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. Guanine was replaced with 4 different counter bases (inosine, 2-aminopurine, 5-nitroindole, and nebularine), showing distinct hydrogen bonding patterns with target cytosine, which was incorporated at the -1 position with respect toK in the K-modified photo-responsive oligodeoxyribonucleotides to ascertain the role of hydrogen bonding in deamination under physiological conditions. Among the counter bases, inosine showed the highest acceleration towards the photo-induced deamination reaction.
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. Thus, non-enzymatic and non-invasive technologies for nucleic acid editing has been reported in this study which might possess some advantages over the older methods of DNA manipulation. 3-cyanovinyl carbazole (CNVK) based nucleic acid editing takes advantage of photo-cross-linking between a target pyrimidine and the CNVK to afford deamination of cytosine and convert it to uracil. This method previously required the use of high temperatures but, in this study, it has been optimized to take place at physiological conditions. Different counter bases (inosine, guanine and cytosine) complementary to the target cytosine were used, along with derivatives of CNVK (NH2VK and OHVK) to afford the deamination at physiological conditions.
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. The major drawback of this method, the requirement for high temperature, has been addressed, and deamination has previously been achieved at 37 °C but with low efficiency. Here, efficient deamination has been accomplished under physiological conditions by using a short complementary photoactive ODN with a 5'-phosphate group in the -1 position with respect to the target cytosine. It is hypothesized that the free phosphate group affects the microenvironment around the target cytosine by activating the incoming nucleophile through hydrogen bonding with the water molecule, thus facilitating nucleophilic attack on the cytosine C-4 carbon. The degree of deamination observed in this technique is high and the effect of the phosphate group is to accelerate the deamination reaction.
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. Through substitution of the groups at the 5′-position of the cytosine, the effect of hydrophilicity was understood. It was then used to compare the various modifications of the photo-cross-linkable moiety as well as the effect of the counter base of the cytosine to edit both DNA and RNA. Furthermore, we were able to achieve cytosine deamination at 37 °C with a half-life in the order of a few hours.
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