UltraFast PhotoInduced double duplex DNA invasion into a 400-mer dsDNA target.

Background: Natural DNA restriction enzymes bind duplex DNA with high affinity at multiple sites; however, for some of the artificial chemical-based restriction moieties, invasion of the double-strand for efficient cleavage is an obstacle. We have previously reported photo-induced double-duplex invasion (pDDI) using 3-cyanovinylcarbazole (K)-containing probes for both the target strands that photo-crosslink with pyrimidine bases in a sequence-specific manner on both the strands, stabilizing the opened double-strand for cleavage. The drawback of the pDDI was low efficiency due to inter-probe cross-linking, solved by the inclusion of 5-cyano-uridine at -1 position on the complimentary strand with respect to K in both probes.

Acceleration of the Deamination of Cytosine through Photo-Crosslinking.

Herein, we report the major factor for deamination reaction rate acceleration, i.e., hydrophilicity, by using various 5-substituted target cytosines and by carrying out deamination at high temperatures.

Ultra-acceleration of Photochemical Cytosine Deamination by Using a 5'-Phosphate-Substituted Oligodeoxyribonucleotide Probe Containing a 3-Cyanovinylcarbazole Nucleotide at Its 5'-End.

Genes are the blueprints for the architectures of living organisms, providing the backbone of the information required for formation of proteins. Changes in genes lead to disorders, and these disorders could be rectified by reversing the mutations that caused them. Photochemical methods currently in use for site-directed mutagenesis employ the photoactive 3-cyanovinylcarbazole ( K) nucleotide incorporated in the oligodeoxyribonucleotide (ODN) backbone.

Study of Photochemical Cytosine to Uracil Transition via Ultrafast Photo-Cross-Linking Using Vinylcarbazole Derivatives in Duplex DNA.

Gene therapies, including genome editing, RNAi, anti-sense technology and chemical DNA editing are becoming major methods for the treatment of genetic disorders. Techniques like CRISPR-Cas9, zinc finger nuclease (ZFN) and transcription activator-like effector-based nuclease (TALEN) are a few such enzymatic techniques. Most enzymatic genome editing techniques have their disadvantages.

Effect of substitution of photo-cross-linker in photochemical cytosine to uracil transition in DNA.

Genome editing is an important technique for protein engineering, treatment of genetic disorders, and production of non-native proteins. A shortcoming of current enzymatic and chemical methods for genome editing is their limited applicability for in vivo studies. In addition, non-enzymatic methods, such as photochemical DNA editing using 3-cyanovinylcarbazole (K), require high temperatures to affect cytosine to uracil transformations.

Effect of nucleobase change on cytosine deamination through DNA photo-cross-linking reaction via 3-cyanovinylcarbazole nucleoside.

Photo-chemical deamination of cytosine using 3-cyanovinylcarbazole nucleoside (K) mediated photo-cross-linking is a technique for site-directed mutagenesis. Using this technique in vivo requires the elimination of a high-temperature incubation step; instead, incubation should be carried out under physiological conditions. To improve the reactivity of K mediated photo-cross-link induced deamination of cytosine under physiological conditions, an evaluation of base pairing in cytosine was carried out with respect to its deamination.