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Crosslinker-Modified Oligonucleotides

Custom reactive functional group oligo modification for downstream conjugation to proteins, peptides, polymers, nanoparticles, and assay surfaces.

Crosslinker-modified DNA and RNA oligonucleotides engineered for bioconjugation, biomolecular labeling, and surface immobilization. Supported chemistries include NHS ester, maleimide, carbodiimide, aldehyde-reactive, and photoreactive crosslinkers for controlled oligonucleotide–biomolecule coupling.

DNA / RNA / siRNA / ASO / SSO / PNA site-directed handle installation crosslinker-ready oligos protein / antibody conjugation support fit-for-purpose purification
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Overview Crosslinker classes Design considerations Representative crosslinkers Workflow QC Applications Related services FAQ Recommended reading Contact

Overview

Crosslinker-modified oligonucleotides are DNA or RNA molecules functionalized with reactive chemical groups that enable controlled covalent conjugation to proteins, antibodies, peptides, polymers, nanoparticles, surfaces, or other biomolecules. By incorporating specialized crosslinking reagents onto the oligonucleotide during synthesis or through post-synthetic modification, these constructs provide defined chemical handles that participate in widely used bioconjugation chemistry workflows.

Most crosslinking reagents contain two or more reactive groups capable of forming covalent bonds with functional groups commonly found in biomolecules, including primary amines, sulfhydryls, carboxyl groups, and carbonyl or aldehyde groups. Crosslinkers are therefore typically classified according to both their reactive chemistry and their linker architecture, such as homobifunctional, heterobifunctional, and photoreactive crosslinkers used for controlled biomolecular assembly.

When these chemistries are applied to oligonucleotides, they enable the construction of a wide range of hybrid biomolecular assemblies including antibody–oligonucleotide conjugates, protein–DNA conjugation systems, surface-immobilized nucleic acid probes, and photo-crosslinkable nucleic acid probes used in DNA-protein crosslinking, biomolecular interaction studies, and diagnostic assay development.

These modifications are commonly used to prepare SMCC-modified oligonucleotides, maleimide-functional oligos, NHS-activated oligos, photoreactive DNA probes, and other custom bioconjugation intermediates used in antibody conjugation, nanoparticle assembly, surface immobilization, and advanced nucleic-acid-based detection platforms.

Crosslinker-modified oligonucleotide architecture showing oligo, linker, crosslinker, and peptide conjugation

Example architecture of a crosslinker-modified oligonucleotide enabling conjugation to peptides or proteins through heterobifunctional linker chemistry.

homobifunctional crosslinkers heterobifunctional crosslinkers amine-reactive groups sulfhydryl-reactive groups aldehyde-reactive groups photoreactive crosslinkers

Reactive functional group map

This table summarizes commonly used crosslinkers that can be incorporated into oligonucleotides to introduce reactive functionality. These modified oligos serve as intermediates for downstream conjugation to proteins, peptides, polymers, nanoparticles, surfaces, or other biomolecules.

Crosslinker class Representative oligo-installed handles or reagents Targets on conjugation partner Typical downstream constructs Main design concern
Homobifunctional DSS, BS3, DSP, DTSSP, EGS, BS(PEG)n Amine-bearing proteins, surfaces, assemblies Immobilized probes, multicomponent assay builds, networked capture systems Spacer length and over-crosslinking risk
Heterobifunctional SMCC, Sulfo-SMCC, EMCS, MBS, BMPH, BMPS, SPDP Amine plus thiol or disulfide-exchange partners Antibody-oligo conjugates, peptide-oligo conjugates, nanoparticle coupling Reaction sequence and chemoselectivity
Amine-reactive (activated ester chemistry) NHS ester, Sulfo-NHS ester, TFP ester formats Lysines, terminal amines, amino-functional surfaces Protein labeling, carrier conjugation, bead or slide attachment Hydrolysis and aqueous stability
Sulfhydryl-reactive Maleimide, iodoacetyl, bromoacetyl, pyridyldithiol Cysteine-containing proteins, peptides, engineered thiols Site-directed protein-DNA builds, reducible capture systems Free thiol availability and thioether stability
Carboxyl-to-amine EDC, NHS, Sulfo-NHS activation schemes Carboxylated materials, proteins, polymers, particles Surface immobilization, hydrogel coupling, material functionalization Substrate pH tolerance and activation efficiency
Aldehyde-reactive Hydrazide, aminooxy, oxime-forming handles Oxidized glycans, aldehyde-bearing biomaterials Glycoprotein-oligo conjugates, carbonyl-directed labeling Carbonyl generation and reaction kinetics
Photoreactive Sulfo-SANPAH, SDA, Sulfo-SDA, LC-SDA, SDAD, aryl azide, diazirine Nearby biomolecules under UV activation Photoaffinity probes, interaction capture, proximity labeling Light dose, nonspecific capture, probe positioning

