoid immunostimulatory motifs.
  • QC: HPLC/PAGE; LC-MS as needed.
siRNA — Quick Recipe

Baseline duplex with stability and safety tweaks. Adjust modification density by matrix (serum/lysate/cell) and delivery route.

Duplex Layout
  • Length: 21-mer with 2-nt 3′ overhangs (dTdT common) on both strands.
  • Guide (antisense): 5′-phosphate; target-complementary.
  • Passenger (sense): Optional single mismatch to bias loading to guide.
  • Alt: 27-mer Dicer-substrate if your workflow prefers Dicer processing.
Chemical Stabilization
  • 2′-Mods: Mix 2′-OMe and 2′-F on both strands (e.g., enrich 2′-OMe in guide seed positions to curb off-targets).
  • PS pattern: 1–3 terminal PS linkages per end (both strands) to slow exonucleases.
  • Overhangs: dTdT or 2′-OMe-UU for added stability.
  • Dyes/handles: Add only on passenger strand termini if needed.
QC & Packaging
  • QC: HPLC on both strands; LC-MS confirmation where applicable.
  • Anneal: Equimolar strands; verify by native PAGE.
  • Format: Tubes or plates; RNase-free buffers; provide guide/passenger labels.
Gapmer — Quick Recipe

RNase H–competent ASO: high-affinity 2′-modified wings protect the ends; a DNA core recruits RNase H. Tune %LNA/2′-mods and PS patterning.

Layout
  • Backbone: PS across full length (default).
  • Wings: 2–4 nt each side with 2′-MOE / 2′-OMe / LNA.
  • Core: DNA for RNase H recruitment.
  • Length: 14–20 nt typical.
Parameters
  • Tm target: assay temp + 10–15 °C (buffer-specific).
  • PS stereochem: consider stereo-enriched/defined PS.
  • End-caps: 3′-inv-dT or spacer for exonuclease control.
  • Over-LNA caution: avoid long runs.
QC & Packaging
  • QC: HPLC/PAGE; LC-MS where applicable.
  • Stability: Serum/lysate challenge to compare layouts.
  • Format: Tubes/plates; barcoding; RUO → GMP-like docs.
Aptamer — Quick Recipe

Start with a validated/SELEX motif; then stabilize, truncate, and format for use. Buffer (Mg2+/K+) and folding conditions are critical.

Design & Layout
  • Length: 25–70 nt typical; truncate to minimal binding core.
  • Folding: validate in final buffer; include Mg2+ (1–5 mM); for G4 motifs ensure K+ (50–100 mM).
  • Multimerization: dimer/branched constructs can boost avidity.
Stabilization
  • Ends: 3′-inv-dT or 3′/5′ capping; optional spacers.
  • Sugar mods: 2′-OMe / 2′-F / LNA mixmers in non-critical positions.
  • Backbone: light PS at termini if needed (avoid over-stabilizing binding face).
Functionalization & QC
  • Handles: Biotin, dyes, PEG, cholesterol—place away from binding region.
  • QC: HPLC; LC-MS where applicable; binding assay (KD).
  • Format: Tubes/plates; RUO → GMP-like docs and kitting.

FAQ

Is “nuclease resistance” a modification or a function?

function/property. It is achieved by applying specific modification types (e.g., 2′-mod sugars, PS backbones, PMO/PNA).

What’s the fastest way to stabilize an ASO?

Start with a PS backbone + 2′-OMe/MOE/LNA wings (gapmer). Tune LNA content to manage off-targets and maintain potency.

When should I choose PMO or PNA?

Use when maximum nuclease resistance and neutrality are required (e.g., in vivo delivery studies). Consider conjugation for uptake.

What is BNA and how does it compare to LNA?

BNA (bridged nucleic acid) refers to LNA-like chemistries (e.g., cEt, ENA, AmNA) that pre-organize the ribose for higher affinity and nuclease resistance. They offer LNA-class stability with tunable safety profiles.

What is a thiomorpholino oligonucleotide?

A morpholino-class backbone variant incorporating thio features; like PMO, it is neutral and highly nuclease-resistant. It’s explored for splice modulation and delivery conjugation strategies.

Can Bio-Synthesis produce multi-gram stabilized oligonucleotides?

Yes. We support bench to multi-gram with phase-appropriate documentation and QC, and we can prepare tech-transfer packages for GMP-like manufacturing.

Do you offer stereodefined PS backbones?

We can discuss stereo-enriched or defined PS strategies on request, balancing potency, off-target protein binding, and manufacturability.

Which is better for extreme stability: PMO or PNA?

Both are enzyme-proof. PMO is widely used for splice-modulating ASOs; PNA shows ultra-strong binding but often benefits from delivery conjugates.

Speak to a Scientist



References & Further Reading

  • Deleavey GF, Damha MJ. Designing chemically modified oligonucleotides for targeted gene silencing. Chem Biol. 2012;19(8):937–954. doi:10.1016/j.chembiol.2012.07.011
  • Bennett CF, Swayze EE. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Ann Rev Pharmacol Toxicol. 2010;50:259–293. doi:10.1146/annurev.pharmtox.010909.105654
  • Prakash TP. An overview of sugar-modified oligonucleotides for antisense therapeutics. Chem Biodivers. 2011;8(9):1616–1641. doi:10.1002/cbdv.201100081
  • Obika S, Uneda T, Sugimoto T, et al. 2′-O,4′-C-methylene bridged nucleic acid (2′,4′-BNA): synthesis and triplex-forming properties. Bioorg Med Chem. 2001;9(4):1001–1011. doi:10.1016/S0968-0896(00)00325-4
  • Seth PP, et al. cEt bridged nucleic acids in antisense designs. J Med Chem. 2010. doi:10.1021/jm101207e
  • Summerton J, Weller D. Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 1997;7(3):187–195. doi:10.1089/oli.1.1997.7.187
  • Nielsen PE, Egholm M, Berg RH, Buchardt O. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science. 1991;254(5037):1497–1500. doi:10.1126/science.1962210
  • Egli M, Manoharan M. Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res. 2023;51(6):2529–2573. article
  • Bio-Synthesis, Inc. Company pages on stabilized oligonucleotides, LNA/2′-OMe/PS designs, and QC workflows. biosyn.com

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