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Click DNA/RNA Chemical Ligation Oligonucleotide

Build covalent DNA/RNA junctions without enzymes using azide–alkyne click (CuAAC/SPAAC), TCO–tetrazine, maleimide–thiol, NHS–amine, and oxime/hydrazone chemistries. We design & manufacture reactive oligos with full QC.

Click (CuAAC / SPAAC) TCO–Tetrazine (iEDDA) Maleimide–Thiol NHS–Amine Oxime / Hydrazone

Overview

Bio-Synthesis engineers chemical-ligation–ready oligonucleotides (DNA/RNA, LNA, PS/PN/PM backbones) featuring azide, alkyne (incl. 3′-propargyl-5-Me-dC), DBCO for SPAAC, TCO–tetrazine (iEDDA), thiol/maleimide, NHS–amine, and aldehyde/aminooxy/hydrazide handles for enzyme-free DNA/RNA ligation. These click-ready oligos enable head-to-tail joining, circularization, splint-guided assembly, and probe/adaptor construction without T4 ligase.

Choose from CuAAC click DNA ligation (azide ↔ alkyne), copper-free SPAAC (DBCO–azide), TCO–tetrazine ligation, robust maleimide–thiol thioether formation, or oxime/hydrazone DNA ligation (aldehyde ↔ aminooxy/hydrazide). We optimize spacer length (TEG/PEG), junction orientation, and buffer conditions to maximize yield while preserving activity for qPCR probes, NGS adapters/padlock probes, ASO/siRNA conjugates, biosensors/surface immobilization, and nanostructures.

Delivery includes RUO→GMP-like pathways with UPLC/HPLC, LC-MS, optional endotoxin testing, and full documentation. Ask us to combine ligation handles with spacers & linkers, terminal caps, or reactive handles to streamline your design.

Formats
Tubes • 96-well plates
Scale
µmol → multi-gram
QC
UPLC/HPLC • LC-MS
Supply
RUO → GMP-like

Products & Notes

Product / Modification Description Typical use Code
5′-Azide (DNA/RNA) Terminal azide for CuAAC or SPAAC ligation. Ligation to 3′-alkyne or DBCO; label attachment. [5′-N3]
3′-Azide (DNA/RNA) 3′-end azide handle for click. Opposes 5′-alkyne/DBCO for head-to-tail assembly. [3′-N3]
Internal Azide-dU / Azide-dC Base analogs bearing azide on nucleobase/sugar. Internal cross-links, branched constructs. [N3-dU]
5′-Alkyne (Hexynyl) Terminal alkyne for CuAAC with azides. Enzyme-free junctions; label/protein coupling. [5′-Alkyne]
3′-Propargyl-5-Me-dC 3′ alkyne via propargylated 5-methyl-dC for click ligation. Click to azide oligo or azide-tagged cargo; directional 5′→3′ build. [3′-Prg-5-Me-dC]
DBCO (5′ / internal) Strained alkyne for rapid copper-free SPAAC with azides. Cell-friendly ligation; avoids copper. [DBCO]
Technical Notes
  • CuAAC: pH 7–8, CuSO4/TBTA (or THPTA) + ascorbate; remove copper post-reaction if needed.
  • SPAAC: DBCO–azide proceeds without copper; ideal for peptides/proteins and live cells.
  • Use TEG/PEG spacers to reduce sterics and boost yield.

Product / Modification Description Typical use Code
TCO (5′ / internal) Trans-cyclooctene handle. Rapid iEDDA ligation & macrocyclization. [TCO]
Tetrazine (5′ / internal) Tetrazine partner for TCO. Bioorthogonal ligation in complex media. [Tz]
Technical Notes
  • Very fast kinetics enable ligation at low concentrations.
  • Confirm dye compatibility—some pairs can quench.

Product / Modification Description Typical use Code
5′-Thiol / 3′-Thiol Terminal sulfhydryl. Thioether formation; Au surface immobilization. [5′-SH], [3′-SH]
Maleimide (5′ / internal) Michael acceptor for thiols. Directional ligation to thiol-oligo; peptide conjugates. [Mal]
5′-Amino / 3′-Amino Primary amine. Amide coupling (NHS, EDC/sulfo-NHS); surface derivatization. [5′-NH2], [3′-NH2]
NHS-Ester Oligo Pre-activated ester. Fast amide ligation to amines. [NHS-Oligo]
Technical Notes
  • Maleimide–thiol: pH 6.5–7.5; quench residual maleimide after ligation.
  • NHS–amine: pH 7.5–8.5 in amine-free buffers; use fresh to avoid hydrolysis.

Product / Modification Description Typical use Code
Aldehyde-Oligo Electrophilic aldehyde tag. Oxime/hydrazone with aminooxy/hydrazide. [-CHO]
Aminooxy-Oligo Oxime-forming nucleophile. Reversible yet stable bonds; probe assembly. [ONH2]
Hydrazide-Oligo Hydrazone-forming partner. Adaptive linkages; reduce post-ligation for permanence. [Hyd]
Technical Notes
  • Typical pH 4.5–6.5; anilinium catalysts accelerate at neutral pH.
  • Optional reduction locks the junction irreversibly.
Explore related chemistries
Services at a glance
  • Custom design review
  • RUO → GMP-like production
  • QC: UPLC/HPLC, LC-MS
Need help selecting the best ligation pair?

