Login / Register Order My Items
Contact Us
800.227.0627
Contact Us Quote Order Login / Register
Affinity & Hybridization Control

High-Affinity and Hybrdication Control Oligonucleotide for Research and Therapeutic

Increase duplex stability, enforce specificity, or soften secondary structure using LNA/BNA/cEt and 2′‑RNA variants (2′‑OMe, 2′‑F, 2′‑MOE), paired with backbone frameworks (PS, PM, PN) and high‑selectivity base analogs. Built for probes, antisense (RNase H or steric‑block), and siRNA.

LNA • cEt • ENA
2′‑OMe • 2′‑F • MOE
ZNA® (spermine)
5‑propynyl‑dC/dU
5‑Me‑dC
RUO → GMP‑like
Overview What is it? Sugar Mods Base Mods Charge/Backbone Related Services FAQ

High-Affinity Modified Base Synthesis

Lewisville, TX LNA • cEt • ENA 2′‑OMe • 2′‑F • MOE ZNA® (spermine) RUO → GMP‑like

Bio‑Synthesis Inc. designs and manufactures affinity‑enhanced oligonucleotides—hybrid molecules that combine base‑stacking, sugar‑locking and charge‑modulating chemistries to push hybridization performance beyond natural limits. We integrate LNA/cEt/ENA bridged sugars, 2′‑OMe / 2′‑F / 2′‑MOE RNA analogs, and ZNA® cationic conjugates to deliver short, high‑Tm probes and therapeutic‑grade oligos with exceptional duplex stability, specificity, and mismatch discrimination.

From research‑grade qPCR probes and aptamer scaffolds to clinical‑bound ASO and siRNA candidates, our team provides end‑to‑end support—sequence design and Tm modeling, synthesis and purification, method development, and RUO → GMP‑like documentation packages.

Service At‑a‑Glance
  • High‑Affinity Design: LNA/cEt/ENA wings, 2′‑OMe/2′‑F patterning, ZNA® loading; probe shortening & mismatch control.
  • Synthesis & Scale‑Up: 50 nmol → multi‑gram with ISO‑aligned SOPs.
  • Purification: Desalt, PAGE, HPLC; optional LC‑MS verification.
  • Customization: Dyes, quenchers, biotin; PEG/lipid (e.g., cholesterol), GalNAc; click‑ready handles.
  • QC & Documentation: HPLC, LC‑MS, melt profiling; RUO/GLP/GMP‑like doc sets & CoA templates.
  • Stability: Serum/lysate challenge studies; accelerated/real‑time stability with interim reporting.
  • Kitting & Packaging: Tubes/plates, barcoding, cold‑chain; OEM/private‑label supply programs.
  • Tech Transfer: Method development/validation and transfer packages for scale‑up.
Formats
Tubes • Plates • Kitting
Scale
50 nmol → multi‑gram
QC
HPLC/PAGE • LC‑MS
Supply
RUO → GMP‑like

What Are Affinity‑Enhanced Oligonucleotides?

Affinity‑enhanced oligos combine sugar locking (LNA/cEt/ENA; 2′‑OMe/2′‑F/MOE), base‑stacking enhancers (5‑propynyl‑dC/dU, 5‑Me‑dC, ΨiC) and charge modulation (ZNA®) to deliver high‑Tm, shorter probes and tighter specificity.

  • Benefits: faster on‑rates, higher duplex Tm, improved mismatch discrimination.
  • Outcome: robust assays at shorter lengths and stringent wash temperatures.
  • Used in: qPCR, molecular beacons, siRNA, gapmer ASO, aptamer and MERFISH/Imaging application.

