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2′→5′ Linked Oligonucleotides

Custom 2′→5′ linked oligonucleotides for RNA biology, RNase L pathway studies and alternative backbone architecture research.

3′-rA / rG / rU / rC 3′-dA / dG / dC / dT Backbone Modification RNA & DNA Backbone Engineering Custom Linkage Placement

Alternative Backbone Connectivity for RNA and DNA Research

Bio-Synthesis provides custom 2′→5′ linked oligonucleotides containing site-specific RNA or DNA backbone linkages for structure-function research, nuclease recognition, ribozyme studies, 2-5A/RNase L pathway models and prebiotic chemistry investigations.

Natural DNA and RNA primarily use 3′→5′ phosphodiester linkages. A 2′→5′ linkage changes the backbone geometry by connecting the 2′ hydroxyl of one sugar to the 5′ position of the next nucleotide, which can affect hybridization, folding, enzyme recognition and degradation behavior.

2′→5′ linked oligonucleotides are commonly classified as backbone-modified oligonucleotides because the modification changes phosphodiester connectivity rather than the nucleobase itself. Researchers use these constructs to study alternative backbone architectures, enzymatic recognition, RNA structure and nucleic acid evolution.

Bio-Synthesis supports both RNA and DNA 2′→5′ linked building blocks, including 3′-rA, 3′-rG, 3′-rU, 3′-rC and 3′-dA, 3′-dG, 3′-dC, 3′-dT. These can be incorporated into custom oligos as single-site linkages, multiple isolated linkages or modified segments depending on sequence feasibility.

Backbone Geometry Comparison

natural 3′→5′ vs modified 2′→5′ linkage

Natural Connectivity

3′ P 5′

Standard 3′→5′ phosphodiester linkage used by most biological DNA and RNA.

2′→5′ Modified Connectivity

2′ P 5′

Alternative backbone linkage used to probe geometry, folding and enzyme recognition.

The linkage position is small chemically but important structurally. Placement and number of 2′→5′ bonds should be selected around the intended readout.

2′→5′ Linked RNA and DNA Building Blocks

Bio-Synthesis supports the following 2′→5′ linked products for custom oligonucleotide synthesis. These are commonly used for site-specific linkage substitution and mixed-backbone oligo designs.

Supported 2′→5′ Linked Product Structures

Visual reference for RNA and DNA 2′→5′ linked building blocks. Displayed at native image size to avoid browser upscaling blur.

2′→5′ linked oligonucleotide backbone modification structures including 3′-rA, 3′-rG, 3′-rU, 3′-rC, 3′-dA, 3′-dG, 3′-dC and 3′-dT building blocks
Product structure guide: DNA series includes 3′-dA, 3′-dT, 3′-dG and 3′-dC; RNA series includes 3′-rA, 3′-rU, 3′-rG and 3′-rC, each configured for 2′→5′ linked oligonucleotide synthesis.

Available 2′→5′ Linked Products

Use RNA building blocks for RNA folding and 2-5A biology; use DNA building blocks for chimeric and backbone-engineered constructs.

ProductSeriesBaseTypical Research Use
3′-rA (2′→5′ linked) RNA Adenosine RNA folding, ribozyme studies, 2-5A pathway models
3′-rG (2′→5′ linked) RNA Guanosine RNA backbone architecture and enzymatic recognition
3′-rU (2′→5′ linked) RNA Uridine RNA structure, prebiotic chemistry and duplex studies
3′-rC (2′→5′ linked) RNA Cytidine RNA folding, structure-function and nuclease studies
3′-dA (2′→5′ linked) DNA Deoxyadenosine DNA/RNA chimeras and alternative backbone designs
3′-dG (2′→5′ linked) DNA Deoxyguanosine Backbone engineering and nuclease-resistance studies
3′-dC (2′→5′ linked) DNA Deoxycytidine Hybrid constructs and synthetic biology research
3′-dT (2′→5′ linked) DNA Thymidine DNA analog designs and modified linkage studies

Design note: Product notation such as 3′-rA (2′→5′ linked) indicates the modified phosphodiester connectivity used during oligonucleotide assembly. Final sequence feasibility depends on length, placement, neighboring residues and requested purification/QC.

Most Requested 2′→5′ Designs

Common starting points for feasibility review and quote requests.

A(2′→5′)A / 2-5A models

Oligoadenylate pathway and RNase L-related studies.

Single-site linkage scan

Compare one modified linkage against a natural 3′→5′ control.

RNA/DNA chimeras

Mix RNA and DNA residues for backbone-architecture studies.

Labeled 2′→5′ probes

Combine with dye, quencher, biotin or click handles when feasible.

Design Suggestions for 2′→5′ Linked Oligos

The most successful designs treat 2′→5′ linkages as structural experiments. Start with the biological question, then decide how many modified linkages are needed and where they should be placed.

Recommended Design Strategies

Plan a control sequence and one or more modified sequences so the effect of the 2′→5′ linkage can be interpreted clearly.

Single-site substitution

Best first step for measuring local structure, Tm or nuclease effects.

Clustered linkages

Useful when testing modified segments or prebiotic-style backbone mixtures.

Hybrid backbone

Combine 3′→5′ and 2′→5′ regions to compare backbone-dependent behavior.

Matched controls

Run the same sequence with natural 3′→5′ connectivity for comparison.

POS

Placement Matters

Internal 2′→5′ linkages can alter helix geometry more strongly than terminal substitutions. Avoid placing modified linkages directly in critical binding motifs unless that is the intended experiment.

Tm

Validate Hybridization

2′→5′ linkages may affect melting temperature, duplex shape and mismatch behavior. Include Tm or binding validation for probe, antisense or aptamer designs.

