Ultrafast Oligonucleotide Photo Cross Linking

The nucleoside analogue 3-cyanovinyl carbazole, ^CNVK, enables ultrafast reversible photo-cross-linking of oligonucleotides.

Recently a new non-enzymatic and non-invasive technologies for nucleic acid editing has been reported that takes advantage of the photo-cross-linker 3-cyanovinyl carbazole (^CNVK). 3-Cyanovinyl carbazole (^CNVK) when incorporated into oligonucleotides photo-cross-links a target pyrimidine and the ^CNVK to afford deamination of cytosine converting it to uracil.

Figure 1: 3-Cyanovinyl carbazole (^CNVK) nucleoside analog. This nucleoside is now available as a cyanoethyl (CE) phosphoramidite allowing the incorporation of the analog into oligonucleotides during automated solid phase oligonucleotide synthesis.

Cross-linking and photo-cross-linking reactions are used widely in biochemistry, biology, chemistry, and molecular biology for the study of molecular interactions. Many new cross-linking reagents have been developed and synthesized in the last two decades. Advances made in analytical instrumentation now allow the analysis of cross-linked products at ever lower levels of detection. For example, various photo-cross-linking approaches can be used to study the interactome of cells.

The interactome refers to the network of molecular interactions in living cells, such as protein-protein, protein-nucleic acid, or protein-carbohydrate interactions.

Photo-cross-linking is used fo the study of protein-protein or protein-oligonucleotide interactions in living cells. In this approach, first photo-cross-linkers are incorporated in the desired proteins or oligonucleotides within cells, next, cells are exposed to UV radiation to induce the cross-linking between molecules in contact to the cross-linking functional groups. Following cross-linking, the target molecule of interest and its cross-linked complexes are isolated using immune-purification and analyzed using mass spectrometry. Often enzymatic digestions are used to allow for easier detection of the cross-linked products and to define molecular interphases.

The new photo-cross-linker 3-cyanovinylcarbazole (^CNVK) nucleoside allows the study of oligonucleotide interactions.

Yoshimura and Fujimoto in 2008 descibed a ultrafast reversible photo-cross-linking reaction for the manipulation of DNA in situ using ^CNVK which was applied to the selection of RNA in 2009 by the same research group.

When ^CNVK is incorporated into oligonucleotides, a very rapid cross-link is formed to a pyrimidine base on the complementary strand when irridated at 366 nm.

According to Yoshimura and Fujimoto, photo-cross-linking to thymine takes only 1 second but takes 25 second to cytosine.

Complete reversal of the cross-link is achieved by irridation at 312 nm for 3 minutes.  

Figure 2: Photo-cross-linking reaction of the 3-Cyanovinyl carbazole (^CNVK) oligonucleotide with a thymidine on a complementary oligonucleotide strand. The photoadduct ^CNVK-T is shown as the reaction product as described by Yohimura and Fujimoto in 2008 and 2009. 

Figure 2: Photo-cross-linking reaction of the 3-Cyanovinyl carbazole (^CNVK) oligonucleotide with a cytosine at 366 nm. Heading the cross-link to 90 ^oC for 3.5 hours causes deamination of the cytosine to form a uracil in the complementary strand (Yoshimura et al. 2009). Reversal of the cross-link at 312 nm results in a complementary strand with a uracil in place of the cytosine. 
 
In 2010 Fujimoto et al. reported the cross-linking reaction of ^CNVK incorporated into oligonucleotides to a dC residue in duplex DNA. The heating of the cross-link to 90 °C for 3.5 hours resulted in a deamination reaction of the cytosine base now forming a uracil moiety in the complementary strand. Reversal of the cross-link at 312 nm resulted in a DNA strand in which dC is replaced with dU.  The transformation is specific for a dC residue opposite a ^CNVK residue. However, any further adjacent dC residues are unaffected by the reaction. Also, oligonucleotides containing ^CNVK residues can be cross-linked to adjacent RNA strands. 

In 2013, this approach was applied to SNP-based genotyping and in 2015 a threoninol linker was introduced in the oligonucleotide strand.

Sethi et al. in 2018 reported that the ultrafast reversible DNA inter-strand photo-cross-linking reaction of oligodeoxynucleotides (ODNs) containing ^CNVK can take place at physiological conditions. in order to find the best combination of photo-active nucleobase and complementary base of the target, a variety of combinatios were studied. The study concluded that the best combination of counter-base and photo-cross-linker will  result in the highest conversion of C→U and in the least time at physiological conditions.

Reference

2008.   Y. Yoshimura, and K. Fujimoto; Ultrafast reversible photo-cross-linking reaction: toward in situ DNA manipulation. Org Lett. 2008 Aug 7;10(15):3227-30. doi: 10.1021/ol801112j. Epub 2008 Jun 27.

2009.   Y. Yoshimura, T. Ohtake, H. Okada, and K. Fujimoto; A new approach for reversible RNA photocrosslinking reaction: application to sequence-specific RNA selection. ChemBioChem, 2009, 10, 1473-6.

2010. Fujimoto K1, Konishi-Hiratsuka K, Sakamoto T, Yoshimura Y.; Site-specific cytosine to uracil transition by using reversible DNA photo-crosslinking.Chembiochem. 2010 Aug 16;11(12):1661-4. doi: 10.1002/cbic.201000274.

2012. Wong and Jameson; Chemistry of protein and Nucleic Acid Cross-Linking and Conjugation. 2nd Edition. CRC Press. 2012.

2013.   Kenzo Fujimoto, Asuka Yamada, Yoshinaga Yoshimura, Tadashi Tsukaguchi, and Takashi Sakamoto; Details of the Ultrafast DNA Photo-Cross-Linking Reaction of 3-Cyanovinylcarbazole Nucleoside: Cis–Trans Isomeric Effect and the Application for SNP-Based Genotyping. J. Am. Chem. Soc., 2013, 135 (43), pp 16161–16167.  DOI: 10.1021/ja406965f. Publication Date (Web): October 2, 2013. Copyright © 2013 American Chemical Society kenzo@jaist.ac.jp  :J. Am. Chem. Soc.  135, 43, 16161-16167

2013. Pham, N. D., Parker, R. B., & Kohler, J. J. (2013). Photocrosslinking approaches to interactome mapping. Current Opinion in Chemical Biology, 17(1), 90–101. http://doi.org/10.1016/j.cbpa.2012.10.034. 

2015.  Sakamoto T, Tanaka Y, Fujimoto K.; DNA photo-cross-linking using 3-cyanovinylcarbazole modified oligonucleotide with threoninol linker.   Org Lett. 2015 Feb 20;17(4):936-9. doi: 10.1021/acs.orglett.5b00035. Epub 2015 Feb 5. 

2018. Sethi, S.; Nakamura, S.; Fujimoto, K.; Study of Photochemical Cytosine to Uracil Transition via Ultrafast Photo-Cross-Linking Using Vinylcarbazole Derivatives in Duplex DNA. Molecules 2018, 23, 828.

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