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Cross Link Modified Bases

The ability to cross link bases in double or triple stranded DNA or cross link bases in oligonucleotides with proteins or other types of molecules is essential for probing of nucleic acid secondary structure or the study of protein-nucleic acid complex. The example of using psoralen, an intercalator cross link bases into an oligonucleotide permits site-specific targeting of the cross-link into double-stranded and triple-stranded DNA. Halogenated bases is another class of UV cross linking modified bases used for probing the molecular structure of protein-DNA complexes. Bio-Synthesis offers cross-linker bases modifications which can be incorporated into 5’, 3’ or internally.

Contact Bio-Synthesis for oligonucleotide modification for cross linking.

Modified Bases for Cross Linking

Cross-linker base modifications generally fall into two categories, nucleic acid intercalators (for example, psoralen) and halogenated bases (for example, 5-Br-dC). Psoralen is one of the most commonly used intercalator cross-linkers used to probe nucleic acid secondary structure at specific sites with thymidine in both duplex and triplex DNA. Psoralen can form either monoadducts with one adjacent thymidine or diadducts with two thymidines when particular UV wavelength are used to cross-link in duplex DNA1. The Intercalation occurs on the same or complementary strands. For triplex DNA, psoralen C6-modified homopyrimidine oligos are used to bind to a complementary homopurine-homopyrimidine duplex, thereby forming a triplex that can be cross-linked together at the triplex-duplex junction point2. Psoralen-modified oligos have been used to demonstrate the existence of triple-helix-directed gene modification and nucleotide excision repair mechanism in DNA interstrand cross-link repair3-4.

Bio-Synthesis offers design assistance for your single or dual-labeled DNA, LNA probes or Molecular Beacons probes. Using sophisticated algorithms, BSI can design dual-labeled probes and molecular beacons that will dramatically improve the success of your assays. Primers and probes on multiple sequences are designed in a single search run and are screened for all possible secondary structures to ensure optimal signal strength. Tm is calculated using nearest neighbor thermodynamic theory and highly accurate Santa Lucia values. For effective primer and probe design for your Multiplex or SNP genotyping assays, contact your local BSI Representative for more information.

Halogenated bases are another type of UV cross-linker bases used for probing molecular structure of protein-DNA complexes. 5-Br-dC and 5-Br-dG have been incorporated into dG-dC oligos capable of easily changing into the Z-conformation. This property allowed oligos to function as probes for detecting and studying Z-DNA binding proteins5. Substituting 5-Br-dU at several thymine positions of oligos allowed them to be used to characterize the binding of Nuclear Factor BA1 with DNA6.

References :
  1. Pieles, U., Englisch, U., Nucleic Acids Res. (1989), 17: 285-299.
  2. Takasugi, M., Guendouz, A., Chassignol, M., Decout, J.L., Lhomme, J., Thuong, N.T., Helene, C., Proc. Natl. Acad. Sci. USA (1991), 88: 5602-5606
  3. Barre, F-X., Ait-Si-Ali, S., Giovannangeli, C., Luis, R., et al. Proc. Natl. Acad. Sci. USA (2000), 97: 3084-3088.
  4. Wang, X., Peterson, C.A., Zheng, H., Nairn, R.S., Legerski, R.J., Lei, L. , Mol. Cell. Biol. (2001), 21: 713-720. Herbert, A.G.; Rich, A., Nucleic Acids Res. (1993), 21: 2669-2672.
  5. Kardassis, D.; Zannis, V.I.; Cladaras, C. , J. Biol. Chem.. (1990), 265: 21733-21740.

Custom Modified Oligonucleotide Synthesis for Cross Linking

Product specifications:
  • Scale: 100 nmol, 250 nmol,1 umole or greater
  • Purification options: Desalted, PAGE, HPLC, dual HPLC
  • Oligo lengths: 5-100 bases
  • TAT: 2 - 4 working days
  • Delivery: lyophilised in tube format by default
  • Concentration adjustment and additional aliquoting available
  • Quality control by OD measurement and MALDI-TOF MS
Product Modification code 5' Internal 3' Function
2-Fluoro deoxy inosine dl (convertible dG) [2-F dl] Y Y Y Convertible dG nucleotide, used as precursors in the studies of cross-linking at G position(s), between oligos, or between an oligo and an enzyme.
5-Bromo deoxycytosine (Br dC) [5-Br dC] Y Y Y A photoreactive halogenated base, used to facilitate the determination of DNA structure by X-ray crystallography.
5-Bromo deoxyuridine (Br dC) [5-Br dU] Y Y Y A photoreactive halogenated base, used to facilitate the determination of DNA structure by X-ray crystallography.
5-lodo deoxycytosine dC [5-l dC] Y Y Y A photoreactive halogenated base, used to facilitate the determination of DNA structure by X-ray crystallography.
5-lodo deoxyuridine dU [5-l dU] Y Y Y A photoreactive halogenated base, used to facilitate the determination of DNA structure by X-ray crystallography.
Psoralen C6 [Psoralen C6] Y Y Y A heterocyclic compound, used as a ss/ds DNA intercalating or cross-linking reagent, for probing nucleic acid secondary structure
6-Thio-dG [6S-dG] Y Y Y Effective at crosslinking DNA to protein.
4-Thio-dT [4S-dT] Y Y Y Effective at crosslinking DNA to protein.
4-Thio-dU [4S-dU] Y Y Y Effective at crosslinking DNA to protein.
Storage conditions

Oligonucleotides are stable in solution at 4oC for up to 2 weeks. Properly reconstituted material stored at -20oC should be stable for at least 6 months. Dried DNA (when kept at -20oC) in a nuclease-free environment should be stable for years.

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