Selection Guide

Family ΔTm / Affinity Mismatch Disc. Nuclease RNase H Typical Uses
LNA/BNA/cEt ↑↑ per base ↑↑ ↑ Gapmer wings only Short probes; ASO wings; SNP clamps
2′‑OMe ↑ ↑ ↑ No (silent) siRNA/ASO tuning; RNA probes
2′‑F ↑↑ (RNA) ↑ ↑ No (silent) siRNA; high RNA affinity
2′‑MOE ↑ ↑ ↑↑ No (silent) ASO steric‑block; aptamers
PS ~ ~ ↑↑ Yes (with DNA gap) ASO gapmers; splice modulation
PM/PN ~ to ↓ ↑ ↑↑ No Steric‑block antisense; uptake/PK tuning
PNA/PMO ↑↑ (PNA); ↑ (PMO) ↑↑ ↑↑ No Clamps; in vivo steric‑block
Tip: To hit a precise Tm in short probes, mix a few LNA/cEt bases with 2′‑OMe or DNA; add 5‑propynyl‑dC/‑dU for extra lift.

Sugar Modifications (Affinity‑Enhancing)

Modification / Abbreviation Mechanism of Affinity Enhancement ΔTm/substitution Application
2′‑O‑Methyl (2′‑OMe) Improves base stacking & reduces backbone flexibility; increases duplex stability & nuclease resistance +0.5 to +1.0 °C ASO, siRNA, aptamer stabilization, qPCR
2′‑O‑Methoxyethyl (2′‑MOE) Enhances hydration shell & base stacking; reduces RNase attack +1.0 °C ASO therapeutics, gapmers
2′‑Fluoro (2′‑F) Increases sugar puckering (C3′‑endo), enhancing A‑form duplex +1.0 °C siRNA, ASO
Locked Nucleic Acid (LNA) Locks sugar in C3′‑endo (A‑form); increases rigidity and affinity +2 – +8 °C ASO, gapmers, probes, aptamers
Bridged Nucleic Acid (BNA) Ring‑bridged ribose analogs up to +7 °C ASO, RNAi
cEt (Constrained Ethyl) Similar to LNA; improved safety and affinity +2 – +4 °C ASO, gapmer
ENA (Ethylene‑Bridged Nucleic Acid) Hybridizes tighter with RNA; nuclease resistant +4 °C Antisense, siRNA
ZNA® (Zip Nucleic Acid) Neutralizes phosphate charges; reduces repulsion +1 – +10 °C (tunable) qPCR, primers, probes
GNA (Glycol Nucleic Acid) Compact structure; high pairing selectivity +2 °C Antisense, molecular design
TNA (Threose Nucleic Acid) Forms A‑type duplex with RNA +1 °C Prebiotic analog, aptamer
UNA (Unlocked Nucleic Acid) Increases flexibility; may reduce Tm slightly but can improve mismatch discrimination −1 °C (context‑dependent) siRNA, aptamer fine‑tuning
5′‑C‑ or 4′‑C‑Substituted Ribose Enhances stacking and hydrophobicity variable ASO, aptamer
2′‑O‑Aminopropyl (2′‑O‑AP) Adds cationic charge, enhances electrostatic attraction +1 – +3 °C Probes, modified primers
2′‑O‑Allyl / 2′‑O‑Propargyl Improved stacking, compatible with click chemistry +0.5 °C Hybrid probes, conjugation platforms
Technical Notes
LNA/BNA/cEt Loading
  • Gapmers: 3–5 residues per wing; avoid contiguous >4 in short probes.
  • Short probes: reduce length as ΔTm increases to keep specificity.
2′‑OMe vs 2′‑F vs MOE
  • 2′‑F: higher per‑base ΔTm, A‑form bias; mix with 2′‑OMe for balance.
  • MOE: good wings for therapeutics; pair with PS.
Aptamers
  • Prefer inserts in stems, not loops/binding face.
  • Validate folding in final Mg2+/K+ buffer.

