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Bioorthogonal Probes — Oligo Modifications

Build DNA/RNA probes that react only with their intended partners using bioorthogonal chemistries: azide–alkyne CuAAC, copper‑free SPAAC (DBCO/BCN), TCO–tetrazine iEDDA, aldehyde–aminooxy oxime/hydrazone, Staudinger ligation, and SuFEx handles. We design, synthesize, and QC your oligos from research to GMP‑like supply.

ISO 9001:2015 / ISO13485:2016 45+ Years of Expertise U.S.A. Facilities-Texas GLP/GMP-Aligned Photocaged Nucleotides Photo-Cleavable Linkers Photocleavable Biotin Light-Triggered Release DNA / RNA Compatible Click (CuAAC / SPAAC) TCO–Tetrazine (iEDDA) Oxime / Hydrazone Staudinger SuFEx
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Overview Products Related Chemistry Design Highlights FAQ Speak to Scientist

Overview

Bio-Synthesis manufactures bioorthogonal oligonucleotides for enzyme-free, chemoselective DNA/RNA labeling and ligation—spanning azide–alkyne click (CuAAC), copper-free SPAAC (DBCO/BCN/DIBAC), TCO–tetrazine (iEDDA), oxime/hydrazone (aldehyde ↔ aminooxy/hydrazide), Staudinger ligation (azide ↔ phosphine), and SuFEx handles, with optional SPANC (nitrone–cyclooctyne) for added orthogonality. Designs are optimized for copper-free live-cell compatibility, site-specific probe assembly, and two-color / dual-route labeling.

Typical use cases include orthogonal dual labeling (e.g., SPAAC + TCO–Tz), templated strand ligation, surface/nanoparticle conjugation, protein/peptide coupling, and in-situ pull-downs. Provide sequences, target chemistry, buffer/pH, and scale; we’ll recommend the best handles, spacers (TEG/PEG), and purification/QC to maximize yield and preserve activity.

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

Also searched as: bioorthogonal probes, DBCO oligo SPAAC, TCO–tetrazine oligo, azide–alkyne click DNA, oxime/hydrazone DNA ligation, Staudinger ligation oligo, SuFEx oligo, SPANC oligo, copper-free click oligonucleotide.

Products & Notes

CuAAC (azide–alkyne) delivers high-yield triazole formation in aqueous buffers using CuSO₄/TBTA or THPTA with ascorbate (pH ~7–8), making it ideal for robust, high-conversion conjugations. SPAAC uses strained cyclooctynes (DBCO/BCN/DIBAC/DIFO) to react with azides without copper, enabling live-cell and protein-friendly labeling and enzyme-free strand joining. Both reactions are highly chemoselective and orthogonal to native biochemistry, and can be paired with TCO–tetrazine for two-route multiplex designs. Design: add short PEG/TEG spacers near the junction to reduce sterics and nonspecific sticking; for copper-sensitive cargos use SPAAC, or after CuAAC include a copper-removal step before downstream assays.

Modification Description Typical use Code
5′‑Azide / 3′‑Azide Terminal azide for CuAAC or SPAAC. Head‑to‑tail ligation; label coupling. [5′‑N3], [3′‑N3]
Internal Azide‑dU / Azide‑dC Base or sugar azide placement. Internal crosslinks; branched constructs. [N3‑dU]
5′‑Alkyne (Hexynyl) Terminal alkyne for CuAAC. Labeling; enzyme‑free junctions. [5′‑Alkyne]
DBCO (5′ / internal) Strained cyclooctyne for SPAAC. Copper‑free ligation; live‑cell friendly. [DBCO]
BCN / DIBAC / DIFO Alternative strained alkynes. Fast SPAAC with azides; orthogonality. [BCN]
Technical Notes
  • CuAAC: pH 7–8 with CuSO4/TBTA(or THPTA)+ascorbate; remove copper post‑reaction if needed.
  • SPAAC: DBCO/BCN + azide proceeds rapidly without copper; ideal for peptides/proteins & live systems.

