RaptGen-Assisted Generation of an RNA/DNA Hybrid Aptamer against SARS-CoV-2 Spike Protein.

Optimization of aptamers in length and chemistry is crucial for industrial applications. Here, we developed aptamers against the SARS-CoV-2 spike protein and achieved optimization with a deep-learning-based algorithm, RaptGen. We conducted a primer-less SELEX against the receptor binding domain (RBD) of the spike with an RNA/DNA hybrid library, and the resulting sequences were subjected to RaptGen analysis. Based on the sequence profiling by RaptGen, a short truncation aptamer of 26 nucleotides was obtained and further optimized by a chemical modification of relevant nucleotides. The resulting aptamer is bound to RBD not only of SARS-CoV-2 wildtype but also of its variants, SARS-CoV-1, and Middle East respiratory syndrome coronavirus (MERS-CoV). We concluded that the RaptGen-assisted discovery is efficient for developing optimized aptamers.

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. 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.

The Inhibition Effect of Photo-Cross-Linking between Probes in Photo-Induced Double Duplex Invasion DNA.

Double duplex invasion (DDI) DNA is a useful antigene method that inhibits expression of genomic DNA. We succeeded in performing photoinduced-DDI (pDDI) using ultrafast photo-cross-linking. 5-Cyanouracil (CN U) has been used in pDDI to inhibit photo-cross-linking between probes, but its importance has not been clarified. Therefore, in this study, we evaluated the effect of spacer (S) and d-spacer (dS) that exhibit photo-cross-linking ability similar to that of CN U. CN U exhibited the highest pDDI efficiency, and S, dS, and T were not very different. The photo-cross-linking inhibitory effect was better with S and dS than with thymidine (T). Conversely, the thermal stability was significantly lower with S and dS than with T. The results suggest that the pDDI efficiency is determined by the balance between the photo-cross-linking inhibitory effect and the thermal stability, which is the introduction efficiency for double-stranded DNA. Therefore, CN U, which has a photo-cross-linking inhibitory effect and a high Tm value, showed the highest inhibitory efficiency.

The effect of 5-substituent in cytosine to the photochemical C to U transition in DNA strand.

Nucleobase editing is a powerful tool in genetic disease therapy. We have reported the photochemical transition of cytosine to uracil using an ultrafast DNA photo-cross-linking. In this study, we used cytosine derivatives such as methylcytosine, hydroxymethylcytosine, and trifluoromethylcytosine to evaluate the effect of 5-position substitution of cytosine on deamination. The conversion of cytosine to uracil was the fastest, and the conversion of trifluoromethylcytosine to trifluoromethyluracil was the slowest. The order was correlated with the hydrophilicity of the double strand containing these cytosine derivatives.

Photochemical RNA Editing of C to U by Using Ultrafast Reversible RNA Photo-crosslinking in DNA/RNA Duplexes.

RNA editing, which is used to edit nucleobases in RNA strands; is more feasible for use in medical applications than DNA editing. We previously reported the photochemical conversion of cytosine to uracil, which required photo-crosslinking, deamination, and photo-splitting. Here, we evaluated the influence of the bases surrounding the target cytosine on the conversion of cytosine to uracil in the RNA strand. The photo-crosslinker 3-carboxyvinylcarbazole(OHV K), which is more hydrophilic than 3-cyanovinylcarbazole(CNV K), 3-carboxyamidevinylcarbazole(NH2V K), and 3-methoxy carbonylvinylcarbazole(OMeV K), induced faster deamination of cytosine. Furthermore, inosine, which forms two hydrogen bonds with cytosine, was the most efficiently paired base for accelerating photochemical RNA editing. Upon evaluation of the conversion from cytosine to uracil in RNA, the use of oligodeoxynucleotides containing OHV K and inosine and the polarity of the bases surrounding the target cytosine were found to be crucial.

Effect of linker length on photo-cross-linking position mediated by click chemistry via [2 + 2] photocycloaddition†.

Ultrafast reversible DNA/RNA photo-cross-linking is a powerful tool for regulating the target strand in living cells. In particular, 3-cyanovinylcarbazole (CNVK) and 3-cyanovinylcarbazole modified by D-threoninol (CNVD) can photo-cross-link to pyrimidine bases within a few seconds of photoirradiation. However, these photo-cross-linkers can only cross-link to the counter base if it is adjacent to the 5'-side (-1 position). In this study, we synthesized novel photo-cross-linkers with varying linker lengths capable of photo-cross-linking with pyrimidine bases at locations other than the -1 position via click chemistry. The photo-cross-linking site was dependent on linker length.

