Modifications that block ligation reactions in oligonucleotide synthesis typically interfere with the enzymatic activity of DNA ligase by altering key functional groups required for the reaction. The most common modifications that block ligation are found at the 3'-hydroxyl (-OH) group and the 5'-phosphate (-P) group.
3'-End Modifications
DNA ligase requires a free 3'-hydroxyl group to form the new phosphodiester bond. Modifying this group effectively terminates the chain and prevents ligation.
Dideoxynucleotides (ddNTPs) are well-known blockers, lacking both the 2'- and 3'-hydroxyl groups on the ribose sugar, preventing the formation of the phosphodiester bond, since there is no 3'-OH to attack the 5'-phosphate of the incoming nucleotide.
A C3 Spacer is a short, three-carbon chain added to the 3'-end. It is non-nucleosidic and lacks a hydroxyl group, thus physically blocking the ligase from attaching another oligonucleotide.
3'-Phosphate (-P): A phosphate group on the 3'-end blocks the ligation reaction by preventing the nucleophilic attack by the hydroxyl group.
Inverted Dideoxythymidine (Inverted ddT): A dideoxythymidine placed at the 3'-end in an inverted orientation (3'-3' linkage) creates a stable, irreversible block.
3'-Amino Modifier: An amino group at the 3' end instead of a hydroxyl group also blocks ligation. This modification can be used as a reactive handle for attaching other molecules, but it also serves as an effective chain terminator.
5'-End Modifications
DNA ligase also requires a 5'-phosphate group on the incoming oligonucleotide. If this group is absent or modified, ligation cannot occur.
The Absence of a 5'-Phosphate is the most straightforward method. Oligonucleotides are typically synthesized with a 5'-hydroxyl group and require a kinase enzyme to add a phosphate group for ligation. If the 5'-phosphate is not present, ligation is blocked.
5'-Modifications: Attaching large, bulky molecules like biotin, a dye, or an amine to the 5'-end can sterically hinder the ligase enzyme, preventing it from accessing the 5' end to perform the ligation.
Normally custom synthesized oligonucleotides come with 5'-OH and 3'-OH, unless requested to have a 5'-Phosphate.
Modifications That Prevent Ligation
| Modification | Position | Blocking Mechanism | HPLC Compatibility | Notes |
| 3′-Phosphate (-P) | 3′-end | Blocks ligase activity | ✓Compatible with RP-HPLC | Common in blocking unwanted elongation |
| 3′-Inverted dT (3′-3′ linkage) | 3′ end | No 3′ OH; terminates strand | ✓ Stable under HPLC | Effective irreversible block |
| 3′-Biotin / C3 Spacer | 3′ end | No hydroxyl; steric block | ✓ Stable through HPLC | Useful for streptavidin capture |
| 3′-Amino Modifier (C6-NH2) | 3′ end | No 3′ OH; reactive handle | ✓ Compatible with RP and IEX HPLC | Used for downstream conjugation |
| 3′-Hexanediol (Spacer 18) | 3′ end | Flexible hydrophilic blocker | ✓ RP-HPLC compatible | Also increases solubility |
| 3′-Dideoxynucleotide (ddNTP) | 3′ end | Terminator; no 3′ OH | ✓ HPLC stable | Used in Sanger sequencing |
| No 5′-Phosphate | 5′ end | Ligation requires 5′-P | ✓ Does not affect HPLC | Use when 5′ ligation must be blocked |
| 5′-Biotin / Amine / Dye | 5′ end | Steric hindrance; label blocks ligation | ✓ Stable during HPLC | Also enables detection or pull-down |
| 2′-O-Methyl, 2′-F, LNA | Internal or ends | Conformational changes reduce ligation | ✓ Stable in RP-HPLC | Depends on sequence context |
| PEG Spacer (e.g., Spacer 9/18) | Any position | Bulky, flexible; impairs enzyme binding | ✓ RP-HPLC compatible | Spacer between moieties or to block activity |
Reference
Oligo-chain-terminator-modifications
Chen X, Zhong S, Zhu X, Dziegielewska B, Ellenberger T, Wilson GM, MacKerell AD Jr, Tomkinson AE. Rational design of human DNA ligase inhibitors that target cellular DNA replication and repair. Cancer Res. 2008 May 1;68(9):3169-77. [PMC]
Zhou W, Ding J, Liu J. An Efficient Lanthanide-Dependent DNAzyme Cleaving 2'-5'-Linked RNA. Chembiochem. 2016 May 17;17(10):890-4. [Wiley]
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