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Custom Circular Oligonucleotide Synthesis

High-purity circular DNA and RNA oligonucleotides engineered for rolling circle amplification, padlock probes, molecular inversion probes, in situ assays, biosensors, NGS workflows and synthetic circular RNA research.

Enzymatic Ligation Click Circularization Padlock & MIP Designs HPLC/PAGE Purification LC-MS / MALDI-TOF Exonuclease Resistance Testing

Circular Oligos Built for Stable, High-Specificity Assays

Circular oligonucleotides are single-stranded DNA or RNA molecules whose 5′ and 3′ ends are covalently joined to form a closed ring. By eliminating free termini, circular oligos can improve resistance to exonuclease degradation and support highly specific probe-based and amplification-based workflows.

Bio-Synthesis supports custom circular oligonucleotide synthesis from design review through precursor synthesis, circularization, purification, analytical QC and delivery. Projects may include simple circular DNA/RNA, dumbbell oligonucleotides, padlock probes, molecular inversion probes, RCA templates, labeled circular probes and custom modified constructs.

Circular Oligonucleotide
Closed-loop topology
Nuclease
Resistant
Rolling Circle
Amplification

Why Circular Oligonucleotides Matter

Circularization changes the oligo from a linear strand into a covalently closed architecture. That structural change is useful when assays need stronger nuclease resistance, ligation-dependent specificity, continuous polymerase extension or spatially localized signal amplification.

Stb

Higher Stability

Closed circular structures remove free ends, increasing resistance to exonuclease digestion and improving functional lifetime in nuclease-rich environments.

RCA

Signal Amplification

Circular templates can support rolling circle amplification, enabling long concatemeric products and sensitive detection formats.

Spec

Greater Specificity

Padlock probes and MIPs use target-dependent ligation, helping distinguish closely related targets, SNPs and low-abundance sequences.

Flex

Custom Architectures

Designs can include DNA, RNA, dumbbell structures, fluorophores, quenchers, spacers, affinity tags and click-compatible handles.

Circular Oligonucleotide Technology & Benefits

This section should stay on the page because it explains the science and benefits behind the service. The redesign presents the same information as a technology selector instead of long text blocks.

Select a circular oligo technology category to view details
Structure Types at a Glance
RCA / probes
circle / dumbbell
topology
circularity
Structure Best Use Design Notes
Simple circular ssDNA or ssRNA RCA templates, synthetic controls, stability studies 5′ and 3′ ends are covalently closed; sequence and secondary structure are reviewed for circularization feasibility.
Dumbbell oligonucleotides Therapeutic research, nuclease-resistant constructs, molecular probes Stem-loop architecture can increase stability and enable defined terminal loop features.
Padlock probes SNP detection, in situ assays, RCA, target-specific ligation Arms hybridize adjacent to the target sequence; ligation creates the circular probe for amplification.
Molecular inversion probes Target enrichment, multiplexed detection, NGS workflows Useful when target capture, gap-fill and circularization are part of the assay design.
Circularization Methods at a Glance
Enzymatic
native junction
Click
bioorthogonal
Design focus
closure chemistry
QC focus
purity + identity

Enzymatic Circularization

Enzymatic ligation can create a native phosphodiester junction. It is commonly selected for padlock probes, MIPs and RCA workflows where polymerase compatibility is important.

  • T4 DNA ligase with splint-assisted ligation
  • Single-stranded DNA ligase approaches where feasible
  • RNA ligase approaches for selected circular RNA designs

Click Chemistry Circularization

Click circularization can provide robust chemical closure when enzyme-free methods or bioorthogonal handles are preferred.

  • CuAAC: azide + alkyne closure
  • SPAAC: DBCO + azide copper-free closure
  • Application compatibility reviewed case by case
Benefits at a Glance
Stability
exonuclease resistance
Signal
RCA amplification
Specificity
target ligation
Fit
diagnostics / assays

Nuclease Resistance

Closed-loop topology can reduce susceptibility to exonucleases compared with linear oligos.

Amplification Efficiency

Circular templates support continuous extension by strand-displacing polymerases in RCA.

Assay Confidence

Target-dependent ligation strategies help improve specificity in detection and genotyping assays.

Specification Planning at a Glance
Length
feasibility review
Backbone
DNA / RNA
Mods
labels + handles
Release
HPLC / PAGE / MS
Specification Available Options
Sequence length Commonly 20–150+ nt depending on design; longer or complex constructs require feasibility review.
Backbone DNA, RNA and selected modified backbones depending on circularization method and application.
Modifications 5′ phosphate, fluorophores, quenchers, biotin, spacers, PEG, amine, thiol, azide, alkyne, DBCO and custom labels.
Purification HPLC, PAGE or project-specific purification strategy.
Scale Research scale through milligram and gram-scale feasibility depending on construct complexity.

Where Circular Oligos Add the Most Value

Instead of another wall of cards, this section prioritizes the highest-value use cases and groups the rest as supporting applications.