We’ll recommend handles, spacers, and buffers to maximize yield and preserve activity.

Design • Chemistry • Applications

Design Strategy

  • Termini pairing: 5′-Azide × 3′-DBCO (SPAAC) or 5′-Alkyne × 3′-Azide (CuAAC) for head-to-tail joins.
  • Sterics control: add short TEG/PEG spacers at the junction to improve yield.
  • Copper-sensitive payloads: prefer SPAAC (DBCO–Azide) or TCO–Tetrazine.
  • Reversible → permanent: use oxime/hydrazone then reduce to lock the linkage.
  • Formats: single/duplex, splint-guided assembly, circularization, branched constructs.

Chemistries

  • CuAAC (Azide ↔ Alkyne) – classic click DNA ligation.
  • SPAAC (DBCO–Azide) – copper-free, cell-friendly.
  • TCO–Tetrazine (iEDDA) – ultra-fast ligation at low µM.
  • Maleimide–Thiol – stable thioether bonds.
  • Oxime/Hydrazone – Aldehyde ↔ Aminooxy/Hydrazide (tunable, reducible).

Popular handles: 5′/3′-Azide, 5′/3′-Alkyne, 3′-propargyl-5-Me-dC, DBCO, TCO, Tetrazine, Thiol, Maleimide, Aldehyde, Aminooxy, Hydrazide.

Applications

  • Head-to-tail joins, macro/circularization, splint-guided assembly.
  • qPCR probes, NGS adapters & padlock probes, linkers/adaptors.
  • ASO/siRNA conjugation and targeted delivery ligands.
  • Biosensors & surface immobilization (Au/thiol, NHS–amine coupling).
  • DNA/RNA nanostructures and branched architectures.
Speak to a Scientist Browse Products & Notes Read FAQs

FAQ

Which chemistry for copper-sensitive systems?

Use SPAAC (DBCO–Azide) or TCO–Tetrazine.

Can I ligate head-to-head or head-to-tail?

Yes—combine handles on desired termini; add short spacers to improve yield.

Do you offer 3′-propargyl-5-Me-dC and azide ends?

Yes—pair 3′-propargyl-5-Me-dC with 5′-Azide or DBCO for efficient CuAAC/SPAAC ligation.

Can I ligate RNA to DNA with click chemistry?

Yes. Azide/alkyne and DBCO–azide pairs work with DNA, RNA, and mixed DNA↔RNA junctions. For RNA, we typically recommend copper-free SPAAC or TCO–Tetrazine to avoid copper exposure and preserve labile modifications.

How do I minimize self-dimerization or oligomerization?

  • Use orthogonal termini (e.g., 5′-azide on strand A × 3′-DBCO on strand B) and avoid having both reactive partners on the same end.
  • Work at µM concentrations, add short spacers (TEG/PEG) at the junction, and maintain slight stoichiometric excess of the limiting strand.
  • Include end blockers on unused termini (e.g., 3′-deoxy or 3′-phosphate) to prevent unintended polymerization.

What buffers and pH do you recommend?

  • CuAAC: pH 7.0–8.0 with CuSO4/TBTA (or THPTA) + ascorbate.
  • SPAAC (DBCO–Azide): neutral buffers (pH ~7.0–7.5), salt 50–200 mM NaCl or PBS.
  • TCO–Tetrazine: neutral buffers; very fast even at low µM.
  • Oxime/Hydrazone: pH 4.5–6.5 (anilinium catalysts allow near-neutral conditions).

How do I confirm ligation success?

  • LC-MS mass shift and HPLC/UPLC retention changes for product vs starting strands.
  • Native/denaturing PAGE band shift; fluorescent label co-migration when applicable.
  • Sequence-dependent melting temperature (Tm) increase at the junction.

What purification do you suggest after CuAAC?

Use HPLC/UPLC purification and a copper removal step (e.g., chelators or desalting). For sensitive cargos, prefer SPAAC to avoid copper altogether.

Can you supply pre-activated NHS ester or maleimide oligos?

Yes. We can deliver NHS-activated or maleimide-bearing oligos ready for amide or thiol coupling. Note that NHS esters are moisture-sensitive—plan for fresh use or we can assemble the conjugate in-house.

Can I combine click handles with dyes or quenchers?

Yes—space the reactive handle and dye by ≥3–5 nt to reduce quenching and sterics. For copper-sensitive fluorophores, use SPAAC or TCO–Tetrazine.

How should I store and handle reactive oligos?

  • Store dry at −20 °C, protected from light and moisture.
  • For maleimide or NHS species, avoid prolonged aqueous storage; prepare reactions immediately after resuspension.
  • Ship S-protected thiols; deprotect immediately prior to coupling.

Speak to a Scientist

Tell us about your chemical ligation project. We’ll recommend the most suitable chemistry (CuAAC, SPAAC/DBCO, TCO–Tetrazine, Maleimide–Thiol, Oxime/Hydrazone), spacers, and purification/QC.

Please avoid confidential details; we can arrange an NDA if needed.

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