Base Modifications (Stacking/Pairing Enhancers)

Modification / Abbreviation Mechanism of Affinity Enhancement ΔTm/Substitution Application
5‑Methyl‑dC (5mC) Enhances stacking, mimics natural CpG methylation +0.5 °C DNA stabilization, antisense
5‑Propynyl‑dC / dU Increases base stacking & duplex rigidity +1 – +2 °C PCR probes, ASO
5‑Hydroxymethyl‑dC (5hmC) Hydrogen bonding, hydration shell +0.5 °C Epigenetic analogs, hybridization control
Pseudoisocytidine (ΨiC) Enhances A‑form pairing with RNA +1 °C RNA pairing improvement
2‑Aminopurine (2‑AP) Enhances H‑bonding & stacking +0.5 °C Fluorescent reporter, hybrid stability
LNA‑A, LNA‑C, LNA‑G, LNA‑T Combined sugar/base locking +2 – +8 °C High‑affinity probes
C‑5 Halogenated Pyrimidines (5‑Br‑U, 5‑I‑U) Increase base polarizability, stacking +0.5 – +1 °C Stabilizing duplex, photo‑reactive
L‑DNA / L‑RNA (mirror‑image) Not hybridizing to native DNA/RNA but improves nuclease resistance; used in aptamer “Spiegelmers” — Therapeutic aptamers
Benzimidazole / Imidazopyridine Bases π‑stacking & minor‑groove interactions +1–3 °C Artificial bases, affinity tuning
Pyrrolo‑dC / Iso‑G / Iso‑C Enhanced hydrogen bonding, base pairing +1 °C Synthetic genetics, probes
Technical Notes
Base‑Stacking & Labeling
  • 5‑Propynyl‑dC/dU near labels restores ΔTm after dye/quencher insertion.
  • Avoid clustering propynyl next to strong quenchers to preserve dynamics.
Fine Tuning
  • 5‑Me‑dC: modest ΔTm; combine with LNA/2′‑mods for small adjustments.
  • ΨiC: stabilizes RNA‑like duplex character; verify in final salt matrix.

Backbone Frameworks (Stability, PK, & Mode of Action)

Backbone Description RNase H Typical use Notes Code
PS (phosphorothioate) Nuclease resistance↑; small Tm↓/linkage Yes (DNA gap) ASO gapmers; splice modulation Protein binding↑; tune patterning [PS]
Stereopure‑PS Defined Rp/Sp stereochemistry Yes (DNA gap) Next‑gen ASO design Fine‑tune PK/protein contacts [PS‑Rp/Sp]
PM (methylphosphonate) Neutral backbone; nuclease↑↑ No Steric‑block antisense; uptake Chiral; Tm↓ per linkage [PM]
PN (3′–5′ phosphoramidate) Nuclease↑; altered charge No Steric‑block; aptamers Often raises RNA affinity [PN]
PNA Peptide backbone; neutral; very high affinity No Clamps; SNP detection Salt/temperature behaviors differ [PNA]
PMO Morpholino; neutral backbone No In vivo steric‑block Well‑known in zebrafish, etc. [PMO]

Charge / Backbone (Terminal & Scaffold)

Modification / Abbreviation Mechanism of Affinity Enhancement ΔTm/Substitution Application
ZNA® (Zip Nucleic Acid) Neutralizes phosphate charges; reduces repulsion +1 – +10 °C (tunable) qPCR, primers, probes
Technical Notes
ZNA® — Principle & Tuning
  • Cationic spermine units reduce phosphate repulsion → faster hybridization and higher Tm.
  • ΔTm scales with the number of spermine units; optimize per oligo length and GC content.
  • Typical increase: +1 – +10 °C per modification series.
Starter Loads & Placement
  • ≤18-mer: 1 unit • 19–24-mer: 2 units • 25–30-mer: 2–3 units.
  • Place units at termini or spaced evenly for short probes.
  • Avoid excessive cationic load to maintain solubility and enzyme compatibility.

Verify performance empirically via melt curves; adjust Mg2+/Na+ buffer accordingly.

Design Suggestions
  • Charge Tuning: Start with 1–2 units; titrate until the desired Tm is reached.
  • Placement Strategy: Favor terminal conjugation; distribute units if the sequence < 20 nt.
  • Enzyme Performance: Confirm polymerase compatibility with ZNA-modified primers.
  • Specificity Control: Keep probe lengths compact; confirm by melt-curve discrimination.
Benefits & Design Trade-Offs
Benefits Challenges / Considerations
  • Higher duplex Tm at shorter probe lengths.
  • Improved hybridization kinetics (lower electrostatic barrier).
  • Tunable affinity through spermine unit count & placement.
  • Over-loading may cause solubility loss or non‑specific interactions.
  • Requires enzyme optimization for PCR/qPCR workflows.
  • Fine-tune unit number vs. GC % and ionic strength.