Crosslinker classes & representative items

Expand each class to see representative reagents, reaction partners, applications, and design notes for oligo modification.

amine-to-amine sulfhydryl-to-sulfhydryl surface assembly

Homobifunctional crosslinkers carry the same reactive group on both ends and are useful when the oligo must be matched to a partner presenting the same chemical functionality or when building networks and assemblies. Common amine-to-amine examples in this category include DSG, DSS, BS3, BS(PEG)9, DSP, DTSSP, and EGS.

Representative items Reactive chemistry Typical oligo applications Notes
DSS / BS3 Amine-to-amine Surface coupling, protein network assembly, multicomponent assay constructs Useful where two primary amine-bearing partners are involved; water solubility differs by reagent
BS(PEG)9 Amine-to-amine Higher-accessibility immobilization and lower-aggregation formats PEG spacer can improve reach and reduce steric crowding
DSP / DTSSP Amine-to-amine, cleavable disulfide Temporary conjugate architectures and cleavable capture systems Chosen when post-conjugation cleavage is desirable
EGS / DSG Amine-to-amine Spacer-optimized assembly of biomolecular constructs Spacer length selection is application-dependent

amine-to-thiol controlled stoichiometry antibody-oligo builds

Heterobifunctional crosslinkers provide two different reactive groups, enabling sequential and more controlled conjugation. This class is especially valuable for preparing oligonucleotides that can undergo sequential, directional coupling to two different partner functionalities such as amines and thiols.

Representative items Reactive chemistry Typical oligo applications Notes
SMCC / Sulfo-SMCC NHS ester to maleimide Antibody-oligo conjugates, protein-DNA constructs, nanoparticle attachment Classic amine-to-thiol format; sulfonated analog improves water compatibility
SPDP / LC-SPDP / Sulfo-LC-SPDP NHS ester to pyridyldithiol Reversible thiol-linked protein-oligo conjugation Useful when disulfide exchange or cleavable thiol chemistry is desired
EMCS / MBS / BMPH / BMPS / Sulfo-LC-SPDP Amine-reactive to thiol-reactive Peptide-oligo, enzyme-oligo, and custom bifunctional conjugates Selected based on spacer type, water solubility, and downstream stability needs

NHS ester Sulfo-NHS lysine targeting

Amine-reactive chemistries are commonly used when the conjugation partner contains accessible primary amines, such as lysines or terminal amines on peptides and proteins. These are foundational chemistries for converting an oligo into a downstream-ready reagent for amine-bearing proteins, peptides, carriers, and surfaces.

Representative items Reactive chemistry Typical oligo applications Notes
NHS ester handles Primary amine coupling Protein labeling, surface immobilization, carrier conjugation Common entry point for coupling to lysine-rich targets
Sulfo-NHS ester formats Primary amine coupling, water-compatible Aqueous bioconjugation and protein attachment workflows Often preferred when organic solvent exposure must be minimized
TFP, STP, or related activated esters Primary amine coupling Custom biomolecule and surface functionalization Alternative activated esters may be selected based on stability or handling goals

maleimide pyridyldithiol cysteine targeting

Sulfhydryl-reactive groups are used for conjugation to thiol-containing partners such as cysteine-engineered antibodies, proteins, or peptides. Maleimide, haloacetyl, and disulfide-exchange chemistries remain among the most widely used options in this class for cysteine-directed oligo conjugation.