RNA fluorescence in situ hybridization hybridisation using photo-cross-linkable beacon probes containing pyranocarbazole in living E. coli.

Understanding intracellular nucleic acids is very important for analysing RNA function and for the diagnosis of genetic diseases. In this study, we demonstrated RNA fluorescence in situ hybridisation in living cells. The described method does not a washing procedure, which affects the detection sensitivity for RNAs with secondary structures and, therefore, is a major limitation of conventional approaches. Ultrafast RNA photo-crosslinking using pyranocarbazole accelerated the invasion of FISH probes, enabling them to target RNAs with secondary structures. Thus, the newly developed method successfully increased the detection sensitivity by 5.4-fold following photo-irradiation at 400 nm for 120 s. In addition, we optimised the beacon probe for detecting target nucleic acids under physiological conditions at 37 °C.

Reversible photo-cross-linking of the GCN4 peptide containing 3-cyanovinylcarbazole amino acid to double-stranded DNA.

Interaction analysis in vivo greatly promotes the analyses and understanding of biological functions. The interaction between DNA and peptides or proteins is very important in terms of readout and amplifying information from genomic DNA. In this study, we designed and synthesized a photo-cross-linkable amino acid, l-3-cyanovinlycarbazole amino acid (l-CNVA), to double-stranded DNA. Reversible photo-cross-linking between DNA and peptides containing CNVA, having 3-cyanovinylcarbazole moieties capable of photo-cross-linking to nucleic acids, was demonstrated. As a result, it was shown that the GCN4 peptide, containing CNVA, can be photo-cross-linked to DNA, and its adduct was photo-split into the original peptide and DNA with 312 nm-irradiation. This is the first report that reversibly manipulates photo-crosslinking between double stranded DNA and peptides. In addition, this reversible photo-cross-linking, using l-CNVA, is faster and with higher yield than that using diazirine and psoralen.

Strong Inhibitory Effects of Antisense Probes on Gene Expression through Ultrafast RNA Photocrosslinking.

We have reported the photochemical regulation of the intracellular antisense effect of antisense probes containing a photo-responsive artificial nucleic acid, 3-cyanovinylcarbazole nucleoside (CNV K). Here we focus on the importance of the photocrosslinking rate on the inhibitory effect on gene expression using photocrosslinkable antisense probes (pcASOs). The inhibitory effect of pcASOs on GFP gene expression was dependent on the photocrosslinking rate of 3-cyanovinylcarbazole with d-threoninol (CNV D), CNV K, or psoralen. The ultrafast RNA photocrosslinking induced the formation of a thermally irreversible covalent bond between pcASOs and the target RNA. These ASOs strongly inhibited gene expression only when the photocrosslinking rate was faster than the random walk of branch migration. In addition, pcASOs containing CNV D or CNV K targeted the RNAs with secondary structures. These results indicate the regulatory effect of photocrosslinker and photoirradiation energy using pcASOs on the gene expression level.

DNA photo-cross-linking using a pyranocarbazole-modified oligodeoxynucleotide with a d-threoninol linker.

An alternative photo-cross-linker having a d-threoninol skeleton instead of the 2'-deoxyribose backbone in 3-cyanovinylcarbazole (CNVK) was investigated to improve the photoreactivity of photo-cross-linkers; the photo-cross-linking rate of 3-cyanovinylcarbazole with d-threoninol (CNVD) was found to be greater than that of CNVK. Therefore, in this study, a novel photo-cross-linker having pyranocarbazole (PCX) and d-threoninol instead of the 2'-deoxyribose backbone in PCX (PCXD) was developed. The PCXD in double-stranded DNA photo-cross-linked to a pyrimidine base at the -1 position of a complementary strand similar to PCX. Furthermore, the photoreactivity of PCXD was significantly higher than that of PCX. The introduction of d-threoninol improved the reactivity of pyranocarbazole to cytosine, the use of PCXD may extend the applicability of the photo-cross-linking reaction for DNA manipulation. In particular, this novel photo-cross-linker can contribute to the photochemical regulation of gene expression or biological events in a living cell.

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 (CNV 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.

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. 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.

DNA Photo-cross-linking Using Pyranocarbazole and Visible Light.

This report presents a novel photo-cross-linker that can cross-link to pyrimidines in nucleic acids under visible light irradiation (λ > 400 nm). This method offers ultrafast photo-cross-linking without any cytotoxicity due to UV irradiation.

Multiplexed detection of nucleic acids using 19F NMR chemical shift changes based on DNA photo-cross-linking of 3-vinylcarbazole derivatives.