Ready to design your Affinity Enhancing Oligonucleotide?

Ordering checklist
  • Sequence & Targets — Final sequence(s) with IDs/length; application (qPCR/ASO/siRNA/aptamer/ISH); target Tm and assay buffer/temperature.
  • Chemistry & Mods — LNA/cEt/ENA and 2′-OMe/2′-F plan; PS pattern; ZNA® units/placement; base mods (5-propynyl, 5-Me-dC); caps/spacers; dyes/quencher/biotin/PEG.
  • Scale, Purity & QC — Scale (50 nmol → multi-gram); Desalt/PAGE/HPLC; QC (UV, LC-MS if applicable); extra tests & acceptance criteria.
  • Format & Packaging — Tubes vs plates (include map); buffer & concentration; aliquots; dry vs lyo; barcodes/labels; kitting/special handling.
  • Docs, Logistics & Timelines — RUO→GMP-like docs (CoA, methods); stability needs; due date & ship temp; PO/quote #; ship-to & billing contacts.
Speak to a Scientist

FAQ

What are affinity-enhanced oligonucleotides?

Affinity-enhanced oligonucleotides are custom DNA/RNA sequences engineered with chemistries that raise duplex stability (high-Tm) and sharpen specificity. Common approaches include LNA/cEt/ENA (bridged sugars), 2′-OMe/2′-F (RNA analogs), and ZNA® (cationic spermine conjugation) to reduce electrostatic repulsion and accelerate hybridization—ideal for qPCR probes, antisense, siRNA, and aptamers.

Which modification increases duplex stability most effectively?

Per-base, LNA/cEt/ENA typically deliver the largest ΔTm gains and strong mismatch discrimination. ZNA® adds tunable, global stabilization via spermine units, often enabling shorter probes without sacrificing Tm. The best choice depends on sequence length, GC%, enzyme step (PCR/RT), and application (probe vs. ASO/siRNA).

Can LNA and ZNA modifications be combined?

Yes. Many designs pair LNA/cEt (localized per-base affinity and specificity) with ZNA® (global charge tuning) to reach target Tm at compact lengths. Start modestly (e.g., 1–2 ZNA units) and verify with melt curves; avoid over-stabilization that can impact enzyme kinetics or raise non-specific binding.

Do you offer GMP-grade oligo manufacturing?

We provide ISO-aligned workflows, phase-appropriate QC (HPLC/PAGE, LC-MS) and RUO → GMP-like documentation packages. For full GMP needs, we can scope enhanced documentation and tech-transfer support; contact us to align on quality requirements and timelines.

How should I design a Gapmer antisense oligonucleotide?

Gapmers: Use LNA/cEt/ENA wings (3–5 nucleotides per side) around a DNA core to enable RNase H activity. Incorporate phosphorothioate (PS) linkages throughout and target an assay temperature about +10–15 °C above hybridization temperature for optimal potency and selectivity.

What’s the best modification pattern for siRNA design?

siRNA: Pattern 2′-OMe and 2′-F alternately to balance safety, potency, and A-form stability. Add 1–3 phosphorothioate (PS) linkages at both termini, and avoid heavy LNA loading in the guide seed region to maintain effective RISC loading and minimize off-target binding.

How can I optimize probe or qPCR oligo designs?

Probes/qPCR: Shorten probe length while maintaining target Tm using LNA or ZNA® modifications. Incorporate 5-propynyl bases near dyes or quenchers to restore stacking and maximize signal-to-noise (S/N) performance for hydrolysis and beacon assays.

How can I improve aptamer stability and folding?

Aptamers: Add sparse 2′-OMe or LNA substitutions within stem regions to enhance nuclease resistance while preserving 3D folding. Avoid modifying the binding face, and validate structural integrity in the final Mg²⁺/K⁺ buffer to maintain target affinity and activity.

LNA vs ZNA: which should I choose for high‑Tm probes?