Representative items Reactive chemistry Typical oligo applications Notes
Maleimide, haloacetyl, or thio-selective oligo handles Thiol-selective Conjugation to cysteine-containing peptides, proteins, and antibody fragments Common for site-directed bioconjugation when free thiols are available
Iodoacetyl / bromoacetyl formats Thiol-reactive alkylation Protein and peptide attachment with robust thioether formation Can be considered when maleimide is not the preferred chemistry
Pyridyldithiol handles Disulfide exchange Cleavable or exchange-enabled conjugation systems Useful for redox-responsive or reversible formats

EDC NHS surface coupling

Carboxyl-to-amine chemistry activates carboxyl groups for coupling to primary amines and is useful for attaching oligos to carboxyl-bearing materials, proteins, polymers, or surfaces. Representative reagents in this category include EDC, NHS, and Sulfo-NHS.

Representative items Reactive chemistry Typical oligo applications Notes
EDC Carbodiimide activation Coupling to carboxylated beads, polymers, proteins, or surfaces Often paired with NHS chemistry to improve efficiency
NHS / Sulfo-NHS Activated ester stabilization Improved carboxyl-to-amine conjugation workflows Sulfo variant improves aqueous handling
Project-defined carboxyl-activated handles Carboxyl-to-amine coupling Custom material, hydrogel, and surface functionalization Attachment site and oligo stability should be evaluated case by case

hydrazide aminooxy glycoprotein targeting

Aldehyde-reactive chemistries are useful when the conjugation partner contains aldehydes introduced chemically or present after glycan oxidation. These formats are commonly considered for glycoprotein labeling, carbohydrate conjugation, and specialized biomaterial coupling.

Representative items Reactive chemistry Typical oligo applications Notes
Hydrazide-modified oligos Aldehyde to hydrazone formation Glycoprotein conjugation and oxidized carbohydrate attachment Useful when targeting aldehydes generated by periodate oxidation
Aminooxy-modified oligos Aldehyde to oxime formation Stable aldehyde-based conjugation workflows Often selected for more controlled carbonyl coupling formats
Project-specific carbonyl-reactive handles Carbonyl-selective Customized biomolecule and material functionalization Reaction conditions depend on substrate stability and pH tolerance

aryl azide diazirine photo-crosslinking

Photoreactive crosslinkers become reactive after UV exposure and are useful for proximity capture, photoaffinity labeling, and interaction mapping. Representative reagents in this class include Sulfo-SANPAH, SDA, Sulfo-SDA, LC-SDA, Sulfo-LC-SDA, SDAD, and Sulfo-SDAD.

Representative items Reactive chemistry Typical oligo applications Notes
Sulfo-SANPAH / SANPAH NHS ester / aryl azide Surface functionalization and photoactivated attachment Useful for immobilization workflows involving light-triggered capture
SDA / Sulfo-SDA / LC-SDA / Sulfo-LC-SDA NHS ester / diazirine Photoaffinity probes and interaction mapping Spacer variants allow control over reach and aqueous compatibility
SDAD / Sulfo-SDAD Amine-reactive / photoactivatable, cleavable options Crosslinking studies with downstream cleavage or analysis needs Selected when both UV capture and cleavage options are valuable

Design Considerations

Designing effective crosslinker-modified oligonucleotides requires careful consideration of the target biomolecule, available reactive functional groups, and the desired conjugation strategy. Crosslinkers are typically selected to react with functional groups commonly present on biomolecules, including primary amines, sulfhydryls, carboxyl groups, and aldehydes. Matching the crosslinker chemistry to the correct reactive handle on the oligonucleotide and the target molecule ensures efficient and controlled covalent coupling.

