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Cleavable Oligonucleotide Probes

Custom cleavable linker-modified oligonucleotides and fluorescent readout probes for MERFISH, cyclic imaging, spatial transcriptomics, affinity capture-release workflows, signal amplification, and multiplex molecular detection applications.

Disulfide Cleavable Dyes Photocleavable Linkers MERFISH Readout Probes HPLC / LC-MS QC Published Spatial Biology Workflows
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Overview Technologies Selection Guide MERFISH Readout Design Notes Products Applications Reading FAQ Contact
Overview

Controlled Signal Removal Through Cleavable Linker Design

Bio-Synthesis manufactures cleavable oligonucleotide probes that use triggerable linker chemistry to attach, image, release or reset fluorescent labels after target detection. These constructs are especially important for high-plex imaging workflows where the same sample must be reused across many sequential hybridization and imaging rounds.

Cleavable probes are commonly designed as dye-labeled readout oligonucleotides that hybridize to an encoding or docking sequence. After imaging, the dye or reporter is removed by a trigger such as disulfide reduction, photocleavage, enzyme treatment, pH change or a custom bioorthogonal cleavage reaction.

Applications include MERFISH readout probes, cyclic imaging, spatial biology, multiplex RNA imaging, capture-and-release assays, signal amplification constructs and custom oligo architectures requiring controlled activation, release or fluorescence reset.

Use the Cleavable Probe Selection Guide to compare disulfide, photocleavable, enzyme-cleavable, acid-labile, base-labile and click-to-cleave strategies.

DNA / LNA-Mix / 2′-OMe Options 5′, 3′ & Internal Dye Sites Dye Loading Report CoA & Lot Records Panel Scale Support
Spatial Biology Imaging Cycle

1. HYBRIDIZE

Readout probe binds barcode or target-docking sequence.

2. IMAGE

Fluorescent dye reports the current barcode bit or target state.

3. REGISTER

Signal is recorded across channels and imaging rounds.

4. CLEAVE

Trigger removes dye, reporter, affinity tag or linker payload.

5. RESET

Low-background sample is ready for the next round.

10-10,000+ barcodes

Panel support is design-dependent for high-plex MERFISH and cyclic imaging builds.

<10% CV fluor load

Typical batch-to-batch dye-loading uniformity target for consistent readout rounds.

RUO to GMP-like scale

Documentation and scale-up paths for R&D through regulated development builds.

Cleavable Linker Technologies

Cleavable Linker Technologies We Support

Each cleavage strategy offers a different trigger, timing profile and sample-compatibility profile. Bio-Synthesis can help align the linker chemistry with your imaging cycle, capture-release workflow or controlled activation requirement.

SS

Disulfide-Cleavable Probes

Reductive cleavage using DTT or TCEP for dye removal and cyclic imaging resets.

MERFISHCyclic FISHDye-Agnostic
PC

Photocleavable Probes

Light-triggered o-nitrobenzyl, DMNB or related photocleavable linker options.

On-Scope ResetFast CyclesAutomation
cPC

Coumarin Photocleavable Linkers

Violet/near-UV responsive linkers that can reduce UV exposure depending on setup.

Delicate SamplesSpatial Control
EC

Enzyme-Cleavable Probes

Esterase- or protease-responsive designs for biologically compatible trigger systems.

Tissue-FriendlyBiocompatible
AL

Acid-Labile Linkers

Mild-acid cleavage strategies for robust cells, materials and release workflows.

pH TriggerMaterials
BL

Base-Labile Linkers

Mild-base release options for specialty orthogonal cleavage workflows.

OrthogonalCustom Release
CtC

Click-to-Cleave Designs

Custom bioorthogonal strategies where a secondary reaction initiates cleavage.

AdvancedCustom
2X

Dual-Dye & Branched Readouts

Signal-boosting readout probes with multi-dye or multi-arm cleavable architectures.

High SNRBranched
Cleavable Probe Selection Guide

Cleavable Probe Selection Guide

Use this table to compare cleavage triggers, timing, advantages, design constraints and best-fit workflows before selecting a cleavable linker for readout probes, imaging cycles or capture-release constructs.

