Yoshinaga Yoshimura

Ultrafast reversible photo-cross-linking reaction: toward in situ DNA manipulation.

We describe a novel ultrafast reversible DNA interstrand photo-cross-linking reaction via 3-cyanovinylcarbazole nucleoside ( (CNV)K). Oligodeoxynucleotide (ODN) containing (CNV)K can be photo-cross-linked by irradiation at 366 nm for 1 s, and the photo-cross-linked ODN can be split by irradiation at 312 nm for 60 s.

Details of the Ultrafast DNA Photo-Cross-Linking Reaction of 3-Cyanovinylcarbazole Nucleoside: Cis–Trans Isomeric Effect and the Application for SNP-Based Genotyping

To clarify the cis-trans isomeric effect on the ultrafast DNA photo-cross-linking reaction of 3-cyanovinylcarbazole nucleoside ((CNV)K) in the DNA duplex, which gives a single photodimer on the reversed-phase HPLC chromatogram, the kinetics of the cis-trans photoisomerization of (CNV)K in double-stranded DNA was evaluated. Since the photoisomerization rate constant for cis to trans isomerization in double-stranded DNA was significantly larger than that for trans to cis isomerization, and the thermodynamic stability of the trans isomer was higher than that of the cis isomer, it was strongly suggested that the trans isomer of (CNV)K is a reactive species of the photo-cross-linking reaction. (1)H-(1)H NOESY analysis of the photoadduct consisting of (CNV)K and T also supported the trans-mediated photo-cross-linking reaction of (CNV)K. By using this ultrafast photo-cross-linking reaction for the molecular beacon-based SNPs typing, four individual Japanese rice strains were clearly distinguishable with simple photoirradiation and fluorescence imaging using double-stranded target DNAs.

Template‐Directed DNA Photoligation in Rapid and Selective Detection of RNA Point Mutations

In recent years, DNA ligation has attracted attention to the application of specific detection of DNA or RNA sequences. Direct analysis of RNA sequences can indicate the relative abundance of specific mRNAs in cellular extracts. Although RNA-template-directed chemical ligation and enzymatic ligation have been reported, RNA-template-directed photoligation has not been extensively investigated. Early studies of the enzyme T4 DNA ligase have shown that the enzyme could join oligodeoxynucleotides (ODNs) hybridized to RNA templates, albeit with a substantially lower efficiency than DNA-templatedirected ligations. Photoligation would require no added reagents to carry out the reaction and would be controllable with space and time by the choice of proper irradiation methods. DNA-template-directed reversible photoligation with 5vinyl-2’-deoxyuridine (U) has already been reported as a tool for DNA manipulations. DNA templated-directed photoligation proceeded through a [2+2] cycloaddition between the double bond of the U side chain and the C5=C6 double bond of thymine. We now report on a highly efficient RNA-templatedirected photoligation of ODNs through 5-carboxyvinyl-2’-deoxyuridine (U). We further demonstrate that the RNA-template-directed photoligation can be extended to solid-state assays. The availability of photoligation also allowed us to detect RNA point mutations. An ODN containing U, ODN1(U) (5’-d(UGCGTG)-3’), was synthesized by using the cyanoethylphosphoramidite of U, according to conventional DNA synthesis. ODN1(U) was characterized by nucleoside composition and MALDI-TOF-MS (calcd 1876.3381 for [M H] ; found 1876.3477). We determined the feasibility of RNA-template-directed photoligation through ODN1(U). When ODN1(U) and ODN(T) (5’-d(TGTGCT)-3’) were irradiated at 366 nm for 30 min in the presence of template RNA(A) (5’-r(CACGCAAGCACA)-3’; Scheme 1), HPLC showed the appearance of a peak relating to ODN(U-T) in 98% yield along with the disappearance of the ODN1(U) and ODN(T) peaks (Figure 1). MALDI-TOF-MS indicates that the isolated ODN(U-T) obtained from HPLC purification was a photoligated product of ODN1(U) and ODN(T) (calcd 3677.51 for [M+H] ; found 3677.82). Also, when ODN1(U) and ODN(T) were irradiated at 366 nm for 30 min in the presence of template ODN(A) (5’-d(CACGCAAGCACA)-3’), we observed a peak corresponding to ODN(U-T) in 74% yield, as determined by HPLC. On the other hand, when an RNA template (5’-r(CACGCAGGCACA)-3’) was used in photoligation, the 3’-terminal C reacted with photoexcited U to produce a photoligated product ODN(U-C) as efficiently as 3’-terminal T. Interestingly, photoligation rates on the RNA template were higher than the corresponding ODN template (Figure 2). We compared the CD spectrum of two hexamers, ODN(T) and ODN1(U), and the template RNA(A) with that of the two hexamers and the template ODN(A) (Figure 3). An A-form duplex shows an intense positive peak around 270 nm, a small nega-

Site-Specific Cytosine to Uracil Transition by Using Reversible DNA Photo-crosslinking

