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DNA Cross-Linking & Ligation Oligonucleotides

Photo-reactive, radical-generating, click-ready and ligation-ready oligonucleotides for interstrand cross-links, protein–DNA capture, proximity ligation, non-enzymatic ligation, NGS adapters and structure–function studies.

Psoralen • CNVK • Thio-Bases 5-Br-dU • 5-I-dU • 8-Br-dG Benzophenone • Diazirine • Azido-dU 5′-P • 5′-App • Click Handles

Design Cross-Linking and Ligation Oligos Around the Reaction

Bio-Synthesis supports custom DNA cross-linking and ligation oligonucleotides for structural biology, repair pathway mapping, proximity capture, protein–DNA interaction studies, chemical ligation and adapter workflows.

Bio-Synthesis offers a broad portfolio of DNA cross-linking and ligation modifications including psoralen cross-linkers, CNVK photocrosslinkers, thio-bases, halogenated radical bases, benzophenone and diazirine photocapture chemistries, as well as 5′-phosphate, 5′-App and click-ready handles for enzymatic and chemical ligation workflows.

Our team can help evaluate placement, spacer length, duplex context, light wavelength, coupling chemistry, matched controls, purification, ICL verification, LC-MS compatibility and documentation from discovery-scale RUO supply to larger development programs.

Reactive Handle → Activation → Cross-Link / Ligation Readout
Reactive oligo Activation ICL / capture / ligation

Choose the Chemistry by Geometry, Activation and Readout

This section reorganizes the important live-site technology details into a decision guide so customers can select the right reactive base, linker, light condition and QC approach.

Select a design topic to view technical guidance
Placement strategy determines yield, specificity and background.

Interstrand Cross-Links

Psoralen and CNVK often perform best at TA/AT steps in stable duplex regions.

Protein–DNA Capture

Place benzophenone or diazirine at suspected contact bases; avoid termini unless end-effects are desired.

Spacer Geometry

C2, C6 and TEG spacers can tune distance, steric access and duplex compatibility.
Activation must match the photochemistry and minimize nonspecific damage.

Psoralen

320–400 nm, often 365 nm; monitor mono-adduct to ICL conversion.

CNVK / BP / Diazirine

350–365 nm activation; CNVK is fast, while diazirine carbene is short-lived.

Halogenated Bases

302 nm commonly used for radical routes; oxygen scavengers may tune adduct profiles.
Enzymatic and chemical ligation require different handles and constraints.

Enzymatic Ligation

Use 5′-phosphate or 5′-App; avoid bulky lesions directly at the nick site.

Click Ligation

Use propargyl, azide, alkyne, DBCO/TCO/tetrazine pairs for CuAAC, SPAAC or IEDDA workflows.

Oxime / Thiol Routes

Formyl + aminooxy gives oxime ligation; maleimide requires pH and thiol-protection planning.
Controls and QC make reactive oligo data interpretable.

Controls

No-UV controls, scrambled-site controls, unmodified duplexes and time/dose titration.

Verification

Denaturing PAGE, native/denaturing comparison, HPLC/UPLC and LC-MS where compatible.

Readouts

Gel shifts, mass-shift mapping, qPCR blockade, fluorescence readouts and ligation efficiency.
Spacer and geometry choices often determine whether the chemistry can physically reach the intended target.
Base
C2

Close Contact

tight positioning, short reach

C6

Standard Reach

general cross-linking and conjugation

TEG

Flexible Access

reduced steric hindrance

PEG

Extended Distance

bulky partners and surfaces

Spacer & Geometry Design Notes

Short spacers help preserve positional accuracy for cross-link formation. Longer C6, TEG or PEG spacers can improve accessibility for proteins, surfaces, fluorophores, click partners and bulky conjugates.

When to Use Short Linkers

Use C2 or direct base placement when geometry must be close and cross-link position needs high specificity.

When to Use C6 / TEG

Use C6 or TEG when protein access, click ligation or conjugation yield may be limited by steric hindrance.

Stacking-Based Designs

Pyrene and perylene analogs can support proximity-driven interactions through π-stacking in high-Tm duplex regions.

