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High-Purity Circular DNA and RNA
Oligonucleotides for Advanced Molecular

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Custom Circular Oligonucleotide Synthesis

Nuclease‑resistant closed‑circular DNA/RNA for padlock probes, RCA, and advanced molecular assays.

Overview Advantages Applications Synthesis Options Modifications Closure Methods QC How to Order FAQ Case Study Contact

Overview

Circular oligonucleotides are single‑stranded DNA or RNA whose 5′ and 3′ ends are covalently joined to create a closed ring. The circular topology improves exonuclease resistance, stability, and performance in assays such as padlock/MIP and rolling circle amplification (RCA).Bio‑Synthesis provides end‑to‑end design, circularization (enzymatic or chemical), labeling, and QC—delivered ready for your downstream workflow.

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At-a-Glance

  • Formats: simple circles (ssDNA/ssRNA), dumbbells, padlock/MIP designs
  • Junction types: native phosphodiester (enzymatic) or triazole (click)
  • Typical length: 5–200 nt (padlocks 80–120 nt; longer on review)
  • Purification: HPLC standard; PAGE optional
  • QC: LC‑MS/MALDI‑TOF (size‑dependent), analytical HPLC, optional exonuclease challenge
  • Scales: 1 mg to gram quantities

Key Advantages

High Stability

Closed circles lack free ends, improving resistance to Exo I/III and many nucleases.

Assay Performance

RCA‑ready templates and padlock probes enable ultra‑sensitive detection and C2CA workflows.

Design Flexibility

Multiple junction chemistries, labels, linkers, and blocking motifs for diverse platforms.

Applications

  • Padlock/MIP probes with
    ligation‑dependent capture
  • Rolling circle amplification (RCA/C2CA)
  • NGS library/sample barcoding adapters
  • qPCR/HRM clamping and SNP
    discrimination (via padlocks)
  • Signal amplification in in situ assays
  • Template circles for polymerase readout
  • Aptamer stabilization by circularization
  • DNA nanotechnology & sensors
  • Controls for circRNA workflows (synthetic circles)

Custom Synthesis Options

Parameter Options
Length 30–200 nt standard; >200 nt upon feasibility review
Topology Simple ring, dumbbell (two hairpins), padlock (5′/3′ arms with target complementarity)
Backbone DNA (standard), RNA (on request), partial PS (phosphorothioate) segments for added stability
Junction Native phosphodiester (ligase‑based) or triazole (CuAAC/SPAAC click)
Blocking Motifs 3′‑inverted dT, terminal spacers, abasic spacers to tune flexibility
Delivery Format Lyophilized; buffer on request
Scalest 1 mg to gram quantities; bulk on request
QC Package LC‑MS/MALDI‑TOF (size‑dependent), analytical HPLC; optional exonuclease‑resistance and RCA function tests

Popular Modifications & Linkers

Category Examples Common Uses
Fluorescent Dyes FAM, HEX, TET, TAMRA, ROX, Cy3, Cy5, ATTO series, Alexa Fluor Probe visualization, in situ readouts
Affinity Tags Azide/Alkyne (click), Amine, Thiol, DBCO Conjugation to surfaces, proteins, or nanoparticles
Reactive Handles Amine, Thiol, Azide, Alkyne (terminal/strained), Maleimide Bioconjugation via amide, thioether, or click reactions
Spacers / PEG AEEA, miniPEG, PEG(n), C6/C12 alkyl, abasic spacers Tune flexibility & reduce sterics at junction or labels
Quenchers BHQ‑1/2/3, Iowa Black, Eclipse Quenched padlock probe designs

Closure & Ligation Methods

Method Junction Chemistry Notes & Applications
Enzymatic ligation (DNA) Native phosphodiester (T4 DNA ligase; with splint) Scar‑minimized junction; padlock/MIP formation; compatible with polymerase‑based RCA.
ssDNA ligase Native phosphodiester (CircLigase™‑type) Direct circularization of ssDNA with 5′‑phosphate; useful for short circles.
RNA ligation Native phosphodiester (RtcB/T4 RNA ligase variants) For synthetic circular RNA controls/templates; sequence‑dependent feasibility.
Click circularization (CuAAC) Triazole junction (5′‑azide + 3′‑alkyne) Enzyme‑free, high‑yield closure; widely used for probe circles; polymerase compatibility may vary by application.
Straint‑promoted click (SPAAC) Triazole (DBCO + azide) No copper; suitable when Cu must be avoided; junction behavior similar to CuAAC.
We offer much more than listed! - get in touch   
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Quality Assurance

  • LC‑MS / MALDI‑TOF for identity (size‑dependent)
  • Analytical HPLC for purity
  • Optional: Exonuclease I/III challenge to confirm circularization
  • Optional: RCA functional test with phi29 polymerase (probe‑dependent)
  • Documentation: RUO by default; GLP/cGMP support on request

Typical Turnaround

Standard labeled circles: 2–3 weeks from order confirmation. Complex padlock panels or RNA circles may require additional time.

Lead time depends on sequence, topology, and closure method; rush options may be available.

How to Order

  1. Provide target concept and desired topology (simple circle, dumbbell, padlock/MIP).
  2. Select closure method (enzymatic vs click) and labels/linkers.
  3. Choose scale, purification, and optional QC/functional tests.
  4. Receive a same‑day quote and timeline.

For padlock probes, include target sequence and intended ligase/polymerase to optimize junction design.