Trigger, Speed & Workflow Compatibility

This selection map preserves the important live-site comparison while making the decision path easier to scan.

Cleavable linker selection guide for fluorescent readout probes and controlled-release oligonucleotide workflows.

Linker Type Trigger Speed Pros Considerations Best-Fit Uses Code
Disulfide 10-50 mM DTT/TCEP 2-10 min Gentle, dye-agnostic, widely adopted Requires reducing step; avoid free thiols before imaging MERFISH and cyclic FISH resets [SS-5′] [SS-3′]
Photocleavable oNB/DMNB UV/violet light <1-2 min Fast on-scope reset and automation friendly Light control needed; evaluate photodamage Time-critical cycles and automated imaging [PC-5′] [PC-3′]
Photocleavable Coumarin Violet/near-UV <1-2 min Lower UV dose than oNB in some setups Optimize fluence and illumination hardware Delicate samples and spatially controlled reset [cPC-5′] [cPC-3′]
Enzyme-Cleavable Esterase/protease 5-30 min Biocompatible trigger options Enzyme sourcing, access and longer cycles Tissue-friendly workflows [EC-5′] [EC-3′]
Acid-Labile pH < 6 5-15 min Simple buffer trigger Check sample and backbone tolerance to pH Robust cells, materials and release assays [AL-5′] [AL-3′]
Base-Labile Mild base 5-15 min Orthogonal to acid-labile release Confirm backbone and modification compatibility Specialty controlled-release designs [BL-5′] [BL-3′]
Click-to-Cleave* Bioorthogonal trigger 1-10 min Spatiotemporal and design-specific control Custom engagement; availability depends on design Advanced and custom workflows [CtC-5′] [CtC-3′]

*Click-to-cleave denotes designs where a secondary click reaction initiates cleavage; available as a custom engagement.

MERFISH Readout Probes

Cleavable Readout Probes for MERFISH & Cyclic Imaging

In MERFISH, barcoded encoding probes bind target RNAs. Across sequential rounds, cleavable dye-labeled readout probes hybridize to barcode docking sequences, reveal a subset of bits, and are then cleaved or reset before the next round.

CODE

Barcodes & Cycles

Design supports Hamming-style codes and multi-color imaging rounds. 8-16+ cycles are common depending on plex, channel count and error tolerance.

SNR

Signal Control

Cleavable linkers help maintain intensity consistency, reduce carryover and preserve tissue morphology across repeated imaging rounds.

DYE

Dye Choices

Options include FITC/Alexa 488, Cy3, Atto 550, Alexa 568, Texas Red, Alexa 594, Cy5/Atto 647N, Alexa 647, Alexa 700 and related channels.

Design tip: Pair readout probe Tm about 5-8 °C above imaging temperature to minimize off-rates while keeping wash times short. Common readout designs are approximately 18-25 nt; LNA or 2′-OMe motifs can support shorter or faster-washing probes.

Design Notes

Cleavable Linker Design Considerations

Use these practical design notes with the Selection Guide to choose a linker strategy that fits the imaging cycle, sample tolerance and downstream analytical requirements.

Disulfide vs Photocleavable Reset

Disulfide-cleavable probes are gentle, dye-agnostic and widely used for MERFISH and cyclic imaging. Photocleavable linkers provide fast on-scope reset when light exposure and sample tolerance are acceptable.

  • DTT/TCEP reset for reductive cleavage
  • UV/violet light for photocleavage
  • Choose around sample sensitivity

Imaging Cycle Optimization

Probe length, Tm, wash conditions, dye brightness and cleavage completeness all affect carryover and consistency across imaging rounds.

  • 18-25 nt readout probes are common
  • Tm typically above imaging temperature
  • Minimize residual signal between rounds

Custom Linker Engineering

Custom probe builds can combine spacers, internal dye sites, mixed backbones, branched formats and orthogonal release handles for specialized workflows.

  • PEG/hexa-EG spacer options
  • LNA or 2′-OMe motifs
  • Branched and multi-dye readouts
Products & Ordering

Representative Cleavable Probe Product Formats

These formats preserve the important live-site ordering information while presenting the options in a more readable table.