Cross-Linking & Ligation Technology Selector

Use the tabs to compare photoreactive cross-linkers, radical bases, conjugation handles, click ligation handles, structural reporters and 2′-OMe variants.

Select a technology category to view product options

Photoreactive Psoralen, CNVK & Thio-Bases

Best forICLs
ActivationUVA / 365 nm
Design focusduplex context
QC focusICL conversion
Category Product / Modification Code Description Application
Psoralen Psoralen C6 [Pso-C6] Psoralen via hexyl linker UVA-induced interstrand cross-links
Psoralen Psoralen C2 [Pso-C2] Shorter C2-linked psoralen Tighter-geometry UVA-induced ICLs
CNVK 3-Cyanovinylcarbazole [CNVK] Ultra-fast 365 nm photocrosslinker Interstrand cross-linking and rapid light activation studies
Thio Base 4-Thio-dU [4S-dU] Photoactive thio-uridine analog UVA cross-linking and photodamage models
Thio Base 4-Thio-dT [4S-dT] Photoactive thio-thymidine analog UVA cross-linking and photodamage models
Thio Base 2-Thio-dT [2S-dT] Thio-thymidine photochemical analog Photochemical probes and oxidative damage studies
Thio Base 6-Thio-dG [6S-dG] Thio-guanine photochemical analog Photochemical probes and oxidative damage studies
Photocapture Benzophenone-dU [BP-dU] Aryl-ketone uridine photocrosslinker Protein–DNA photocapture
Photocapture Benzophenone-dC [BP-dC] Aryl-ketone cytidine photocrosslinker Protein–DNA photocapture
Photocapture Diazirine-dU [Diaz-dU] Carbene-generating uridine photocrosslinker Protein–DNA contact mapping
Photocapture Diazirine-dC [Diaz-dC] Carbene-generating cytidine photocrosslinker Protein–DNA contact mapping
Nitrene Azido-dU [N3-dU] Azide-bearing uridine photo-insertion handle Photo-crosslinking and capture assays
Nitrene 4-Azidophenacyl-dT [AzPh-dT] 4-azidophenacyl thymidine photo-insertion handle Photo-crosslinking and capture assays

Technology note: Use 350–365 nm for CNVK, benzophenone and diazirine, and 320–400 nm for psoralen to reduce unwanted 254 nm damage.

Radical Halogenated & 8-Bromo Purine Bases

Best forradicals
Activation~302 nm
Design focusoxygen / dose
QC focusadduct profile
Category Product / Modification Code Description Application
Bromo Base 5-Br-dU [5-Br-dU] 5-bromouracil Radical cross-linking and mutagenesis models
Bromo Base 5-Br-dC [5-Br-dC] 5-bromocytidine Radical cross-linking
Iodo Base 5′-I-dT [5′-I-dT] 5′-iodinated thymidine analog Photo-radical cross-linking and lesion formation
Iodo Base 5-I-dU [5-I-dU] 5-iodouridine analog Photo-radical cross-linking and lesion formation
Iodo Base 5-I-dC [5-I-dC] 5-iodocytidine analog Photo-radical cross-linking and lesion formation
8-Bromo Purine 8-Br-dG [8-Br-dG] 8-bromoguanine UV-induced cross-links and lesion models
8-Bromo Purine 8-Br-dA [8-Br-dA] 8-bromoadenine UV-induced cross-links and lesion models
Bromo U Bromo-U [Br-U] Generic brominated uracil analog Radical cross-linking panels

Technology note: Oxygen scavengers can modulate radical lifetime and adduct profiles; include no-light and unmodified controls.