LNA gives the biggest per‑base ΔTm and mismatch discrimination; ZNA® is ideal when you want to keep sequence length but raise Tm via spermine loading.

How much ΔTm per LNA or cEt residue?

Design‑ and sequence‑dependent, but typically +2–+8 °C per insertion for LNA‑class residues.

2′‑OMe vs 2′‑F for siRNA?

Mix both: 2′‑F increases A‑form bias and Tm; 2′‑OMe is helpful for safety and reduces off‑targeting, especially in the seed region.

How do you design LNA/cEt wings for gapmers?

Start with 3–5 nt wings on each side, PS backbone throughout, and tune ΔTm to assay temp +10–15 °C.

Do you provide HPLC/LC‑MS and RUO→GMP‑like documentation?

Yes—phase‑appropriate QC (HPLC/PAGE, LC‑MS) and documentation sets, with tech‑transfer packages on request.

Speak to a Scientist


Email Instead

References

  1. 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
  2. Koshkin A.V., et al. LNA (locked nucleic acid): synthesis and high-affinity nucleic acid recognition. J Am Chem Soc. 1998;120(50):13252–13253. doi:10.1021/ja982422x
  3. Martin P. 2′-O-(2-methoxyethyl)-modified ribonucleosides (MOE): synthesis and properties. Helv Chim Acta. 1995;78(3):486–504. doi:10.1002/hlca.19950780303
  4. Deleavey G.F., Damha M.J. Designing chemically modified oligonucleotides for targeted gene silencing. Chem Biol. 2012;19(8):937–954. doi:10.1016/j.chembiol.2012.07.011
  5. Seth P.P., et al. Constrained ethyl (cEt) nucleic acids in antisense design. J Med Chem. 2010;53(21):8309–8318. doi:10.1021/jm101207e
  6. Vasseur J-J., Debart F., Robbins M. Zip Nucleic Acid (ZNA®): cationic oligonucleotide conjugates for enhanced hybridization. Bioconjug Chem. 2010;21(7):1231–1239. doi:10.1021/bc100105m
  7. (Companion ZNA® papers in the same timeframe—synthesis/biophysics series, see issue with Vasseur et al.).
  8. Thomas T., Thomas T.J. Polyamines in cell growth and nucleic acid interactions. Cell Mol Life Sci. 2001;58:244–258. doi:10.1007/PL00000852
  9. (General polyamine–nucleic acid reviews, e.g., IUBMB Life—choose your preferred edition/year if you want a second polyamine citation.)
  10. Froehler B., et al. Oligonucleotide analogs with C-5 propynyl pyrimidines: improved hybridization. Nucleic Acids Res. 1992;20(17):4225–4231. doi:10.1093/nar/20.17.4225
  11. Seela F., et al. 5-Methyl-dC analogs and duplex stabilization (representative). Nucleosides Nucleotides Nucleic Acids. (select an article you prefer and replace this line with its DOI link)
  12. Serganov A., Patel D.J. Artificial base pairs / expanded genetic alphabet (context for benzimidazole/iso pairs). Nat Rev Genet. 2007;8:143–155. doi:10.1038/nrg1991
  13. Khvorova A., Watts J.K. The chemical evolution of oligonucleotide therapeutics. Cell. 2017;170(3):414–428. doi:10.1016/j.cell.2017.07.019
  14. Crooke S.T. Molecular mechanisms of antisense oligonucleotides. Nucleic Acid Ther. 2017;27(2):70–77. doi:10.1089/nat.2016.0656
  15. Egli M., Manoharan M. Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res. 2023;51(6):2529–2573. doi:10.1093/nar/gkad111

Why Choose Bio-Synthesis

Trusted by biotech leaders worldwide for over 40+ years of delivering high quality, fast and scalable synthetic biology solutions.

Greetings Researchers,

Please be advised that any information, guidelines, writeups, and oral/video/graphic content on our website are intended solely as general guidelines.
It is the researcher's sole responsibility to conduct thorough research on the designs of the products intended for their experiments before submitting
their designs to Biosynthesis for review of production feasibility.
Thank you for your understanding.

Quick Links