The development of a template-directed procedure for the artificial site-specific conversion of cytosine into uracil should be a useful genetic technique for gene therapy and genetic manipulation for DNA and RNA based on their genetic code. UV irradiation of DNA produces various photoproducts containing pyrimidine dimer moieties in the 5’-CT-3’, 5’-TC-3’,and 5’-CC-3’ sequences, whereas the hydrolytic deamination of cytosine leads to the formation of uracil through the replacement of the C4 exocyclic amino group by oxygen. 2] The uracil formed by cytosine deamination is potentially mutagenic, changing the coding information during DNA replication and RNA transcription. 4] Although treating DNA with bisulfite, acid, or base can noticeably accelerate the velocity of cytosine deamination, the bisulfite treatment results in target DNA degradation and in multiple cytosine-to-uracil transformations. Activation-induced cytosine deaminase (AID) deaminates cytidine residues in single-strand DNA with site-specificity, but this enzymelinked method requires careful optimization of the conditions, including temperature, pH, and salt concentration, and also has to make allowance for additional procedures such as pretreatment with RNase. Further efforts to create more broadly applicable transition methods that perform suitably rapidly and reliably are therefore needed. As a technique for site-specific mutation, we have reported a template-directed DNA photoligation mediated by 5-carboxyvinyldeoxyuridine (U), through [2+2] cycloaddition between U and cytosine, and have demonstrated the deamination of the photoligated dimer on heat treatment. Changes in the local conformation of the duplex and/or in the hydrophobicity around the cytosine base induced by the [2+2] cycloaddition cause changes in the solvent exposure of the cytosine base. This effect accelerates the deamination of cytosine. With U, the technique requires photoirradiation on a timescale of hours for DNA photoligation. In a recent study, however, we reported that a modified oligodeoxynucleotide (ODN) containing 3-cyanovinylcarbazole nucleoside (K) can be photoreversibly crosslinked with a cytosine through UV irradiation at two different wavelengths, and can effect reversible DNA photo-crosslinking through [2+2] cycloaddition between K and cytosine in seconds under the irradiation conditions. Although this photo-crosslinking with K allows a target cytosine to be crosslinked sequence-specifically, site-specific conversion of cytosine into uracil through reversible DNA photocrosslinking had not previously been investigated. Here we report a new photochemical procedure for the site-specific conversion of cytosine into uracil through the use of an artificial DNA base such as K. The rapid, selective conversion of the target cytosine into uracil was achieved through the photo-crosslinking process and subsequent photolysis of the photo-crosslinked product after heat treatment. The phosphoramidite of K was prepared by a literature method. Modified ODNs containing K were prepared by standard phosphoramidite chemistry with a DNA synthesizer and the phosphoramidite of K as shown in Table 1. ODNs containing K were characterized by MALDI-TOF-MS. Scheme 1 illustrates the method for the site-selective conversion of cytosine into uracil through reversible DNA photocrosslinking. Photoirradiation at 366 nm of a cytosine-containing ODN in the presence of the K-containing complementary ODN afforded the product photo-crosslinked at the target

A New Approach for Reversible RNA Photocrosslinking Reaction: Application to Sequence-Specific RNA Selection

On and off in a flash: We describe a novel, ultrafast, reversible, interstrand RNA photocrosslinking reaction via 3‐cyanovinylcarbazole nucleoside. The interstrand RNA‐photocrosslinking reaction showed a high degree of sequence specificity and can be used in the selection of a target RNA sequence.

Quick regulation of mRNA functions by a few seconds of photoirradiation.

3-Cyanovinylcarbazole nucleoside, which effectively photocrosslinks to the pyrimidine base in complementary RNA strands, was incorporated into antisense oligonucleotides, and we evaluated the photoreactivity and the sequence selectivity to mutated K-ras oligoRNAs, as well as the regulation of the function of K-ras mRNA. We demonstrated that the reverse transcription and the translation activity of K-ras mRNA were quickly suppressed by a few seconds of photoirradiation with the addition of the photoresponsive antisense ODN.

Sequence specific interstrand photocrosslinking for effective SNP typing

We describe a simple and inexpensive SNP typing method by using sequence specific interstrand photocrosslinking via p-carbamoylvinyl phenol nucleosides. Interstrand photocrosslinking showed a high degree of single nucleotide specificity as high as 10(3)-fold and more, and can be used in the diagnostic detection of DNA sequences.

Development of a rapid and reversible photocrosslinking of RNA

We describe a novel reversible interstrand photocrosslinking reaction of RNA. In this system, a modified oligodeoxynucleotide (ODN) containing 3-cyanovinylcarbazole nucleoside ((CNV)K) reacts by photoirradiation at 366 nm for 1 s with pyrimidine residue of a complementary template RNA to yield a crosslinked product in 94% yield.

Development of template-directed reversible DNA photocrosslinking.

We describe a novel reversible DNA interstrand photocrosslinking reaction via the artificial nucleoside (X). Oligodeoxynucleotide (ODN) containing X can be photocrosslinked by irradiation at 366 nm for 1 s, and the photocrosslinked ODN can be split by irradiation at 312 nm for 60 s.

SNP genotyping by DNA photoligation: application to SNP detection of genes from food crops

Abstract We describe a simple and inexpensive single-nucleotide polymorphism (SNP) typing method, using DNA photoligation with 5-carboxyvinyl-2′-deoxyuridine and two fluorophores. This SNP-typing method facilitates qualitative determination of genes from indica and japonica rice, and showed a high degree of single nucleotide specificity up to 10 000. This method can be used in the SNP typing of actual genomic DNA samples from food crops.