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Ligation Blocking Oligonucleotides (3′ & 5′ End Blockers)

Purpose-built end-capped and sterically hindered oligos to prevent DNA/RNA ligation at nicks or junctions—ideal for selection, library prep control, synthetic biology gates, and enzymology assays.

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Overview Services Design Highlights Products FAQ Speak

Overview

Bio-Synthesis designs and manufactures ligation blocking oligonucleotides to give you precise control over ligase activity. From 3′-end blockers (3′-phosphate, inverted dT, Spacer C3/C7, 3′-amino, dideoxy) to 5′-end blockers (no 5′-P, 5′-biotin/5′-amino) and internal steric blocks (abasic sites, HEG/Sp18 near junctions), we deliver assay-ready constructs with robust QC.

  • Application-aligned control: lock junctions to prevent unwanted ligation in library prep, rolling-circle assays, and selection workflows.
  • Assay-ready formats: single oligos, annealed duplexes, or plated libraries with barcodes and LIMS-ready labels.
  • Comprehensive QC: HPLC/UPLC purification and LC-MS mass confirmation; CoA with sequence + modification map.
ISO 9001 / 13485 RUO → GMP-like µmol → Multi-gram Design-to-QC
1
Design & Consultation

Choose the right block type and placement relative to the nick; strand context and duplex design.

2
Synthesis & Purification

Specialty coupling for non-nucleosidic spacers; HPLC/UPLC purification or PAGE on request.

3
Analytics & Verification

LC-MS mass confirmation; duplex annealing validation; custom release QC analyses

4
Scale & Documentation

From µmol to multi-gram production; RUO→GMP-like documentation and traceability.

Products — Ligation Blocking Oligonucleotides & Modifications

Product / Modification Description Function Application Code
3′-Phosphate Terminal phosphate at 3′ end Removes free 3′-OH Ligation block; extension stop [3P]
3′-Inverted dT Thymidine linked in reverse orientation Hard 3′ terminator Strong ligation/polymerase block [idT-3]
3′-dideoxy N (ddN) dideoxynucleotide at 3′ end No 3′-OH Sanger-style termination; ligation block [3ddN]
3′-Amino Primary amine at 3′ end Blocks ligation; functional handle Blocking + conjugation [3NH2]
3′-Spacer C3 / C7 Non-nucleosidic alkyl spacer Removes hydroxyl; adds sterics Hard block; geometry control [SpC3-3]/[SpC7-3]
Technical Notes
  • Strength order (typical): idT ≈ ddN ≥ 3′-P > 3′-NH2 ≈ Spacer C3/C7 (context dependent).
  • Handling: Store capped oligos at −20 °C; avoid repeated freeze-thaw to preserve end integrity.

Product / Modification Description Function Application Code
No 5′-Phosphate Deliver as 5′-OH (unphosphorylated) Missing 5′-P prevents ligation Baseline 5′-block [No-5P]
5′-Biotin Bulky biotin cap at 5′ Steric hindrance at junction Dual function: capture + block [Bio-5]
5′-Amino Primary amine at 5′ Introduces bulk; no 5′-P Blocking + conjugation [5NH2]
5′-Inverted dT Thymidine in reverse orientation at 5′ Steric obstruction Hard steric block [idT-5]
Technical Notes
  • Enzymatic rescue: If you need ligation later, you can re-phosphorylate a 5′-OH with T4 PNK.
  • Capture workflows: 5′-biotin enables streptavidin capture while keeping the junction blocked.

Product / Modification Description Function Application Code
Abasic Site (dSpacer / rSpacer) Non-informational abasic analog Distorts helix at junction Ligation suppression; structural studies [dSp]/[rSp]
HEG / Sp18 (–O–CH2–CH2–O– repeats) Flexible PEG-like spacer Steric gap; geometry control Reduce junction access [HEG]/[Sp18]
Phosphorothioate near nick PS linkage at junction Can reduce ligase efficiency Context-dependent blocking [PS@junction]
Technical Notes
  • Positioning: Start 1 nt from the nick and adjust outward until you balance blocking vs hybrid Tm.
  • Tm management: Compensate flexible spacers with GC content or length to maintain duplex stability.

Need help picking the best ligation block?