In many oligonucleotide conjugation workflows, heterobifunctional crosslinkers are preferred because they contain two different reactive groups, enabling directional coupling between an oligonucleotide and a specific biomolecular partner. For example, NHS-ester / maleimide crosslinkers such as SMCC can couple an amine-modified oligonucleotide to cysteine residues in proteins or peptides. Alternative strategies include carbodiimide-mediated EDC/NHS coupling, hydrazide-based aldehyde reactions, and photoreactive crosslinkers for proximity-based conjugation.

Reactive Group Compatibility

Select crosslinkers that match available functional groups on both the oligonucleotide and target molecule, such as amine, sulfhydryl, carboxyl, or aldehyde groups.

Linker Architecture

Homobifunctional crosslinkers react with identical functional groups, while heterobifunctional reagents enable directional conjugation between two different biomolecular handles.

Spacer Length

Spacer arms control steric accessibility and flexibility between the oligonucleotide and the conjugated biomolecule, which can influence hybridization efficiency and binding interactions.

Reaction Environment

Reaction conditions such as buffer composition, pH, solvent compatibility, and temperature can significantly affect crosslinker stability and conjugation efficiency.

Representative Crosslinkers for Oligonucleotide Functionalization

A wide variety of crosslinking reagents can be used to functionalize oligonucleotides for downstream bioconjugation workflows. These reagents are typically selected based on reactive chemistry, spacer length, and compatibility with the functional groups present on both the oligonucleotide and the target biomolecule.

Crosslinker Reactive Groups Typical Application Oligonucleotide Functionalization Example
SMCC NHS ester / Maleimide Antibody–oligonucleotide conjugates Amine-modified oligo converted to a maleimide-bearing intermediate for coupling to cysteine-containing proteins
Sulfo-SMCC NHS ester / Maleimide Water-compatible protein conjugation 5′-amine oligonucleotide converted to a maleimide-activated oligo for aqueous conjugation workflows
SPDP NHS ester / Pyridyldithiol Reversible disulfide conjugates Oligonucleotide linked to thiol-containing peptides or proteins through cleavable disulfide chemistry
DSS NHS ester / NHS ester Amine-to-amine crosslinking Oligonucleotide immobilization to amine-functionalized surfaces or protein assemblies
BS3 NHS ester / NHS ester Water-soluble amine crosslinking Surface conjugation of DNA probes to proteins, carriers, or polymer supports
EDC Carboxyl / Amine coupling Carbodiimide-mediated conjugation Carboxyl-modified oligonucleotide linked to amine-containing proteins, particles, or materials
Sulfo-SANPAH NHS ester / Photoreactive azide Surface immobilization UV-activated attachment of oligonucleotides to biomaterial or assay surfaces
SDA NHS ester / Diazirine Photoaffinity labeling Photo-crosslinkable DNA probes used for interaction mapping and proximity capture experiments
Hydrazide Linkers Aldehyde / Hydrazide Glycoprotein and carbohydrate conjugation Hydrazide-modified oligonucleotides coupled to oxidized glycans or aldehyde-bearing biomolecules

Workflow: from design to delivery

1) Define the chemistry

Review oligo format, attachment position, conjugation partner, and whether the chemistry should be permanent, reversible, or photoactivated.

2) Install the handle

Functionalize the oligo with the requested crosslinker-compatible group or linker architecture using the selected synthesis strategy.

3) Purify and confirm

Apply fit-for-purpose purification and analytical confirmation to support downstream conjugation and research use.

Typical downstream uses: antibody-oligo conjugates, peptide-oligo constructs, protein labeling, nanoparticle coupling, surface immobilization, pull-down tools, and photo-crosslinking probes.

QC & deliverables

Standard analytical confirmation

  • Analytical HPLC or UPLC profile
  • Mass confirmation when applicable
  • COA and modification summary

Fit-for-purpose purification

  • Desalting or higher-purity workflows as needed
  • Handling aligned to reactive group stability
  • Project-specific delivery format when feasible

Documentation

  • Sequence and modification annotation
  • Requested attachment position summary
  • Notes aligned to downstream conjugation intent

Typical Applications

Antibody–Oligonucleotide Conjugates

Crosslinker-modified oligos enable controlled attachment to antibodies for immuno-PCR, spatial biology, and multiplex detection platforms.

