5′-, 3′-, internal-, diphosphate and triphosphate oligonucleotide modifications for ligation, circularization, NGS, CRISPR, padlock probes and RNA research.
Phosphorylated oligonucleotides contain a phosphate or polyphosphate group at the 5′ end, 3′ end, or internal position of DNA or RNA. These modifications are essential for many enzymatic workflows, including DNA ligation, adapter construction, cloning, padlock probe circularization, rolling circle amplification, CRISPR donor preparation and next-generation sequencing.
Bio-Synthesis provides custom phosphorylated oligos ranging from routine 5′ monophosphate and 3′ phosphate modifications to advanced class="text"5′ diphosphate and 5′ triphosphate RNA designs. Standard phosphate modifications are commonly used for ligation, cloning, NGS adapter construction and circularization, while polyphosphate modifications are better suited for RNA biology, capping-related studies and innate immune signaling research.
Design note: Standard 5′ phosphate oligos are commonly used for ligation. Polyphosphate RNA designs such as 5′-pp and 5′-ppp are advanced modifications and should be reviewed for oligo type, length, scale, purification and analytical requirements.
The guide will show the recommended phosphate position, typical notation and design guidance for ligation, circularization, NGS adapters, polymerase blocking or RNA polyphosphate studies.
Use this guide as a first-pass selection tool. Final design depends on sequence, oligo type, enzyme system and downstream workflow.
Join fragments, adapters or probes enzymatically.
Padlock probes, circular DNA and RCA templates.
Adapter ligation and barcoded library workflows.
Prevent polymerase extension from the 3′ end.
5′-ppp RNA, RIG-I and capping studies.
Phosphate plus dyes, amino, thiol or click handles.
Use a 5′ monophosphate when the oligo must serve as a ligation substrate. DNA and RNA ligases typically require a 5′ phosphate and a 3′ hydroxyl to form the phosphodiester bond.
Padlock probes and circular oligonucleotides generally require a ligatable 5′ phosphate. The 5′ phosphate supports enzymatic closure when the 3′ end is properly aligned by a template or splint.
NGS adapters and barcoded oligos often require 5′ phosphorylation to participate efficiently in library-preparation ligation steps.
>Use a 3′ phosphate when the goal is to block polymerase extension from the 3′ terminus or control downstream ligation direction.
Advanced 5′ polyphosphate RNA modifications are useful for RNA biology, capping intermediates, in vitro transcription mimicry and innate immune signaling research.
Phosphate modifications can be combined with dyes, quenchers, amino handles, thiols, azides, alkynes, biotin and other modifications when the sequence design and synthesis route are compatible.
Use the tabs below to compare standard terminal phosphates, advanced polyphosphate modifications and combination designs.
General purification note: HPLC purification is recommended for most phosphorylated oligos used in ligation, diagnostic, circularization, NGS or multiple-modification workflows. Advanced polyphosphate designs may require technical review before quoting.
Click a phosphate category to view available options and applications.
5′, 3′ and internal phosphate
3Diphosphate and triphosphate
Phosphate plus other modifications
Advanced technical review
Typical choice: Choose 5′ phosphate for ligation and adapter construction; choose 3′ phosphate when the goal is extension blocking.
Important distinction: 5′ triphosphate RNA is not simply a stronger version of 5′ phosphate. It has different biological roles, especially in RNA sensing and capping research.
Typical choice: Combination designs are common for padlock probes, capture probes and custom diagnostic constructs. Send a full modification map for review.
Technical review recommended: Advanced phosphate designs should be evaluated for synthesis route, purification, mass confirmation, enzymatic compatibility and storage conditions.
Click a position to visualize the difference between 5′ phosphate, 3′ phosphate, internal phosphate and 5′ polyphosphate RNA.
Ligation handle
Extension block
Specialty design
RNA biology
Best for ligation, adapter construction, cloning, padlock probes and circularization.
Best for blocking 3′ extension and controlling enzymatic reactions.
Used in specialized probe designs, ligation strategies or structural studies.
Used for RNA biology, RIG-I activation and capping studies.
Yes. Phosphate groups can often be combined with many other modifications when the synthesis route, purification plan and final application are compatible.
Phosphorylated probes with FAM, Cy dyes, Alexa Fluor®, ATTO and specialty dyes.
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Combine phosphate with amino handles for post-synthetic conjugation.
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Use phosphate with thiol handles for maleimide or gold-surface chemistry.
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Azide, alkyne and copper-free click handles for ligatable conjugation designs.
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Phosphorylated capture oligos for streptavidin workflows.
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Use C3, C6, PEG or cleavable spacers to control distance and orientation.
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5′ phosphate is often central to ligation-mediated circularization.
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Custom phosphate-containing oligos for advanced conjugation workflows.
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For cloning, adapter ligation, padlock probes and circularization.
For RNA biology, innate immunity and capping-related studies.
Phosphorylated DNA, RNA, modified RNA, siRNA-style and custom oligonucleotide constructs.
5′ phosphate, 3′ phosphate, internal phosphate and custom modification maps.
Diphosphate, triphosphate RNA and multi-modified constructs for specialized research.
HPLC purification, mass confirmation, UV-Vis analysis and project-specific QC documentation.
Custom DNA oligos with phosphate and other modifications.
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RNA oligonucleotides with phosphate and advanced modification options.
Circular DNA/RNA constructs and ligation-mediated circularization.
Ligation workflows for custom oligonucleotide assembly.
Reactive amine handles for post-synthetic conjugation.
Azide, alkyne and copper-free click handles.
Dye-labeled phosphorylated probes and custom reporter oligos.
Spacer and linker modifications for custom probe architecture.
Synthesis route, phosphate placement, enzyme compatibility, purification, mass confirmation and combined modification feasibility.
Selected background topics for phosphorylated oligonucleotide design, ligation chemistry, circular probes, NGS adapter workflows and RNA polyphosphate biology.
Suggested design note: References are provided for scientific background. Final phosphorylated oligo design should be evaluated within the sequence, phosphate position, enzymatic workflow, purification method and QC requirements.
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