3-Cyanovinylcarbazole Phosphoramidite (CNVK)

5'-O-(4,4'-Dimethoxytrityl)-1'-(3-cyanovinylcarbazol-9-yl)-2'-deoxy-β-D-ribofuranosyl- 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite

Product Specifications

Formula:
C50H53N4O6P
M.W.:
836.95
F.W.:
396.33
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Description

When 3-cyanovinylcarbazole nucleoside (CNVK) is incorporated into an oligonucleotide, very rapid photo cross-linking to the complementary strand can be induced at one wavelength and rapid reversal of the cross-link is possible at a second wavelength.  Neither wavelength has the potential to cause significant DNA damage.  Irradiation of a duplex containing a single incorporation of CNVK at 366nm led to 100% cross-linking to thymine base in 1 second, although complete cross-linking to cytosine takes 25 seconds.1  A 30 second irradiation time should cover all situations.  In addition, it was demonstrated that the purine bases were unreactive to cross-linking, allowing differentiation between pyrimidines and purines at the target site.  The authors also determined the effect of sequence contexts around the CNVK site and demonstrated that the identity of bases on either side of the cross-linking site has little effect on the reaction.  Once cross-linked, the UV melting temperature of the duplex was raised by around 30 °C relative to the duplex before irradiation.  Complete reversal of the cross-link takes place at 312nm in 3 minutes.  This facile reversal reaction is, therefore, accomplished with no damage to normal DNA.

In a later publication, a further application of this cross-linking technique was investigated.2  When CNVK was cross-linked with a dC residue in duplex DNA, heating at 90°C for 3.5 hours led to deamination of the cytosine base to form uracil in the complementary strand.  Reversal of the cross-link at 312nm led to a DNA strand in which dC had been converted to dU.  The authors showed that this transformation is specific for the dC residue opposite the CNVK and any further adjacent dC residues are unaffected.  Similarly, the authors have shown that CNVK can be cross-linked to an adjacent RNA strand.3

Details

Usage

  • Coupling: Regular. The use of UltraMILD monomers are preferred. (Catalog Numbers: dA: 10-1601-xx, dC: 10-1015-xx, dG: 10-1621-xx, dT: 10-1030-xx). To avoid any exchange of the iPr-Pac group on the dG with acetyl, use the UltraMild Cap Mix A (40-4210-xx/ 40-4212-xx).
  • Deprotection: If UltraMILD reagents were used, deprotect in 0.05M Potassium Carbonate in Methanol for 4 hours at Room Temperature OR for 2 hours at Room Temperature in 30% Ammonium Hydroxide. If standard bases were used, deprotection in Ammonium Hydroxide at Room Temperature for 24-36 hours will give acceptable yields. For photo cross-linking conditions please see http://www.glenresearch.com/GlenReports/GR23-14.html.
Specifications
Diluent Anhydrous Acetonitrile
Storage Freezer storage, -10 to -30°C, dry in the dark. Light sensitive material.
Stability 1-2 days


References

(1) Y. Yoshimura, and K. Fujimoto, Org Lett, 2008, 10, 3227-30.
(2) K. Fujimoto, K. Konishi-Hiratsuka, T. Sakamoto, and Y. Yoshimura, ChemBioChem, 2010, 11, 1661-4.
(3) Y. Yoshimura, T. Ohtake, H. Okada, and K. Fujimoto, ChemBioChem, 2009, 10, 1473-6