Rolling Circle Amplification & Padlock Probe Assays

Circular templates and ligation-dependent probes are especially useful when the assay needs strong signal generation, high specificity, and spatially localized detection.

RCA templates
C2CA workflows
Padlock probes
In situ RCA
MIP / NGS

Molecular Inversion ProbesTarget capture, gap-fill, circularization and sequencing-compatible enrichment workflows.

circRNA

Synthetic circRNA ResearchCircular RNA controls and templates where sequence, length and modification feasibility allow.

Biosensors

Biosensors & DNA NanotechnologyStable looped constructs for programmable assemblies, sensors and molecular devices.

Aptamers

Aptamer StabilizationClosed structures that may improve nuclease resistance and functional persistence.

Build the Right Circular Oligo Configuration

The design section is now an interactive configuration lab. Customers can move through topology, closure chemistry, modifications and release requirements without scanning a page full of repeated cards.

Select a design layer to view options and guidance
Simple circlePadlock probeDumbbell / MIP

Select the topology around the assay mechanism.

Simple circular DNA/RNA works well for circular templates and stability studies. Padlock probes and molecular inversion probes are better when target-dependent ligation, gap-fill or sequencing workflows are required.

Simple circular ssDNA / ssRNA

RCA templates, synthetic controls and closed-loop stability studies.

Dumbbell oligonucleotides

Stem-loop constructs for nuclease-resistant probes and research architectures.

Padlock probes and MIPs

Target-dependent ligation, SNP discrimination, in situ assays and target enrichment.

Enzymatic
ligation
Click
closure

Match closure chemistry to downstream compatibility.

Enzymatic circularization is preferred when a native phosphodiester junction and polymerase compatibility are important. Click circularization is useful for enzyme-free closure, specialty handles or bioorthogonal designs.

Enzymatic ligation

Splint-assisted ligation, ligase-compatible termini and native junction design.

Click circularization

CuAAC or SPAAC approaches using azide, alkyne, DBCO or related handles.

Feasibility review

Sequence structure, length, junction placement and modification burden should be reviewed before quoting.

Dye Q Bio N3

Add only the modifications the assay truly needs.

Fluorophores, quenchers, affinity tags, spacers and reactive handles can be added, but each modification should be checked for circularization and purification compatibility.

Detection

FAM, Cy3, Cy5, TAMRA, HEX, ROX, quenchers and custom dye feasibility.

Capture and spacing

Biotin, digoxigenin, C3 spacers, HEG, PEG and project-specific linkers.

Conjugation handles

Amine, thiol, azide, alkyne, DBCO and other click-compatible modifications.

1Purify precursor
2Circularize & clean up
3Confirm purity / identity
4Optional exonuclease or RCA

Define release testing before synthesis begins.

Complex circular constructs need release criteria that match the application. A padlock probe, RCA template and circular RNA control may require different QC evidence.

Purification

HPLC, PAGE or project-specific cleanup depending on length, structure and modifications.

Identity and circularity

Mass confirmation where compatible, circular/linear tracking and optional exonuclease challenge.

Functional options

RCA or assay-specific validation can be added when performance evidence is needed.

From Sequence Review to Circular Oligo Delivery

A simpler visual workflow keeps the live-site process but reduces card fatigue across the page.

01

Design Review

Sequence, topology, target region, modifications, scale and assay constraints.

02

Precursor Build

Linear DNA/RNA synthesis with required termini, labels, spacers or handles.

03

Circularization

Enzymatic ligation or click closure with method-matched cleanup.

04

QC & Delivery

Purity, identity, circularity checks, optional RCA validation and documentation.

Quote tip: For padlock probes and MIPs, include the target sequence, ligase/polymerase plan and readout method so arm design and junction placement can be reviewed.

QC Strategy for Circular Oligos

Circular oligos require confirmation that the product is not just synthesized, but successfully circularized, purified and fit for the intended assay.

Analytical Control Matrix

QC packages may include analytical HPLC, PAGE purification, LC-MS or MALDI-TOF identity confirmation, exonuclease resistance testing, OD260 quantitation, documentation and optional RCA performance checks.

Purity Assessment

Analytical HPLC, UPLC, PAGE or method-matched release testing for circularized products.

Identity Confirmation

LC-MS or MALDI-TOF where size and chemistry permit, with documentation for release.

Circularity Check

Exonuclease resistance testing can help distinguish circular material from linear precursor.

Functional Validation

Optional RCA or assay-specific validation may be added when the application requires performance evidence.

Linear vs Circular Tracking

Track precursor, ligation product and purification outcome to reduce ambiguity.

Application-Driven QC

Padlock, MIP and RCA projects may require different release criteria.

Documentation

CoA, concentration, purity, identity, format and custom documentation options.

Circular 91-mer ssDNA Validation Study

The live-site case study is now presented as proof first, with the detailed sequence and analytical image available without overwhelming the page.

91-Base Circular ssDNA Synthesis

Project goal: synthesize a high-purity circular 91-mer single-stranded DNA oligonucleotide for gene expression and structural studies.