MOD

Combine Carefully

2′→5′ linkages can often be combined with dyes, quenchers, biotin, amino modifiers, thiol modifiers, click handles or PEG spacers, but compatibility should be reviewed.

Applications for 2′→5′ Linked Oligonucleotides

2′→5′ linked oligos are selected when researchers need to study the effect of non-natural phosphodiester connectivity on structure, stability or biological recognition.

RNA

RNA Structure & Folding

Evaluate how alternative linkage geometry affects secondary structure, tertiary folding and duplex behavior.

2-5A

2-5A / RNase L Research

Build 2′→5′ oligoadenylate models for interferon-induced OAS/RNase L pathway studies.

RIB

Ribozyme Studies

Probe catalytic RNA structure, substrate recognition and cleavage behavior with altered backbone geometry.

NUC

Nuclease Recognition

Compare enzymatic degradation and cleavage profiles against natural 3′→5′ control sequences.

PRE

Prebiotic Chemistry

Study alternative linkage formation and mixed-backbone polymers relevant to origin-of-life research.

SYN

Synthetic Biology

Engineer nucleic acid constructs with non-natural connectivity for specialized structure-function experiments.

Purification, QC and Delivery

PUR

Purification

  • RP-HPLC, IE-HPLC or PAGE purification
  • Purity target matched to application
  • Additional purification for long or multi-modified constructs
MS

Analytical QC

  • ESI-MS or MALDI-TOF identity confirmation
  • Analytical HPLC/UPLC trace when applicable
  • Optional Tm or functional support by request
DEL

Delivery Format

  • Lyophilized oligo in tubes or plates
  • Custom concentration or buffer
  • CoA and modification annotation

Frequently Asked Questions

FAQ

What is a 2′→5′ linked oligonucleotide?
 A 2′→5′ linked oligonucleotide contains one or more phosphodiester bonds connecting the 2′ hydroxyl of one sugar to the 5′ position of the next nucleotide rather than the natural 3′→5′ linkage.
How is a 2′→5′ linkage different from a natural 3′→5′ linkage?
 Natural DNA and RNA primarily use 3′→5′ phosphodiester bonds. A 2′→5′ linkage changes the backbone geometry and can alter folding, hybridization, nuclease recognition and enzymatic processing.
Can Bio-Synthesis make both RNA and DNA 2′→5′ linked products?
 Yes. Supported building blocks include 3′-rA, 3′-rG, 3′-rU, 3′-rC and 3′-dA, 3′-dG, 3′-dC, 3′-dT in 2′→5′ linked formats.
Can multiple 2′→5′ linkages be incorporated into one oligonucleotide?
 Yes. A design can include a single site-specific 2′→5′ linkage, multiple isolated linkages or clustered modified segments depending on sequence feasibility and project goals.
Do 2′→5′ linkages affect Tm and hybridization?
 They can. Effects depend on placement, number of linkages, sequence context and whether the construct is RNA, DNA or a hybrid. Critical applications should include pilot testing and Tm validation.
Are 2′→5′ linkages compatible with dyes, quenchers or bioconjugation handles?
 Many designs can combine 2′→5′ linkages with fluorescent labels, quenchers, biotin, amino modifiers, thiol modifiers, click handles or spacers, but compatibility should be reviewed during design.
What is the relationship between 2′→5′ oligoadenylates and RNase L?
 The OAS/RNase L antiviral pathway uses 2′→5′ linked oligoadenylates, often called 2-5A, to activate RNase L. Modified 2′→5′ oligos are useful tools for studying this pathway.
What QC is recommended?
 HPLC or PAGE purification with mass spectrometry identity confirmation is recommended for most 2′→5′ linked oligos, especially when multiple modified linkages or labels are included.
How are 2′→5′ linked oligonucleotides classified?
 2′→5′ linked oligonucleotides are generally classified as backbone-modified oligonucleotides because the phosphodiester connectivity is altered while the nucleobases remain unchanged. They are commonly used to study alternative backbone architectures, RNA biology and enzymatic recognition.

Need help designing a 2′→5′ linked oligo?

Share your sequence, desired 2′→5′ linked building block, linkage position, number of modified linkages, RNA/DNA format, scale, purification target and QC requirements. Bio-Synthesis can help review sequence feasibility and design controls for your experiment.

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Fast Quote Checklist

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Sequence 2′→5′ product Position Scale QC

Recommended Reading

Selected references for 2′→5′ oligoadenylates, RNase L biology, alternative RNA linkages and prebiotic nucleic acid chemistry.

  1. Silverman RH. Viral encounters with 2′,5′-oligoadenylate synthetase and RNase L during the interferon antiviral response. Journal of Virology. 2007.
    Background on the biological 2-5A/RNase L pathway.
  2. Player MR, Torrence PF. The 2-5A system: modulation of viral and cellular processes through acceleration of RNA degradation. Pharmacology & Therapeutics. 1998.
    Detailed review of 2′→5′ oligoadenylate biology.
  3. Usher DA, McHale AH. Nonenzymatic joining of oligoribonucleotides on a polyuridylic acid template. Science. 1976.
    Classic work relevant to alternative RNA linkage formation.
  4. Engelhart AE, Hud NV. Primitive genetic polymers. Cold Spring Harbor Perspectives in Biology. 2010.
    Context for prebiotic genetic polymers and alternative backbone structures.
  5. Torrence PF, Johnston MI. 2-5A and related 2′,5′-oligoadenylates. Methods in Enzymology. 1981.
    Foundational methods and biology for 2′→5′ oligoadenylate systems.

Note: References provide scientific background and design context. Final oligo design should be evaluated with the target sequence, linkage placement, assay conditions and purification/QC requirements.

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