Tell us your ligase, junction sequence, and assay readout— we’ll recommend placement, purification, and QC.

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Technical Highlight

Ligation blocking oligonucleotides work by removing or masking the reactive ends required for ligase activity (3′-OH and 5′-phosphate) or by introducing steric/structural barriers near the nick. Use this section as a quick reference for mechanism, design rules, and troubleshooting.

How It Works
  • 3′-end blocks: 3′-inverted dT, 3′-dideoxy, 3′-phosphate, 3′-amino, or Spacer C3/C7 remove the free 3′-OH or make it inaccessible, stopping ligation and polymerase extension.
  • 5′-end blocks: Delivering oligos as 5′-OH (no 5′-P) or adding bulky tags (e.g., 5′-biotin, 5′-amino) prevents adenylation/strand joining by ligases.
  • Internal/steric blocks: Abasic sites (dSpacer) or flexible spacers (HEG/Sp18) placed 1–2 nt from a nick reduce ligase access while maintaining hybridization.
Design Rules (Quick Start)
  • Pick the right end: If ligation proceeds from a 3′-OH, choose a 3′ blocker (idT or ddN for the “hardest stop”). If it requires a 5′-P, deliver 5′-OH or add 5′ bulk.
  • Distance to nick: For internal blocks, start 1 nt away from the junction; move to 2–3 nt if Tm drops too much.
  • Dual blocking: In complex pools, combine 3′-idT or 3′-ddN with no 5′-P for robust suppression.
  • Tm management: If HEG/Sp18 or abasic lowers duplex stability, compensate with length or GC content.
  • Future ligation option: You can restore a 5′-OH to ligatable form using T4 PNK to add a 5′-phosphate later.
Goal Recommended Mods Notes
Hard 3′ block + stop extension 3′-inverted dT, 3′-dideoxy Highest blocking; great for preventing carry-over ligation/polymerase.
Moderate 3′ block + handle 3′-phosphate, 3′-amino 3′-NH2 adds a conjugation handle; 3′-P is simple and effective.
5′ ligation suppression 5′-OH (no 5′-P), 5′-biotin, 5′-amino Re-enable later with T4 PNK; 5′-biotin adds capture functionality.
Local steric hindrance dSpacer, HEG/Sp18 near nick Start 1 nt from the junction; adjust outward to balance Tm vs blocking.
Do
  • Include an unblocked control to quantify blocking efficiency.
  • Validate duplex annealing (stoichiometry) before ligation assays.
  • Store at −20 °C; minimize freeze–thaw to preserve end caps.
Don’t
  • Place flexible spacers too close if Tm is marginal — move 1→2 nt.
  • Forget that ddN and idT will also halt polymerase extension.
  • Mix 5′-P and 5′-OH lots inadvertently — label clearly in plates.
Troubleshooting
  • Residual ligation observed: Upgrade to 3′-idT or 3′-ddN; for 5′, ensure oligo is 5′-OH (not inadvertently phosphorylated).
  • Hybridization weakened: Increase length/GC or shift spacer from 1 nt to 2–3 nt away from the nick.
  • Need ligation later: Re-phosphorylate 5′-OH with T4 PNK; replace ddN/idT with 3′-P if extension is required downstream.

FAQ

What’s the strongest 3′ ligation block?

3′-inverted dT and 3′-dideoxy are typically hardest stops, followed by 3′-phosphate and 3′-amino. Context matters; we can help test.

How do I block ligation at the 5′ end?

Use an oligo delivered as 5′-OH (no 5′-phosphate), or add a bulky 5′ tag (biotin/amine). You can re-phosphorylate later with T4 PNK if needed.

Can I combine 3′ and 5′ blockers?

Yes—dual blocking is common in complex libraries; we often pair 3′-idT or 3′-ddN with no 5′-P for robust suppression.

Will spacers or abasic sites affect hybridization?

They can reduce Tm. We’ll tune sequence length/GC or adjust spacer distance from the nick to preserve binding while blocking ligation.

Speak to a Scientist

Tell us about your colorimetric detection project. We’ll recommend the most suitable reporter (HRP/AP, AuNP, DNAzyme, redox), spacers, capture strategy, and purification/QC.

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

You’ll receive a confirmation by email.

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