Design Checklist

  • Length and topology (simple/dumbbell/padlock)
  • Junction chemistry (native vs triazole)
  • Labels/tags and any reactive handles
  • Assay context (RCA, in situ, NGS, sensors)

Circular Oligonucleotide Technology & Benefits

What are circular oligos? Circular oligonucleotides are covalently closed single-stranded DNA/RNA constructs with no free 5′ or 3′ ends. They are produced by enzymatic ligation (e.g., T4 DNA/RNA ligase, CircLigase®) or by chemical “click” ligation to join the termini into a stable loop. The closed topology boosts nuclease resistance and enables powerful isothermal amplification methods.

How Circular Oligos Work
  • Padlock/MIP principle: Linear precursor ends hybridize adjacent to a target sequence; only a perfect match allows ligation to form a circle (high SNP specificity). The circle then serves as a template for detection.
  • Rolling Circle Amplification (RCA): A primer and strand-displacing polymerase generate long concatemers from the circular template, yielding strong, isothermal signal amplification.
  • Circular stabilization: Cyclizing aptamers/ASOs can reduce exonuclease attack and maintain functional folds for binding or blocking applications (research use).
Design Principles
  • Ligation strategy: Splint-assisted T4 ligase for DNA, T4 RNA ligase for RNA; or bio-orthogonal click ligation (CuAAC/SPAAC) for sensitive payloads.
  • Junction planning: Provide a 5′-phosphate; avoid strong secondary structure at the join; use a short splint for efficient, specific closure.
  • Circle size: Typical 30–150 nt circles (padlocks ~80–120 nt). Very small loops may need spacers (AEEA/PEG) to reduce strain.
  • Labels & handles: Add dyes, biotin, amine/azide/alkyne pre-or post-cyclization; confirm compatibility with the ligation chemistry.
  • Backbone tuning: Mix in LNA/BNA or 2′-mods to raise Tm and improve stability as needed for your assay conditions.
Benefits at a Glance
  • High stability: No free ends → improved resistance to exonucleases and longer functional lifetime.
  • Exceptional specificity: Ligation is highly stringent, enabling single-base discrimination (SNP genotyping).
  • Isothermal sensitivity: RCA delivers strong signal without thermal cycling—ideal for point-of-care and imaging assays.
  • Compact, customizable design: Small probes with built-in labels/tags; tunable Tm and spacing via spacers/PEG.
  • Versatility: Works for target detection, capture, imaging, and stabilization of aptamer/antisense constructs (research).
  • Reproducible manufacturing: Fully synthetic workflows, HPLC-purified with MS confirmation.

Extremely small circles can be strain-prone. We model the junction and add flexible linkers where needed to preserve efficiency and specificity.

Use Case Why Circular Helps Typical Setup
Rolling Circle Amplification (RCA) Strong isothermal signal for sensitive detection 80–120 nt circle; primer + φ29 polymerase; fluorescent detection probes
Padlock probe SNP genotyping Ligation only on perfect match → single-base specificity 5′-phosphorylated padlock; splint-guided ligation; RCA/qPCR readout
Molecular Inversion Probes (MIPs) Target capture & library prep with high multiplexing MIP with flanking arms + UMI; gap-fill + ligation; NGS
Circular aptamer stabilization Resists exonucleases; preserves binding conformation Aptamer cyclization via T4 ligase or click; optional PEG spacer
Circular antisense/siRNA prototypes Exploratory increase in durability for research Cyclized ASO/duplex constructs; evaluate potency vs. linear controls
Design My Circular Oligo

FAQ

What sequence lengths are supported?

Most synthesis platforms support circularization for oligos ranging from 20 to 150 nucleotides. Longer sequences may be technically feasible but require optimization of ligation conditions.

Which closure method should I choose?

Enzymatic ligation yields a native phosphodiester junction and is preferred for polymerase‑intensive workflows (e.g., RCA). Click circularization (CuAAC/SPAAC) is enzyme‑free and robust; junction compatibility is application‑dependent.

How do you verify circularization?

In addition to MS/HPLC, we can perform exonuclease challenge (Exo I/III) showing resistance compared to linear controls, and optional RCA function tests.

Can you supply RNA circles?

Yes, feasibility depends on sequence and junction chemistry (RtcB/T4 RNA ligase or click strategies). Share your use case and we will recommend an approach.

Storage recommendations?

Store lyophilized circles at 4 °C (short‑term) or −20 °C (long‑term). In solution, use nuclease‑free buffer, aliquot, and keep at −20 °C.

What is a circular oligo?

A circular oligo is a single-stranded DNA or RNA molecule that has been covalently ligated at its 5′ and 3′ ends to form a closed, circular structure. These molecules are resistant to exonucleases and have enhanced stability compared to linear oligos.

How is circularization achieved?

Circularization typically involves:

  • Synthesizing a linear oligo with compatible ends
  • Phosphorylation (if needed)
  • Ligation using T4 DNA ligase or RNA ligase
  • Purification to separate circular from linear byproducts

What length works best for circular oligos?

Most applications use 30–200 nt. Padlock probes are typically 80–120 nt with ~20–30 nt arms. We can advise based on ligase and target.

What modifications are available for circular oligos?

Common modifications include:

  • Phosphorylation (5′ or 3′)
  • Fluorescent labels (e.g. FAM, Cy5)
  • Biotin or other affinity tags
  • Spacer arms (e.g. C3, hexaethylene glycol)
  • Note: Some modifications may affect ligation efficiency.

Case Study: 91-Base Circular DNA Synthesis

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

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

Outcome:

  • Successfully synthesized and circularized the target 91-base sequence
  • Verified circularity via exonuclease resistance
  • Purified by denaturing PAGE to >95%
  • Delivered ready-to-use, research-grade oligonucleotide with documentation and QA validation
Denature Gel Results:
ESI Mass Spectra:

Linear 91 mer ssDNA, target MW: 28128.1, observed MW: 28126.9

Circular 91 mer ssDNA, target MW: 28110.1, observed MW: 28111.1

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