Representative cleavable dye and reporter-modified oligonucleotide formats.

Product / Modification Description Typical Use Notes Code
5′-Dye Disulfide-Cleavable Dye via disulfide at 5′ terminus Cyclic imaging / MERFISH reset Fast and dye-agnostic [Dye-SS-5′]
3′-Dye Disulfide-Cleavable Disulfide-tethered dye at 3′ terminus Alternative geometry / dual-dye designs Balances sterics and docking orientation [Dye-SS-3′]
Photocleavable Dye oNB/DMNB o-nitrobenzyl-family linker Rapid on-scope reset Protect from stray light [Dye-PC-5′/3′]
Photocleavable Dye Coumarin Violet-sensitive coumarin linker Lower UV dose, setup-dependent Tune illumination [Dye-cPC-5′/3′]
Enzyme-Cleavable Dye Ester or peptide-based cleavage Tissue-compatible workflows Longer cycle time [Dye-EC-5′/3′]
Acid-Labile Dye Cleavage under mild acidity Materials and robust cells Check backbone stability [Dye-AL-5′/3′]
Dual-Dye Cleavable Readout Two dyes per oligo with a cleavable strategy High SNR / two-color rounds Verify crosstalk [2xDye-SS/PC]
Biotin / Reporter Cleavable Cleavable biotin or hapten such as DIG Capture-and-release assays Orthogonal triggers available [Bio-SS/PC-5′/3′]

Customizations: internal dye sites, PEG/hexa-EG spacers, mixed backbones such as LNA or 2′-OMe, desalting-to-HPLC purification, aliquots, kit packaging, CoA, method summary and lot record on request.

Applications & Published Workflows

Applications & Published Workflows for Cleavable Oligonucleotide Probes

Cleavable linker probes support controlled signal reset, release and activation across spatial biology, cyclic imaging and custom biomolecular detection workflows.

MER

MERFISH

Cleavable readout probes for high-plex spatial transcriptomic barcodes and repeated imaging cycles.

Explore Application →

IMG

Cyclic Imaging

Fluorescent oligo probes designed for signal reset, re-hybridization and multi-round imaging workflows.

Explore Application →

REL

Cleavable Affinity Capture

Cleavable linker strategies for target capture, enrichment, purification, controlled release and downstream molecular analysis.

Explore Application →

UV

Photo-Controlled Cleavage

Photocleavable oligo linkers for on-scope, time-controlled or spatially localized signal removal.

Explore Application →

RNA

Spatial Biology & Multiplex RNA Imaging

Sequential imaging and cleavable readout probe workflows for spatial transcriptomics, multiplex RNA detection and high-plex tissue mapping.

Explore Application →

bDNA

Signal Amplification

Branched or multi-arm readout probe formats for higher signal-to-noise and custom probe architectures.

Explore Application →

TIS

Tissue-Friendly Workflows

Enzyme-cleavable and tuned photochemistry options for fixed cells, tissues and morphology-sensitive samples.

Explore Application →

PUB

Published Probe Builds

Disulfide-linked dye readout probes used in multiple published spatial biology and MERFISH workflows.