Sequence and Project Outcome

Sequence

TTC ACA GAG GAA GGG CCA GTA TCC TGT CCA AAC TTG ATG CTC CGG T CGT TTA GCA TAC TAA TCT GAG AGT CCG ACG GTC GTC AGT CAG TCA
  • Successfully synthesized and circularized the target 91-base sequence.
  • Verified circularity by exonuclease resistance and analytical comparison.
  • Purified by denaturing PAGE to greater than 95%.
  • Delivered ready-to-use research-grade circular DNA with documentation and QA validation.

Experimental Validation

Gel mobility and ESI-MS data support the successful conversion from linear 91-mer ssDNA to circular ssDNA.

Gel electrophoresis and ESI-MS validation data for linear and circular 91-mer ssDNA
🔍 Click to Enlarge

Gel image and ESI mass spectra from the 91-base circular ssDNA case study. Click to enlarge; close from the overlay.

Linear MW

Target 28128.1; observed 28126.9

Circular MW

Target 28110.1; observed 28111.1

Purity

>95% by denaturing PAGE

Format

Circular ssDNA, 91 bases

Recommended Production Treatment for 91-mer Circular ssDNA

This section turns the case study into a practical production recommendation, so visitors understand what Bio-Synthesis would typically evaluate, manufacture, purify, and release for a similar circular oligonucleotide project.

Recommended Build & Release Path

01

Linear Precursor

Design ligation-ready 91-mer ssDNA precursor with compatible termini.

02

Circularization

Use enzymatic ligation when a native phosphodiester junction is preferred.

03

Purification

Use denaturing PAGE or HPLC to separate circular product from linear precursor and side products.

04

Verification

Confirm product by gel comparison, exonuclease resistance, and ESI-MS identity.

05

Release

Deliver lyophilized circular ssDNA with purity, MW confirmation, and documentation.

Suggested Treatment Matrix

Oligo Type Circular ssDNA
Length 91-mer
Circularization Method Enzymatic ligation
Purification Denaturing PAGE or HPLC
Identity Confirmation ESI-MS
Circularity Verification Gel electrophoresis and exonuclease resistance
QC Release Purity, MW confirmation, concentration, CoA
Delivery Format Lyophilized, research-grade material
Why gel verification matters

Shows the mobility difference between linear and circular products and helps confirm successful circularization.

Why ESI-MS matters

Confirms the expected molecular weight after ligation and supports identity release.

Why purification matters

Removes unligated linear precursor, truncated oligos, and side products before delivery.

FAQ

What lengths can be circularized?
Many circular oligo projects fall in the 20–150+ nucleotide range, but feasibility depends on sequence, secondary structure, modifications and closure chemistry.
Which circularization method should I choose?
Use enzymatic ligation when a native phosphodiester junction and polymerase compatibility are priorities. Use click circularization when chemical closure, bioorthogonal handles or enzyme-free processing are preferred.
What should I submit for a quote?
Send sequence, topology, target sequence if applicable, closure preference, labels/modifications, scale, purification, QC requirements and intended application.
How is circularity confirmed?
Common options include mass confirmation, analytical purification traces, exonuclease challenge and functional RCA testing when appropriate.
Can circular RNA be synthesized?
Yes, selected circular RNA designs may be feasible. Sequence, length, modifications and ligation strategy should be reviewed before quoting.
Can Bio-Synthesis make labeled circular oligos?
Yes. Fluorophores, quenchers, biotin, spacers and reactive handles can be reviewed for compatibility with circularization and purification.

Start a Circular Oligo Project

Sequence
5′→3′ and target region
Topology
Circle, dumbbell, padlock, MIP
Closure
Enzymatic or click
Labels
Dye, quencher, biotin, handle
Scale
nmol, µmol, mg, gram
QC
HPLC, PAGE, MS, exonuclease, RCA

Ready to start your circular oligonucleotide project?

Share your sequence, target, topology, closure preference, labels, scale, purification and QC requirements. Bio-Synthesis can help evaluate feasibility and recommend the best circularization strategy for your application.

Design Review

Get help selecting topology, ligation method, labels and feasibility path.

RCA Padlock MIP circRNA

QC Package

Add purification, identity, circularity and functional validation options.

HPLC PAGE MS Exonuclease

Scientific References & Further Reading

  1. Rolling circle amplification methods for sensitive nucleic acid detection and circular template amplification.
  2. Padlock probe design literature covering ligation-dependent detection, SNP discrimination and in situ RCA.
  3. Molecular inversion probe workflows for target capture, gap filling, circularization and sequencing applications.
  4. Circular RNA and circular oligonucleotide stability studies describing closed-loop topology and nuclease resistance.
  5. Bio-Synthesis application note opportunity: publish a short guide titled “How to Choose Enzymatic vs Click Circularization for RCA and Padlock Probes.”

Website recommendation: Link these to internal technical notes or external references once the CMS team confirms approved citations.

Why Choose Bio-Synthesis

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