The detection methodology for nucleic acids is a useful tool for the analysis of biological systems and diagnosis of diseases. We demonstrated the feasibility of the detection of any nucleic acids based on large chemical shifts via ultrafast DNA photo-cross-linking and the effects of substitution by 3-vinylcarbazole derivatives. These chemical shifts enable the sequence-specific detection of any strand using hybridization chain reaction.

Photochemical Acceleration of DNA Strand Displacement by Using Ultrafast DNA Photo-crosslinking.

DNA strand displacement is an essential reaction in genetic recombination, biological processes, and DNA nanotechnology. In particular, various DNA nanodevices enable complicated calculations. However, it takes time before the output is obtained, so acceleration of DNA strand displacement is required for a rapid-response DNA nanodevice. Herein, DNA strand displacement by using DNA photo-crosslinking to accelerate this displacement is evaluated. The DNA photo-crosslinking of 3-cyanovinylcarbazole (CNV K) was accelerated at least 20 times, showing a faster DNA strand displacement. The rate of photo-crosslinking is a key factor and the rate of DNA strand displacement is accelerated through ultrafast photo-crosslinking. The rate of DNA strand displacement was regulated by photoirradiation energy.

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 (CNVK), require high temperatures to affect cytosine to uracil transformations. To overcome this limitation, we developed new photo-cross-linkers based on CNVK, 3-methoxycarbonlycarbazole, 3-carboxyvinylcarbazole, and 3-carbonylamidevinylcarbazole. The use of 3-carboxyvinylcarbazole resulted in greater acceleration of the deamination reaction than that achieved with CNVK. The most likely factors affecting the ability of ultrafast photo-responsive nucleosides to accelerate the deamination reaction are polarity and hydrophilicity of the oligodeoxyribonucleotides that contain photo-cross-linker.

Double duplex invasion of DNA induced by ultrafast photo-cross-linking using 3-cyanovinylcarbazole for antigene methods.

In the creation of double duplex formation with genomic DNA, the probe must be able to invade and hybridize to the duplex DNA. We designed new photoresponsive probes containing CNVK and CNU; these probes have a high double-duplex invasion capability upon photoirradiation.

RNA fluorescence in situ hybridization using 3-cyanovinylcarbazole modified oligodeoxyribonucleotides as photo-cross-linkable probes.

Photo-cross-linkable fluorescent oligodeoxyribonucleotides having 3-cyanovinylcarbazole nucleoside were applied to fluorescence in situ hybridization (FISH) based 16S rRNA detection in Escherichia coli cells. As the photo-cross-linked probe/rRNA hybrid was stable under the denaturing condition, decrease of the fluorescence signal through the washing process was prevented. The thermally irreversible hybridization property also enabled stable hybridization with the structured region on the target RNA, and facilitated design of the sequence for the FISH probe. Further development of the method might contribute to quantitative and stable FISH staining.

Sequence-Specific DNA Photosplitting of Crosslinked DNAs Containing the 3-Cyanovinylcarbazole Nucleoside by Using DNA Strand Displacement.

An oligodeoxynucleotide (ODN) containing the ultrafast reversible 3-cyanovinylcarbazole ((CNV) K) photo-crosslinker was photo-crosslinked to a complementary strand upon exposure to 366 nm irradiation and photosplit by use of 312 nm irradiation. In this paper we report that the photoreaction of (CNV) K on irradiation at 366 nm involves a photostationary state and that its reaction can be controlled by temperature. Guided by this new insight, we proposed and have now demonstrated previously unknown photosplitting of (CNV) K aided by DNA strand displacement as an alternative to heating. The photo-crosslinked double-stranded DNA (dsDNA) underwent >80 % photosplitting aided by DNA strand displacement on irradiation at 366 nm without heating. In this photosplitting based on DNA strand displacement, the relative thermal stability of the invader strand with respect to the template strands plays an important role, and an invader strand/template strand system that is more stable than the passenger strand/template strand system induces photosplitting without heating. This new strand-displacement-aided photosplitting occurred in a sequence-specific manner through irradiation at 366 nm in the presence of an invader strand.

Photo-cross-linking using trifluorothymidine and 3-cyanovinylcarbazole induced a large shifted (19)F MR signal.

Photo-cross-linking of trifluorothymidine ((TF)T) using 3-cyanovinylcarbazole ((CNV)K) clearly shifted its (19)F nuclear magnetic resonance (NMR) signal 8 ppm. This (CNV)K mediated ultrafast photo-cross-linking-induced shift can be utilized for miRNA detection by hybridization chain reaction (HCR) to detect 10 nM of a target in a sequence-specific manner.