Protein–DNA Conjugates

Reactive oligos can be coupled to enzymes, binding proteins, or nanobodies to create hybrid biomolecular probes.

Surface Immobilization

Crosslinker-functionalized oligonucleotides support attachment to beads, nanoparticles, biosensor surfaces, and microarray substrates.

FAQ

What are crosslinker-functionalized oligonucleotides?

Crosslinker-functionalized oligonucleotides are DNA or RNA molecules carrying reactive chemical groups that enable covalent coupling to proteins, peptides, polymers, nanoparticles, or surfaces. These reactive handles allow oligos to participate in controlled bioconjugation reactions.

Which crosslinker chemistries are commonly used?

Common chemistries include NHS ester amine-reactive linkers, maleimide thiol-reactive reagents, carbodiimide systems such as EDC/NHS, hydrazide aldehyde-reactive linkers, and photoreactive crosslinkers such as diazirine or benzophenone.

What is the difference between homobifunctional and heterobifunctional crosslinkers?

Homobifunctional crosslinkers contain two identical reactive groups that react with the same functional group. Heterobifunctional crosslinkers contain two different reactive groups, enabling directional coupling between two different biomolecules.

How are crosslinker-modified oligonucleotides used?

These oligonucleotides are used to build antibody–DNA conjugates, protein–DNA conjugates, nanoparticle-oligo systems, surface-immobilized probes, and photo-crosslinkable nucleic acid probes used in diagnostics, biosensors, and molecular biology workflows.

Can crosslinker-modified oligonucleotides be used for surface immobilization?

Yes. Reactive groups such as NHS ester, maleimide, or photoreactive crosslinkers allow oligonucleotides to be covalently attached to glass slides, nanoparticles, hydrogels, polymer supports, or other functionalized surfaces used in biosensors and microarrays.

What information is needed to request a quote?

For the fastest quote, provide the oligonucleotide sequence, desired crosslinker or reactive group, attachment position (5′, 3′, or internal), required purification, quantity, and intended conjugation target such as proteins, peptides, nanoparticles, or surfaces.

More FAQ'sAll FAQ's

Contact & quote request

For the fastest quote on crosslinker-modified oligonucleotides, share the sequence, oligo class, requested crosslinker or functional group, preferred attachment position, quantity, purification target, and intended conjugation partner.

Fast quote checklist

  • Sequence and oligo type: DNA, RNA, siRNA, ASO, SSO, PNA, PMO, or related format
  • Named crosslinker or reactive class: SMCC, SPDP, NHS, maleimide, hydrazide, diazirine, etc.
  • Desired installation position: 5′, 3′, internal, branched, or duplex-specific
  • Conjugation partner and intended application
  • Scale, purification target, and timeline constraints

Fastest path

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Recommended Reading

  • Hermanson, G. T. Bioconjugate Techniques, 3rd Edition. Academic Press, 2013. Comprehensive reference covering crosslinkers, reactive groups, and biomolecular conjugation strategies.
  • Kalia, J.; Raines, R. T. Advances in Bioconjugation. Current Organic Chemistry, 2010.
  • Sinz, A. Chemical Crosslinking and Mass Spectrometry to Study Protein Interactions. Angewandte Chemie International Edition, 2018.
  • Crooke, S. T., et al. Antisense Drug Discovery and Development Technology. Nature Reviews Drug Discovery, 2018.
  • Roberts, T. C., et al. Advances in Oligonucleotide Drug Delivery. Nature Reviews Drug Discovery, 2020.
  • Dormán, G.; Prestwich, G. D. Benzophenone Photophores in Biochemistry. Biochemistry, 1994.
  • Dubinsky, L.; Krom, B. P.; Meijler, M. M. Diazirine Based Photoaffinity Labeling. Bioorganic & Medicinal Chemistry, 2012.

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