Maleimide, Thiol & Amino Conjugation Handles

Best forconjugation
Routethiol / NHS
Design focuspH + protection
QC focusmass shift
Category Product / Modification Code Description Application
Maleimide Maleimide-dT [Mal-dT] Internal thiol-reactive maleimide handle Thiol–maleimide ligation and cross-linking
Maleimide 5′-Maleimide-C6 [5′-Mal-C6] Terminal C6 maleimide handle Terminal thiol-reactive conjugation
Thiol Thiol-dT [Thiol-dT] Internal sulfhydryl handle Maleimide conjugation and disulfide designs
Thiol 5-Mercapto-dC [5-Mercapto-dC] Mercapto cytidine analog Thiol-based ligation and conjugation
Amine 3′-Amino dT [3′-NH2-dT] Terminal primary amine NHS/EDC coupling and capture to surfaces

Technology note: Maleimide coupling commonly uses pH 6.5–7.0; thiols often ship protected and are deprotected under mild reducing conditions.

Click Chemistry, Vinyl & Aldehyde Ligation Handles

Best forchemical ligation
RouteCuAAC / SPAAC
Design focuspartner chemistry
QC focusconjugate identity
Category Product / Modification Code Description Application
Terminal Alkyne 3′-Propargyl-5-Me-dC [3′-Propargyl-5-Me-dC] 3′ terminal alkyne on 5-methyl-dC CuAAC click ligation to azides
Internal Alkyne Propargyl-dU [Propargyl-dU] Internal uridine alkyne handle CuAAC to azide-DNA or azide-protein partners
Internal Alkyne Propargyl-dC [Propargyl-dC] Internal cytidine alkyne handle CuAAC to azide-DNA or azide-protein partners
Vinyl Vinyl-dU [Vinyl-dU] Electrophilic vinyl uridine handle Michael-type ligation and cross-linking
Vinyl Vinylbenzyl-dC [VB-dC] Vinylbenzyl cytidine handle Michael-type ligation and cross-linking
Aldehyde Formyl-dC [Formyl-dC] Aldehyde-bearing cytidine Oxime or Schiff-base ligation
Aldehyde Formyl-dU [Formyl-dU] Aldehyde-bearing uridine Oxime or Schiff-base ligation
Click Pair Azide [N3] Azide bioorthogonal handle CuAAC or SPAAC workflows
Click Pair Alkyne [Alkyne] Terminal or internal alkyne handle CuAAC click workflows
Click Pair DBCO [DBCO] Strain-promoted cyclooctyne Copper-free SPAAC ligation
Click Pair TCO [TCO] trans-Cyclooctene handle IEDDA ligation with tetrazine
Click Pair Tetrazine [Tz] Tetrazine bioorthogonal handle IEDDA ligation with TCO

Technology note: CuAAC requires copper-stabilizing ligands; oxime ligation is usually more hydrolytically stable than imine formation.

Structural, Stacking & Reporter Analogs

Best forreadouts
Routestacking / fluorescence
Design focushigh-Tm segments
QC focusassay compatibility
Category Product / Modification Code Description Application
Structural 2′-deoxy-Pseudouridine [dΨ] Isomerized uridine lacking 2′-OH Structural probes and ligase compatibility studies
Fluorescent Base 2-Aminopurine [2-AP] Fluorescent base analog Stacking and ligation kinetics readout
Damage Mimic 5-F-dU [5-F-dU] 5-fluorouracil deoxy analog Damage mimic and reactivity handle
PAH Base Pyrene-dU [Pyr-dU] π-stacking pyrene base analog Templated non-enzymatic ligation
PAH Base Perylene-dU [Per-dU] π-stacking perylene base analog Templated non-enzymatic ligation

Technology note: Embed stacking analogs in high-Tm duplex regions when proximity or stacking-driven ligation is the goal.

