Author: Kenzo Fujimoto
Japan Advanced Institute of Science and Technology
Asahidai 1-1, Nomi, Ishikawa, Japan
3-Cyanovinylcarbazole nucleoside (CNVK1) and D-threoninol (CNVD2) are members of a novel class of photo-cross-linkers for DNA or RNA strands (Figure 1). Cross-linking technology is useful for the detection, regulation, and manipulation of DNA or RNA. In particular, photo-irradiation induced cross-linking is useful from the perspective of regulation, and photo-cross-linkers such as psoralen and coumarin have been used in traditional methods. However, many improvements remain necessary in terms of photoreactivity, sequence specificity, and so on. We report that CNVK and CNVD have very high photoreactivity and they enable selective photo-cross-linking of target DNA or RNA by photo-irradiation for a few seconds. We expect that they could be successfully applied in the photochemical regulation of nucleic acids in living cells, which has been difficult with traditional methods.
The photo-cross-linkers CNVK and CNVD can be included in DNA or RNA strands according to the usual DMTr-phosphoramidite method. They also have very high photoreactivity and it is possible to photo-cross-link them to a pyrimidine base by photoirradiation for a few seconds at 365nm, as well as to the pyrimidine base in the complementary strand. In addition, CNVK can photo-cross-link to a pyrimidine base via [2+2] photocycloaddition, similar to psoralen and thymidine dimers. Therefore, it is possible to photo-split the cross-link by a 3 min, 312 nm irradiation (Figure 2).
The sequence selectivity of photo-cross-linking of ODN containing CNVK or CNVD was evaluated (Figure 3). CNVK can photo-cross-link with a position of -1 in the target strand. Therefore, we investigated the effect of varying the CNVK pairing base and surrounding bases on photo-cross-linking. Photo-irradiation was performed for 10 seconds using 64 sequences obtained by changing the bases of X and Y and Z to A, T, G, and C and their complementary strands. As a result, it was found that photo-cross-linking occurs only in the case of thymine or cytosine without being affected by the pairing base or adjacent bases. Compared with CNVK, CNVD has faster photo-cross-linking, and for cytosine CNVD is used, and a significant acceleration of photo-cross-linking reaction can be confirmed.
This photo-cross-linking reaction had high sequence selectivity and operability, and it could photo-cross-link a targeted site in a plasmid3. Results of the atomic force microscopy (AFM) imaging indicated that a biotin-modified ODN can be photo-cross-linked to a single-stranded or double-stranded plasmid. It was possible to photo-cross-link only one selective site, even on a long plasmid (Figure 4).
This ultrafast photo-cross-linking reaction is an effective tool for multiple methods involving nucleic acids. For example, DNA strand displacement is a fundamental reaction for both in vivo and in vitro biological events such as genomic DNA replication, transcription, and PCR. It is usually a very fast reaction with enzyme assistance in vivo, but it takes time to proceed in vitro. Therefore, the use of an ultrafast photo-cross-linking reaction would greatly improve stability, and possibly significantly accelerate the DNA strand exchange reaction (Figure 5).4
This ultrafast photo-cross-linking reaction is also capable of photo-cross-linking to RNA5. We evaluated the antisense effect using the photo-cross-linking reaction to mRNA. Ultrafast photo-cross-linking using CNVK or CNVD greatly improved the stability of the duplex, and therefore, when it was used as an antisense nucleic acid, it exerted a large antisense effect. A CNVK probe was converted to phosphorothioate and was introduced into HeLa cells stably expressing GFP. The intensity of GFP fluorescence and the amount of GFP mRNA was decreased by a 10 sec photoirradiation at 365 nm6, which indicated that GFP gene expression was inhibited by photoirradiation. In addition, it is also possible to regulate its antisense effect by the desired timing of photoirradiation (Figure 6).
The stabilization of double-strand formation using ultrafast photo-cross-linking can also be applied to RNA fluorescence in situ hybridization (FISH). In the RNA FISH method, the wash steps are necessary to remove nonspecific signals but can also cause problems such as low detection signal and poor reproducibility. A photo-cross-linkable FISH probe containing CNVK was introduced into the immobilized E. coli, and RNA FISH was performed using 16S rRNA as a target (Figure 7). Fluorescence was confirmed even when the wash steps utilized buffer containing formamide, indicating that stable detection is possible without wash conditions7. In addition, strong fluorescence was confirmed even for targets with undetectable sensitivity.
CNVK and CNVD are photo-cross-linkers capable of photo-cross-linking at an extremely high photo-reactivity compared with conventional optical crosslinkers such as psoralens. This technology could be applied to many fields such as those of antisense nucleic acids and FISH.
1. (a) Y. Yoshimura, K. Fujimoto, Org Lett., 2008, 10(15), 3227-30.
(b) K. Fujimoto, A. Yamada, Y. Yoshimura, T. Tsukaguchi, T. Sakamoto, J. Am. Chem. Soc., 2013, 135(43), 16161-7.
2. T. Sakamoto, Y. Tanaka, K. Fujimoto, Org. Lett., 2015, 17(4), 936-9.
3. K. Fujimoto, K. Hiratsuka-Konishi, T. Sakamoto, T. Ohtake, K. Shinohara, Mol. BioSyst., 2012, 8(2), 491-4.
4. S. Nakamura, H. Hashimoto, S. Kobayashi, K. Fujimoto, ChemBioChem, 2017, 18(20), 1984-9.
5. A. Shigeno, T. Sakamoto, Y. Yoshimura, K. Fujimoto, OBC, 2012, 38, 7820-5.
6. T. Sakamoto, A. Shigeno, Y. Ohtaki, K. Fujimoto, Biomater. Sci., 2014, 2, 9, 1154-7.
7. K. Fujimoto, K. Toyosato, S. Nakamura, T. Sakamoto, Bioorg. Med. Chem. Lett., 2016, 26, 5312-4.
Intellectual property rights
The ultrafast photo-cross-linker has been granted the following patents: US8697357 and US7972792.
Glen Research and Maravai LifeSciences have completed an agreement with Nicca Chemical to begin supplying CNVK Phosphoramidite to the research market worldwide, with the exception of Japan.
For coupling of CNVK Phosphoramidite (Figure 8), regular coupling times are suggested. However, the use of UltraMILD monomers is 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).
For deprotection: If UltraMILD reagents were used, use 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.