Anthracene-DNA conjugates as building blocks of designed DNA structures constructed by photochemical reactions.

We present the photochemical ligation of anthracene-ODN (oligodeoxyribonucleotide) conjugates through cycloaddition of anthracenes. Anthracene-DNA conjugates were synthesized by linking the 5'- or 3'-end of ODNs to the anthracene group. The sequences of the conjugates were designed to hybridize to neighboring sites on the target ODN with their anthracene units facing each other. When the ternary duplex consisting of the two conjugates and the target forms, the conjugates can be dimerized by light irradiation. The dimerization efficiency was affected by the substitution position of the anthracene group and by a one-base displacement in the template sequence. Furthermore, it is demonstrated that the capping of the duplex and connecting third strands in a triplex structure could also be the target of the photochemical ligation.

The effect of local structural disruption on the yield of photochemical ligation between anthracene-oligonucleotide conjugates.

The techniques of chemical ligation have attracted great attention as an alternative to enzymatic joining of DNA ends. Here we introduce the photoligation of anthracene-modified ODN conjugates through anthracene cyclodimer formation. The effect of the positions and the kinds of single base mismatch on the template was evaluated using eight templates with one-base displacements. We found out that the yield of the ligation was affected by mispairing in a position-dependent manner. Such results would be attributed to the disruption of the local structure at the ligation site.

Effect of local structural disruption on the photochemical ligation between anthracene-modified DNA conjugates.

DNA templated ligation reactions has attracted great interest in the field of DNA probing and medical diagnosis. To date some research groups reported a variety of chemical ligations, in which two DNAs with reactive groups that bound adjacently on the complementary strand were connected covalently to form a corresponding longer product. Here we introduced the photoligation of anthracene modified ODN conjugates through anthracene cyclodimer formation. The effect of the position of single base mismatch on the template was evaluated using six sequences with one-base displacements. The yield of the ligation was affected by mispairing in position-dependent manner. It would be due to the disruption of the local structure around the two anthracenes of both reaction partners.

Detection of one base mismatch using photochemical ligation.

We attached anthracene molecules covalently to one end of oligodeoxyribonucleotides to form three pairs of anthracene-ODN conjugates. The sequences of each pair of the conjugates were designed to bind adjacent sequences of the template with the anthracene units directed such that they stacked each other. The conjugates dimerized only in the presence of the template by light irradiation. We found that the ligation yields largely depended on the substituted position of anthracene groups. Sequence selectivity in the dimerization yield was preserved even in the system with highest reactivity. SNP bases could be discriminated rapidly by combining the techniques of MS spectroscopy.

Homogeneous DNA-detection based on the non-enzymatic reactions promoted by target DNA.

Much effort has focused on methods for detecting various genetic differences in individuals, including single nucleotide polymorphisms (SNPs). SNP can be characterized as a substitution, insertion, or deletion at a single base position on a DNA strand. There is expected to be on average one SNP for every 1000 bases of the human genome, and some variations located in genes are suspected to alter both the protein structure and the expression level. Therefore, highly sensitive techniques with a simple procedure would be desirable for a high-throughput screening of millions of SNPs widely dispersed throughout the human genome. In this short review, we consider recently reported unique techniques for genotyping in a homogeneous solution, and organize them in terms of the chemical and physical processes accelerated on DNA.

Colorimetric multiplexed immunoassay using specific aggregation of antigenic peptide-modified luminous nanoparticles.

A rapid immunoassay capable of detecting specific antibodies in one-step procedure is described. Antigenic peptides with cationic (KKKKC) or anionic (DDDDC) pentamer tail were immobilized on luminous nanospheres of 40 nm diameter (Ø) through cystamine and bifunctional linker molecules under various conditions. The numbers of each peptide anchored to a sphere were 5.0 x 10(2) and 0.8-3.8 x 10(3), respectively. A mixture of the antigenic peptides of FAK and c-Myc was immobilized to the spheres with red emission, while that of c-Myc and alpha-catenin was likewise to green spheres. Multiplexed immunoassay was easily achieved by adding the antibodies to a mixed dispersed solution of these spheres under appropriate conditions. Anti-FAK and anti-alpha-catenin antibodies formed aggregates with red and green emissions, respectively. On the other hand, the anti-c-Myc antibody formed aggregates emitting a yellow light. This system enabled us to differentiate three antibodies in one vessel from the definite differences in aggregate color.

Photochemical ligation between anthracene-DNA conjugates and its analytical application to gene analysis.

Anthracene readily forms photoadducts (anthracene dimers). The reaction generally requires close proximity and certain spatial alignment of both reaction partners. DNA could provide an ideal scaffold for accelerating the reaction, photocyclic addition. We synthesized a number of anthracene-DNA conjugates. The sequences of the conjugates, 5'AntODN and 3'AntODN, were designed to bind adjacent sequences of the template with the anthracene units directed such that they stacked with each other. The conjugates were only dimerized in the presence of the template by light irradiation. The efficiency of dimerization was dependent on the structure of the conjugates and affected by one-base displacement in the template sequence.

Photochemical ligation of DNA conjugates through anthracene cyclodimer formation and its fidelity to the template sequences.

Anthracene readily forms photoadducts, anthracene dimers, and this photodimerization reaction has been well characterized. In general, however, the reaction requires close proximity and certain spatial alignment of both reaction partners. DNA could provide an ideal scaffold for accelerating the photocyclic addition. We synthesized a number of anthracene-DNA conjugates. The sequences of the conjugates, 5'AntODNn and 3'AntODNn (the length of methylene linkers: n = 3 or 6), were designed to bind adjacent sequences of the template with the anthracene units directed such that they stacked with each other. The conjugates were only dimerized in the presence of the template by light irradiation. The efficiency was affected by one-base displacement in the template sequence.

Cooperative hybridization of DNA conjugates and its analytical application.

Anthracene readily forms photoadducts (anthracene dimers). This photodimerization reaction has been well characterized. However the reaction generally requires close proximity and certain spatial alignment of both reaction partners. DNA could provide an ideal scaffold for accelerating the photocyclic addition. We synthesized a number of anthracene-DNA conjugates. The sequences of the conjugates, 5'AntODN6 and 3'AntODN6, were designed to bind adjacent sequences of the template with the anthracene units directed such that they stacked with each other. The conjugates were only dimerized in the presence of the template by light irradiation. The efficiency was affected by one-base displacement in the template sequence.