2′-O-Methyl Variants

Best forstability
Routeduplex tuning
Design focusdensity
QC focussequence function
Category Product / Modification Code Description Application
2′-OMe 2′-OMe-2,6-Diaminopurine [2′-OMe-DAP] 2′-O-Me ribose plus DAP base Stabilized pairing and templated ligation
2′-OMe 2′-OMe-5-F-U [2′-OMe-5-F-U] 2′-O-Me-5-fluorouridine Ligation, stacking and probe design
2′-OMe 2′-OMe-5-Me-U [2′-OMe-5-Me-U] Methylated 2′-O-Me uridine Duplex stabilization and affinity tuning
2′-OMe 2′-OMe-5-Me-C [2′-OMe-5-Me-C] Methylated 2′-O-Me cytidine Duplex stabilization and affinity tuning
2′-OMe 2′-OMe-I [2′-OMe-I] Hypoxanthine base on 2′-O-Me ribose Degenerate pairing and templated ligation
2′-OMe 2′-OMe-5-Br-U [2′-OMe-5-Br-U] Brominated 2′-O-Me uridine Photo/radical strategies with added stability
2′-OMe 2′-OMe-2-Aminopurine [2′-OMe-2-AP] Fluorescent base on 2′-O-Me ribose Real-time ligation and stacking readouts

Technology note: 2′-OMe can increase nuclease resistance and Tm; balance density to preserve ligase, polymerase or hybridization function.

Match Cross-Linking Chemistry to the Experimental Goal

The same reactive base can behave differently depending on whether the goal is interstrand cross-linking, protein capture, chemical ligation or adapter/library construction.

Select an application goal to view design recommendations

Interstrand cross-link studies require duplex-compatible placement, controlled light activation and ICL verification.

Recommended Modifications

Psoralen C6/C2, CNVK, 4-thio-dU, 4-thio-dT and selected halogenated bases.

Typical Readout

Denaturing PAGE, LC-MS, native/denaturing comparison, repair processing or qPCR blockade.

Design Focus

TA/AT context, flanking duplex stability, light dose, no-UV control and conversion tracking.

Protein–DNA capture benefits from short-lived reactive groups placed at suspected contact positions.

Recommended Modifications

Benzophenone-dU/dC, diazirine-dU/dC, azido-dU and 4-azidophenacyl-dT.

Typical Readout

Gel shift, pull-down, mass-shift mapping, IP enrichment or sequencing-linked readout.

Design Focus

Contact-site placement, spacer selection, protein compatibility and background controls.

Chemical ligation depends on compatible functional groups, reaction pH and partner chemistry.

Recommended Modifications

Propargyl-dU/dC, azide, DBCO, TCO, tetrazine, formyl-dC/dU, thiol and maleimide handles.

Typical Readout

HPLC shift, LC-MS identity, gel shift, fluorescence labeling or conjugate isolation.

Design Focus

Reaction route, spacer geometry, compatible partner, side reactions and purification method.

Adapter and library workflows need ligation-ready termini and careful avoidance of blocking chemistry near nick sites.

Recommended Modifications

5′-phosphate, 5′-App, 3′ blockers, propargyl handles and ligase-compatible terminal designs.

Typical Readout

Ligation yield, NGS adapter performance, primer compatibility and library conversion efficiency.

Design Focus

Terminal purity, enzyme compatibility, avoid bulky lesions at nick sites and validate adapter context.

Workflow for Cross-Linking & Ligation Oligos

Successful reactive oligo projects connect assay design with placement, activation chemistry, synthesis, purification and analytical release.

01
Reaction Goal Define ICL, protein capture, click ligation, adapter ligation or proximity readout.
02
Technology Choice Select psoralen, CNVK, thio-base, halogenated base, click handle or ligation terminus.
03
Placement Design Review internal vs 5′/3′ placement, spacer length, duplex context and controls.
04
Synthesis Build DNA/RNA constructs with specialty amidites, reactive handles or terminal groups.
05
Purification Use HPLC/UPLC, PAGE or method-matched cleanup for reactive constructs.
06
QC & Delivery Release with purity, mass identity, concentration, CoA and packaging.

QC Strategy for Reactive Oligos

BCross-linking and ligation-ready oligos often require method-matched purification and analytical confirmation because reactive groups can alter stability, chromatography and mass interpretation.

Analytical Control Matrix

QC packages may include HPLC/UPLC purity, LC-MS identity, PAGE, OD260 concentration, ICL conversion support, conjugate mass shift verification, custom packaging and documentation.

HPLC / UPLC

Purity assessment and chromatographic profile for modified oligos.

LC-MS

Mass identity confirmation and conjugate mass-shift verification where compatible.

