Takehiro Ami

Autonomous DNA Computing Machine Based on Photochemical Gate Transition

We report the construction of a one-pot autonomous DNA computing machine based on photochemical gate transition (photocleavage, hybridization, and photoligation), and we performed binary digit additions using this machine. In our method, both photochemical DNA manipulations previously reported, photoligation via 5-carboxyvinyldeoxyuridene (cvU) containing ODN and photocleavage via carbazole-modified ODN, were employed. The binary digit additions were autonomously carried out by one-time irradiation at 366 nm in the single test tube. The fluorescence readout by the DNA chip was in good agreement with the correct answer of binary digit additions. We believe that this system is easily applicable to correlation analysis between SNPs as well as other binary digit processing, such as subtraction.

Click Chemistry as an Efficient Method for Preparing a Sensitive DNA Probe for Photochemical Ligation

Genetic science entered a new stage with the progress of the genome project. As new genes are rapidly being identified, examination of their functions is becoming more important, especially in the field of biology, where the focus is shifting from individual genes and proteins to the interactions between the enormous numbers of proteins and their involvement in various life phenomena. Technology that cuts and ties DNA and DNA enzymes such as DNA ligase is essential to this research. Although enzymatic ligation methods have advantages, there are many limitations. For example, enzyme-linked assays need to include a careful selection of the most suitable conditions, including temperature, pH, and salt concentration, because of the use of enzymes and the fact that ligases have low activities for RNAs. Furthermore, although very short probes have the highest sequence specificity, ligase enzymes are inefficient with short oligonucleotides. For a alternative to enzymatic ligation methods, we have reported a highly efficient and reversible template-directed DNA photoligation procedure. Template-directed photoligation with 5-carboxyvinyl-2’-deoxyuridine (U) can be used for detecting DNA or RNA sequences with high sensitivity. However, in photoligation with U, a long photoirradiation time at 366 nm is required to complete photoligation. To overcome the limitation of photoligation with U, we now report a new photosensitive probe that takes advantage of the electronic structural changes associated with the formation of triazole rings in the Cu-catalyzed version of the azide–alkyne cycloaddition reaction (“click” reaction) discovered by the groups of Meldal and Sharpless. In the field of nucleic acid chemistry, Cu-catalyzed azide–alkyne cycloaddition has been used for labeling oligonucleotides with a range of moieties, such as fluorescein, for example, for coupling oligonucleotides to monolayers, and for templated strand ligation. As an example, we report an efficient procedure for preparing DNA probes for quick SNP typing through photochemical ligation. The nucleoside phosphoramidite of 5-ethynylvinyl-2’-deoxyuridine (U) was prepared, and this monomer was incorporated into an ODN by standard automated DNA synthesis protocols. After deprotection and purification of the oligomer, an ODN containing U—5’-dACHTUNGTRENNUNG(UGCGTG)-3’, or ODN ACHTUNGTRENNUNG(U)—was characterized by MALDI-TOF-MS. Four azides were purchased or synthesized by a method reported in the literature, and Huisgen cycloadditions between the ODN ACHTUNGTRENNUNG(U) and the azides were carried out. After purification of the products, photoresponsive ODNs (Scheme 1) were characterized by MALDI-TOF-MS (see the Supporting Information).

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.

Fluorescence labeling of DNA based on photochemical ligation

Abstract Fluorescent labeling of oligonucleotides has been attracting interest in connection with the development of methods for distinguishing and detecting nucleic acids sequences. And photochemical ligation has the merit of the avoiding the need for additional reagents. Fujimoto et al. reported template directed DNA photoligation using 5-carboxyvinyl-deoxyuridine (CVU). Here, we describe the synthesis and photocrosslinking ability of fluorescent reporter analogue (Cy5) tethered CVU-containing ODN.

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.

Sensitive DNA probe for photochemical ligation prepared in click chemistry

We report a new photosensitive probe, that takes advantage of the electronic structural changes associated with the triazole ring formed in the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition. We succeeded in performing photoligation on the second time scale. Huisgen cycloaddition are employed to various labeling. As an example, we report a procedure for preparing the DNA probe for quick SNP typing.