Custom DNA and RNA probes with azide, alkyne, DBCO, BCN, TCO, tetrazine, aldehyde, aminooxy, hydrazide, phosphine, SuFEx and SPANC handles for selective labeling and conjugation.
Bio-Synthesis manufactures bioorthogonal DNA and RNA probes for enzyme-free, chemoselective labeling, ligation, conjugation and capture workflows.
This page focuses on modifications used to make bioorthogonal oligo probes: CuAAC azide–alkyne handles, copper-free SPAAC handles such as DBCO/BCN/DIBAC, ultrafast TCO–tetrazine iEDDA pairs, aldehyde–aminooxy oxime/hydrazone pairs, Staudinger ligation, SuFEx and nitrone–cyclooctyne SPANC options.
These designs support orthogonal dual labeling, templated strand ligation, surface/nanoparticle conjugation, peptide/protein coupling, pull-downs, live-cell compatible labeling and RUO to GMP-like supply paths.
Bioorthogonal handles let you build oligo conjugates without relying on native biomolecule reactivity.
React azides, alkynes, cyclooctynes, TCO, tetrazines or carbonyl handles with defined partners while minimizing side reactions.
Use copper-free SPAAC or TCO–tetrazine chemistry when copper-sensitive cells, proteins or cargos are involved.
Combine independent reaction pairs such as SPAAC plus TCO–tetrazine for staged or two-color labeling.
Connect oligos to dyes, peptides, proteins, nanoparticles, surfaces, small molecules and affinity capture systems.
The best chemistry depends on copper tolerance, reaction rate, aqueous compatibility, handle size, orthogonality and downstream purification/QC requirements.
CuAAC azide–alkyne
SPAAC DBCO / BCN
TCO–tetrazine iEDDA
Oxime / hydrazone
Staudinger, SuFEx or SPANC
Place compatible handles on opposite strands, termini or conjugate partners, such as azide × DBCO or TCO × tetrazine.
Use TEG, PEG or HEG spacers near bulky handles to improve accessibility, yield and solubility.
Choose SPAAC or TCO–tetrazine for copper-sensitive systems, or remove copper after CuAAC when needed.
Use HPLC/UPLC and LC-MS to confirm intact oligo, modified handle and post-conjugation product where feasible.
Recommended addition: For complex probe builds, include a small matched-control set: unmodified oligo, handle-only oligo, partner-only reagent and final conjugate. This improves troubleshooting for incomplete coupling, hydrolysis, copper sensitivity, hydrophobic sticking and LC-MS interpretation.
Browse click-ready and bioorthogonal handle categories for DNA/RNA labeling, ligation, pull-downs, live-cell probes and conjugate assembly.
CuAAC & SPAAC Handles — azide/alkyne click and copper-free cyclooctyne ligation.
Technical note: CuAAC is high conversion at pH 7–8 with CuSO4/TBTA or THPTA and ascorbate. SPAAC avoids copper and is preferred for live-cell, protein or sensitive cargo systems.
TCO–Tetrazine iEDDA — ultrafast catalyst-free ligation under physiological conditions.
Technical note: Pair with hydrophilic spacers to reduce hydrophobic sticking. Protect TCO from light and avoid prolonged heat to reduce isomerization.
Oxime / Hydrazone Handles — aldehyde, aminooxy and hydrazide chemistry for carbonyl-selective conjugation.
Technical note: Oxime/hydrazone ligation can be catalyzed with anilinium additives near neutral pH and optionally reduced when a more permanent linkage is required.
Staudinger Ligation — mild azide–phosphine reaction for chemoselective labeling.
Technical note: Phosphines oxidize. Use fresh reagent, degassed buffers where possible and confirm conversion analytically.
SuFEx & SPANC Specialty Handles — additional orthogonal routes for robust or staged conjugation.
Technical note: Specialty routes are useful when standard CuAAC/SPAAC/iEDDA handles conflict with other labels or conjugation partners.
Bioorthogonal oligos can be optimized for labeling, ligation, surfaces, nanoparticles, proteins, pull-downs or live-cell compatible workflows.
A practical workflow connects chemistry selection, handle placement, synthesis, conjugation and analytical release.
Define CuAAC, SPAAC, TCO–tetrazine, oxime/hydrazone, Staudinger, SuFEx or SPANC.
Choose 5′, 3′, internal or dual-handle placement with spacer requirements.
Build DNA/RNA probes with modified handles, spacers and optional blockers.
Purify by HPLC/UPLC or PAGE and confirm identity by LC-MS when compatible.
Support post-conjugation analysis, tubes, plates and RUO to GMP-like supply paths.
Bioorthogonal projects require control of handle identity, oligo purity, conversion, residual reagents and downstream compatibility.
QC packages may include HPLC/UPLC purity, LC-MS identity, OD260 concentration, conjugation conversion, residual copper considerations, CoA, plate maps and custom documentation.
Purity assessment and separation of unmodified, modified and conjugated species.
Mass confirmation of click-ready handles and conjugation products where feasible.
Post-CuAAC copper removal and residual reagent control for sensitive assays.
Tubes, 96-well plates, duplexes, normalized concentrations and CoA documentation.
Prefer copper-free SPAAC or TCO–tetrazine and hydrophilic spacer support.
Verify orthogonality and avoid handle cross-reactivity in staged reactions.
Support from research-scale tubes to plate and larger supply requirements.
Connect bioorthogonal probe design with supporting Bio-Synthesis functionalization, conjugation, spacer, labeling, purification and analytical services.
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Compare CuAAC, SPAAC, TCO–tetrazine, oxime/hydrazone, Staudinger, SuFEx and SPANC routes.
Plan spacer placement, reaction conversion, HPLC/UPLC purification, LC-MS identity and residual reagent considerations.
Use these references to support scientific background for click chemistry, copper-free ligation and bioorthogonal labeling.
Suggested page note: References are provided for scientific background. Final bioorthogonal oligo design should be evaluated within the sequence, handle placement, reaction partner, buffer, scale, purification and QC requirements.
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