PAGE / ICL Check

Native vs denaturing comparison and ICL conversion support.

Custom QC

Optional endotoxin, residuals, stability points, plate formatting and barcodes.

Light-Control Review

Plan wavelength, dose/time, no-light controls and non-specific background checks.

Conjugate Identity

Confirm modified oligo profile and attachment where method-compatible.

Scale & Packaging

Discovery µmol to larger supply, tubes, vials, plates, labels and barcodes.

Quality Support for Cross-Linking & Ligation Oligos

Reactive oligonucleotides require controlled synthesis, purification, handling and analytical release methods matched to photochemistry or ligation chemistry.

QMS

ISO-Supported Reactive Oligo Platform

Bio-Synthesis supports design, synthesis, purification, analytical characterization, custom packaging and documentation for cross-linking and ligation oligonucleotide programs.

ISO 9001:2015 Quality management system
ISO 13485:2016 Medical-device quality framework
Analytical QC HPLC/UPLC, LC-MS, PAGE, OD260 and CoA
Custom Programs Photoreactive, radical, click, ligation and adapter support

FAQ

Which base is best for interstrand cross-links?
Psoralen C6/C2 and CNVK are common starting points. Choose placement within stable duplex regions, often near TA/AT steps, and confirm conversion using denaturing PAGE or LC-MS where compatible.
Can cross-linkers be combined with click handles or fluorophores?
Yes, but spacing, reaction compatibility, purification and readout design must be reviewed. Include controls to separate cross-linking, labeling and background effects.
When should I use benzophenone or diazirine?
Use benzophenone or diazirine for protein–DNA photocapture and contact mapping. Diazirine generates a short-lived carbene, so placement near the suspected contact base is important.
What is needed for enzymatic ligation oligos?
Common requirements include 5′-phosphate or 5′-App termini, ligase-compatible sequence context, clean termini and avoidance of bulky modifications at the nick site.
What QC is recommended?
HPLC/UPLC purity, LC-MS identity, OD260 concentration and CoA are common. PAGE or native/denaturing comparison may be added for ICL verification.
What information is needed for a quote?
 Provide sequence, modification, position, intended reaction, light wavelength if applicable, scale, purification, QC requirements, controls and delivery format.

Information Helpful for Reactive Oligo Designs

Goal
ICL, capture, ligation, adapter
Sequence
5′→3′ and placement
Chemistry
psoralen, CNVK, click
Activation
light, pH, enzyme, click
Scale
nmol, µmol, mg, g
QC
HPLC, LC-MS, PAGE, CoA

Need help choosing a cross-linking or ligation strategy?

Share your sequence, reaction goal, modification type, placement, linker preference, light activation conditions, scale, purification and QC requirements. Bio-Synthesis can help evaluate manufacturability and build a reactive oligo workflow around your assay.
Rx

Technology Review

Compare photoreactive, radical, click, enzymatic and chemical ligation strategies.

Psoralen CNVK Click 5′-App
QC

Release Package

Purification, LC-MS, PAGE, analytical purity, concentration, labeling and documentation.

HPLC LC-MS PAGE CoA

Recommended Reading & Literature References

Use this section to support scientific credibility while keeping the page focused on cross-linking chemistry, ligation strategy, synthesis and analytical verification.

  1. Cimino GD, Gamper HB, Isaacs ST, Hearst JE. Psoralens as photoactive probes of nucleic acid structure and function. Annual Review of Biochemistry. 1985.
  2. Fujimoto K, et al. Ultrafast DNA photo-cross-linking by 3-cyanovinylcarbazole modified oligonucleotides.
  3. Hermanson GT. Bioconjugate Techniques. Academic Press.
  4. Kolb HC, Finn MG, Sharpless KB. Click chemistry: diverse chemical function from a few good reactions. Angewandte Chemie International Edition. 2001.
  5. Saito I, Sugiyama H. Photochemical reactions of DNA and nucleic acid analogs.

Suggested page note: References are provided for scientific background. Final reactive oligo design should be evaluated within the sequence, placement, light/chemical activation, linker geometry, purification method and QC requirements.

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