Explore Application →

FAQ

What is a readout probe?
A readout probe is a short dye-labeled oligonucleotide that hybridizes to a docking or barcode sequence on an encoding probe. It is imaged during each round and then removed or reset using a cleavable linker strategy.
What is a MERFISH probe?
MERFISH uses encoding probes that bind target RNAs and readout probes that bind barcode docking sequences. Sequential imaging rounds and error-robust barcodes enable highly multiplexed detection with error correction.
Do you provide branched cleavable readout probes?
Yes. Bio-Synthesis can manufacture branched or multi-arm readout probes, including 2-4 arm concepts, with optional cleavable linkers such as disulfide, photocleavable or enzyme-cleavable formats.
How do I design a MERFISH readout probe?
Start with the codebook, define rounds and channels, choose 18-25 nt readout sequences, minimize cross-talk, choose dye and cleavable linker, and confirm wash and cleavage conditions. Bio-Synthesis can review the codebook and manufacturable bill of materials.
What length and Tm are recommended?
Common readout probes are about 18-25 nt with Tm approximately 5-8 °C above imaging temperature. LNA or 2′-OMe motifs may be used to increase Tm for shorter or faster-washing probes.
How fast is disulfide cleavage versus photocleavage?
Disulfide cleavage with 10-50 mM TCEP or DTT typically completes in a few minutes. Photocleavage can be sub-minute to a few minutes depending on light source, fluence and sample tolerance.
Can the same readout probe be reused across rounds?
Readout designs can be cycled across rounds if carryover is controlled. Cleavable linkers reduce bleed-through and residual signal compared with non-cleavable dye formats.
Which dyes are robust for multi-round imaging?
Atto 550/647N, Alexa 568/594/647 and related dye families are often selected for brightness and stability. Dye choice should match excitation/emission hardware and crosstalk tolerance.
More FAQ'sAll FAQ's
Contact & Quote Request

Ready to design a cleavable readout probe or linker-modified oligo?

Share your sequence, readout length, codebook or docking tags, dye channels, desired cleavage mode, number of imaging rounds, purification target and QC requirements. Bio-Synthesis can help align cleavable linker chemistry, dye selection, spacer architecture, purification and analytical documentation with your workflow.
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Email: info@biosyn.com
Phone: 800-227-0627 (US/CAN)
Phone: +001-972-420-8505 (INT)

Related Services

Cleavable linker and fluorescence probe design options.

Cleavable Linkers Fluorescence Probes Photocleavable Oligos MERFISH Probes

Fast Quote Checklist

Include sequence, dye, cleavage mode, cycles, scale and QC.

Sequence Dye Mode Rounds QC
Referenced in Peer-Reviewed Publications

Key Publications & Literature References

Published Spatial Biology WorkflowsMERFISH & Cyclic Imaging References
Referenced in peer-reviewed spatial biology workflows
PNASNatureScientific ReportsNature NeuroscienceSpatial Biology

Selected references from the live Bio-Synthesis page highlight MERFISH, cyclic imaging and spatial transcriptomics workflows using dye-labeled readout probes, including multiple publications that mention Bio-Synthesis/Biosynthesis readout probes or disulfide-linked probe formats.

  1. Moffitt JR, et al. High-performance multiplexed fluorescence in situ hybridization with error-robust barcoding and clearing. PNAS. 2016;113(39):11046-11051. DOI
  2. Wang G, et al. Multiplexed imaging of high-density libraries of RNAs with MERFISH and expansion microscopy. Scientific Reports. 2018;8:4847. DOI
  3. Zhang M, et al. Molecularly defined and spatially resolved cell atlas of the whole mouse brain. Nature. 2023;615:117-124. Article
  4. Bhattacherjee A, et al. Spatial transcriptomics reveals the distinct organization of inhibitory and excitatory neurons in human cortex. Nature Neuroscience. 2023. Article
  5. Wang C, et al. Imaging-based pooled CRISPR screening reveals regulators of lncRNA localization. PNAS. 2019;116(39):19490-19499. DOI
  6. Luce JJ, et al. Protocol optimization improves the performance of MERFISH readout probes in tissue. Scientific Reports. 2025. Article
  7. Chaves-Perez A, et al. Metabolic adaptations direct cell fate during tissue regeneration. Nature. 2025. Article
  8. Moffitt JR. MERFISH - Imaging protocol. Zhuang Lab / protocols.io reference.

Note: Reference statements should be checked against final journal text before publication. The live page notes several papers that mention readout probes purchased or synthesized by Bio-Synthesis/Biosynthesis and disulfide-linked dye readout probe formats.

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Greetings Researchers,

Please be advised that any information, guidelines, writeups, and oral/video/graphic content on our website are intended solely as general guidelines.
It is the researcher's sole responsibility to conduct thorough research on the designs of the products intended for their experiments before submitting
their designs to Biosynthesis for review of production feasibility.
Thank you for your understanding.

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