Bioinspired Conjugates for Chemical Biology

Various biomolecules created by life activity show their superior potentiality to us. ?In this laboratory, we design and create new supramolecules that further exceed natural materials by learning how natural molecules function in life.? We are now developing new tools for future biotechnology, nucleic acid drugs for next generation, and high-performance nano-materials by making full use of nucleic acid (DNA, RNA) and peptide. We also analyze their functions on the bases of structural biology and physical chemistry. Here, we introduce our several on-going projects.

1. Photoresponsive oligonucleotide

DNA, which is composed of four nucleobases, exhibits increasingly interesting and important functions in the fields of biochemistry, biotechnology, and nanomaterials due to its sequence specific recognition (hybridization) of the counterstrand and formation of a double-helical structure. The rule of hybridization is ingeniously simple: A only pairs with T, and G only with C. Recently, DNA and RNA are regarded as very promising nano-materials for constructing various nano-architectures and nano-machines, which are based on the spontaneous hybridization of DNA (or RNA) with its complementary counterstrand. Hence, if double-stranded DNA can be converted into two single-strands reversibly at will, we can control the mechanical motion of these machines.

In our laboratory, we have for the first time succeeded in the reversible photo-regulation of the formation and dissociation of DNA duplex by designing photoresponsive DNA carrying azobenzene (Angew. Chem. Int .Ed. 1999, 38, 2393-2395.). After modification of azobenzene and sequence design of photoresponsive DNA, we attained perfect photo-control of hybridization and developed new photoresponsive DNA that can be operated with only visible light (λ > 400 nm) which does not damage living cells. With our photoresponsive DNA and RNA, we could successfully photo-control almost all the reactions related to oligonucleotides and nanomaterials composed of DNA and RNA, such as photo-regulation of gene expression, photon-fuelled DNA machine, and so on. In addition, we could also developed photo-responsive T7 promoter and succeeded in the on-off photo-switching of transcription and following translation with this promoter.

As an external stimulus, light has the following advantages: 1) light does not contaminate the reaction system, 2) the excitation wavelength is controllable by designing the photoresponsive molecule, and 3) control of irradiation time and/or local excitation is easy. Hence, our photoresponsive DNA has attracted many researchers in the world, are applied to various systems by others. At present, our photoresponsive oligonucleotide can be purchased from the following Japanese company (only photoresponsive DNA carrying non-modified azobenzenes).

Hokkaido System Science Co., Ltd.
Contact person: Satoshi Kitamura
Tel: +81-11-768-5901
Fax: +81-11-768-5951
Address: 2-1, Shikawa Nishi 2-1, Kita-ku, Sapporo, Hokkaido 001-0932, Japan

Photo-regulation of the formation and dissociation of duplex with azobenzene-tethered oligonucleotide
図2 光応答性プロモーターによる転写反応の可逆的光スイッチング
Photo-regulation of transcription and following translation with azobenzene-tethered T7 promoter.

2. Highly sensitive fluorescent probe that can detect oligonucleotide sequence-specifically.

In the genomic and post-genomic era, there is an ever-increasing demand for the detection of DNA and RNA targets with high sequence specificity and sensitivity. Comprehensive analysis of human genome revealed that there are a huge number of genetic variations such as single nucleotide polymorphisms (SNPs) in human genome. In addition, recent studies have revealed that non-SNP alterations such as insertion/deletion polymorphisms account for about 70% of all variant bases. Detection of such variants is required in order to assess disease risks and drug responses of individuals. Hence, highly sensitive fluorescent probes that can detect DNA and RNA sequence-specifically are required to realize tailor-made medicine.

1) In-Stem Molecular Beacon (ISMB)

Our group has proposed a novel molecular beacon that we call an in-stem molecular beacon (ISMB), which has multiple pairs of a fluorophore and a quencher in its stem. When dyes are introduced into adjacent base pairing positions via D-threoninol, the dyes are stacked in an anti-parallel manner and strongly interact with each other. Thus, the background emission from fluorophore in the closed state is suppressed due to the strong interaction with a quencher. By designing the probe sequence, we can discriminate deletion polymorphism from wild type with emission color.

In-Stem Molecular Beacon (ISMB)

2) Linear probe with multiple fluorophores

We also propose a new quencher-free stemless linear probe involving multiple fluorophores on D-threoninols, each of which is separated by intervening natural nucleotides. In the absence of target, the flexible linear probe does not emit fluorescence due to self-quenching among the weakly interacting fluorophores. Upon hybridization with the target, intercalation of each dye between base pairs results in strong fluorescence emission from the separated dyes. This new design allow for high signal-to-background ratio as well as rapid response.

Linear probe with multiple fluorophores

3. nano-cluster of organic functional molecules

DNA is a supramolecule in which four nucleobases are strictly aligned within a double-helical structure. We have prepared dye clusters with pre-determined size, orientation and sequence by incorporating dyes into DNA. We have also succeeded in preparation of hetero dye cluster. We aim to elucidate novel physical phenomena between different dyes such as hetero excitonic interaction and hetero photocycloaddition reaction.

4. Artificial oligonucleotides

We have synthesized aTNA and SNA by modifying DNA with acyclic backbones. aTNA formed extremely stable duplex with complementary aTNA. In contrast, stable hetero duplex was formed between SNA and DNA or RNA. We are developing nucleic acid drugs and probes by using aTNA and SNA.

5. Insulators

Fluorescent-labeled oligonucleotides are powerful tools for biochemical and biological research. However, most fluorophores are quenched through electron/hole transfer by natural nucleobases. We have prepared artificial bases of cyclohexane derivatives, which can suppress electron transfer. Quantum yields of fluorophores were drastically enhanced by incorporating the artificial bases. Highly-emissive labeling agents and probes would be prepared by using these molecules.

6. Oligonucleotide based drug

RNA interference is the endogenous gene silencing system which can be induced by short interfering RNAs (siRNAs) and has been considered to be a powerful tool which can knock-down the disease-related gene. Although siRNA has potential to be a therapeutic use, we have several issues to be overcome; Delivery system, low stability, off-target effect, and so on. We are developing a novel design of siRNA by introducing the original artificial nucleic acids.