TCO–Tetrazine Click Chemistry Trans-cyclooctene–tetrazine (TCO–Tz) ligation is a powerful bioorthogonal click chemistry reaction widely used for catalyst-free bioconjugation. This inverse-electron demand Diels–Alder (IEDDA) reaction offers exceptionally fast kinetics (up to 2000 M⁻¹ s⁻¹) and near-quantitative yields under physiological conditions. With unmatched speed, selectivity, and biocompatibility, TCO–Tz chemistry enables reliable protein labeling, antibody–drug conjugation (ADC/AOC), nucleic acid modification, and live-cell imaging. Its ability to form stable covalent bonds in complex biological systems makes it an essential tool for drug discovery, diagnostics, and therapeutic development.

Modification Description Typical use Code
TCO (5′ / internal) Trans‑cyclooctene handle. Ultrafast iEDDA ligation; live‑cell compatible. [TCO]
Tetrazine (5′ / internal) Dienophile partner for TCO. Rapid ligation at low µM concentrations. [Tz]
Cyclopropene Tag Small‑tag partner for tetrazine. Minimal sterics; fast cycloaddition. [CPE]
Technical Notes
  • Pair with hydrophilic linkers to reduce hydrophobic sticking in cells or on beads.
  • Excellent for orthogonal two‑color labeling (e.g., SPAAC + TCO–Tz).

Aminooxy-Modified Oligos and Oxime Ligation The chemoselective conjugation of an aminooxy-modified oligonucleotide with an aldehyde forms a stable oxime linkage, similar to hydrazide–aldehyde coupling but yielding oximes instead of hydrazones. These aminooxy oligos are produced by solid-phase synthesis using a 5′-aminooxy modifier 11, which includes a tetraethylene glycol spacer to enhance solubility and reduce hybridization interference. The resulting oxime bond is robust and biocompatible, enabling diverse applications such as fluorescent probe labeling, nucleic acid–protein conjugation, drug delivery systems, site-specific bioconjugation, and diagnostic assay development.

Modification Description Typical use Code
5'Aldehyde C2, Aldehyde-Oligo Electrophilic –CHO handle. Oxime/hydrazone ligation with aminooxy/hydrazide. [–CHO]
5' aminooxy 11, Aminooxy‑Oligo Oxime‑forming nucleophile. Rapid oxime formation to aldehyde partners. [ONH2]
5-Formylindole Aldehyde-functionalized base analog. Site-specific aldehyde introduction for oxime/hydrazone bioconjugation. [Formyl-Ind]
Hydrazide‑Oligo Hydrazone-forming nucleophile. Adaptive linkages; optional post‑reduction. [Hyd]
Technical Notes
  • Typical pH 4.5–6.5 (anilinium catalysts enable near‑neutral conditions).
  • Reduce the oxime/hydrazone post‑ligation for permanence if needed.

Azide–phosphine Staudinger ligation is a metal-free, chemoselective reaction that proceeds in mild, aqueous buffers through an aza-ylide intermediate to give amide-like covalent linkages. With excellent tolerance for proteins and native biomolecules—and no copper required—it is well-suited for tagging oligos in complex mixtures, surface capture, and late-stage bioconjugation. Design: use electron-rich aryl phosphines, minimize oxygen/light exposure, and prepare phosphine-functional oligos fresh to limit oxidation.

Modification Description Typical use Code
Azide‑Oligo Partner for ligation with aryl‑phosphines. Labeling and conjugation without metal catalysts. [N3‑Oligo]
Phosphine‑Oligo Staudinger ligation handle. Selective coupling to azide‑functional partners. [PPh3‑Oligo]
Technical Notes
  • Use degassed buffers and protect phosphines from oxidation; prepare fresh.

SuFEx (sulfur(VI) fluoride exchange) employs –SO₂F or –OSO₂F electrophiles that react cleanly with nucleophiles (e.g., amines, phenols) to form durable S–N/O linkages. These handles are hydrolytically persistent yet water-compatible, enabling modular assembly, surface immobilization, and post-synthetic diversification of oligos. Design: tune base/pH and use hydrophilic linkers to reduce non-specific binding; avoid competing nucleophiles.

Modification Description Typical use Code
Aryl Sulfonyl Fluoride Oligo –SO2F electrophile. Click‑like conjugation via SuFEx partners. [Ar‑SO2F]
Fluorosulfate Oligo –OSO2F handle. Link to amines/phenols via SuFEx chemistry. [OSO2F]
Technical Notes
  • Optimize pH and nucleophile (amine/phenol) for clean conversion; avoid competing nucleophiles.

SPANC (strain-promoted alkyne–nitrone cycloaddition) is a copper-free click between a nitrone and a strained cyclooctyne (e.g., DBCO/BCN), forming stable isoxazolidines rapidly at µM concentrations. It is orthogonal to many azide-based workflows and is useful when SPAAC sites are already occupied or for dual labeling. Design: consider nitrone stability and sterics; short PEG/TEG spacers often improve yield and accessibility.

Modification Description Typical use Code
Nitrone‑Oligo Spin‑trapping nitrone handle. SPANC to DBCO/BCN analogs; orthogonal routes. [Nitrone]
DBCO/BCN Partner Strained alkyne counterpart. Copper‑free cycloaddition with nitrone. [DBCO], [BCN]

Technology • Design • Application

Technology

  • CuAAC (azide ↔ alkyne); SPAAC (DBCO/BCN—copper‑free).
  • TCO–Tetrazine iEDDA — ultrafast ligation at low µM; excellent for live‑cell compatible workflows.
  • Oxime/Hydrazone (aldehyde ↔ aminooxy/hydrazide) — tunable, reducible to permanence.
  • Staudinger ligation (azide ↔ phosphine) — mild, chemoselective labeling.
  • SuFEx (–SO2F) — sulfur(VI) fluoride exchange for robust conjugation in water‑compatible media.

Design

  • Place complementary pairs on opposite strands/termini (e.g., 5′‑azide × 3′‑DBCO).
  • Use short PEG/TEG spacers near junctions for yield and accessibility.
  • For live cells or sensitive cargos, prefer SPAAC or TCO–Tz (no copper).
  • For oxime/hydrazone, consider anilinium catalysts at neutral pH; optionally reduce after ligation.
  • Include 3′ end blockers where needed to prevent unwanted extension.

Application

  • Enzyme‑free strand joining, circularization, templated reactions.
  • Conjugation to peptides, proteins, nanoparticles, and surfaces.
  • Live‑cell labeling, pull‑downs, and proximity assays (copper‑free routes).
  • Dual/orthogonal labeling using CuAAC + TCO–Tz in one construct.

Tip: provide sequences, target chemistry, and environment (buffer, pH, additives) for best design.

FAQ

Which chemistry is best for live‑cell or protein‑sensitive systems?

Use copper‑free routes: SPAAC (DBCO/BCN) or TCO–Tetrazine. Both are fast and biocompatible in aqueous buffers.

Can I combine two orthogonal reactions on one oligo?

Yes. Common pairs are SPAAC + TCO–Tz or CuAAC + Oxime/Hydrazone to enable multiplex labeling or staged assembly.

How should I handle phosphine or TCO reagents?

Phosphines oxidize—prepare fresh and use degassed buffers. TCO can isomerize—store protected from light and avoid prolonged high temperatures.

What purification/QC do you recommend?

We recommend HPLC/UPLC purification and LC‑MS confirmation; additional testing (endotoxin, residual copper) is available on request.

More FAQ'sAll FAQ's

Speak to a Scientist

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

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