파이-파이 상호작용을 이용한 변형 핵산 시스템의 설계 및 응용
- 파이-파이 상호작용을 이용한 변형 핵산 시스템의 설계 및 응용
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- The modified nucleic acid system is one of the fascinating research areas in recent research field. The motivation for this research ranges from a desire to obtain understanding of nucleic acid structures over to the prospect of developing enabling tools for wide-ranging applications in nanotechnology, diagnostics and therapeutics. Organic synthesis is a powerful tool to achieve the desired modified nucleosides and their phosphoramidites, even triphosphates. Chemical modifications of nucleic acid systems continue to produce the strong demand via expanded areas, from biological understanding to nanotechnology.
Part I Design, synthesis and their properties of aromatic fluorophores substituted oligodeoxyadenylates
Oligodeoxynucleotides are fascinating materials for the construction of nano-architectures because of their well-organized and programmable structures that result from base pairing. Most DNA-based approaches toward nano-architectures have been dependent on the base-pairing of natural nucleobases (i.e., Watson–Crick or Hoogsteen). The structures of nucleic acids are stabilized by hydrogen bonding between complementary bases as well as by π–π stacking of nucleobases or between nucleobases and intercalators. Inter- and intramolecular π–π stacking interactions of aromatic rings both play important roles in many chemical and biological processes. Strong π–π stacking interactions in nucleic acids can be used to generate novel secondary structures.
We synthesized the pyrene-labelled deoxyadenosine (PyA) and incorporated it in the oligodeoxyadenylate with different numbers and length. These modified oligodeoxyadenylates featuring PyA units inserted at a specific 1,4-relationship display a wide range of fluorescence emissions: blue, white, and reddish orange. Importantly, these fluorescence emissions can be regulated by adjusting the number of PyA moieties in the oligodeoxyadenylate, without of the need for any other fluorescent nucleosides or materials. Moreover, the complete insertion of six PyA units with 1,4-relationships in the 18-mer oligodeoxyadenylate provided a dominant reddish orange fluorescence emission and high thermal stability under our examined conditions.
We also introduced the different polyaromatic hydrocarbons, naphthalene and anthracene, into the deoxyadenosine and synthesized various modified oligonucleotides involving oligodeoxyadenylates. We compared the fluorescence phenomena and self-assembly ability of PAHA-substituted oligodeoxyadenylates. W found the naphthalenyl moiety as a small polyaromatic hydrocarbon could also form the self-duplex structure in oligodeoxyadenylate, when we inserted six NapA units at a specific 1,4-relationship. Contrary to NapA-substituted oligodeoxyadenylate, the AnA-substituted oligodeoxyadenylates displayed the relatively unstable structures. By using SAXS technique, we could find the self-duplex structure of PyA-substituted oligodeoxyadenylates. Consequently, we could realize that the formation of self-duplex was determined by not only wide area but also the orientation of aromatic fluorophores.
In order to develop the modified oligodeoxyadenylate systems, we also designed and synthesized the acyclic version of PyA by applying GNA moiety. This acyclic PyA was inserted in the GNA homo-oligodeoxyadenylate and DNA/GNA chimeric oligodeoxyadenylates. Significantly, modified oligodeoxyadenylates incorporated GNA moieties did not form the self-duplex structure contrary to DNA version of PyA substituted oligodeoxyadenylate. However, the modified oligodeoxyadenylates possessing GNA moieties showed various fluorescence emissions: bluish green, green, and yellow.
These modified oligodeoxyadenylates displayed the blue-shifted fluorescence emission with natural 18-mer thymidylate. Moreover because the hybrid between modified oligodeoxyadenylates possessing GNA moieties and natural thymidylate had low thermal stability compared with natural duplex, natural oligoadenylate could easily exchange with modified oligodeoxyadenylate. Due to strand exchange, the fluorescence emission of each modified oligodeoxyadenylate was recovered.
Part II Applications of π–π interaction to modified nucleic acid systems
Aromatic fluorophore-labelled nucleic acids have been applied in wide research fields, from the development of diagnostic tools and construction of nano-achitecture. The π–π interaction between aromatic plane of fluorophores and nucleobases occasionally induced the enhanced stability of nucleic acid structure and changed fluorescence emission. Moreover, the π–π interaction between carbon nano-material and fluorescent nucleic acids caused the fluorescence quenching phenomena.
Previously, we reported a QF-MB that exhibits several advantageous features, including a high level discrimination between the target and its single-mismatched congeners and an economical device set-up due to the absence of the quencher. Nevertheless, because of its intrinsic fluorescence, the QF-MB exhibited a low S/B ratio of ca. 50%. The oxidized functional groups of GO, a highly oxidized form of graphene, allow it to be suspended in aqueous and organic solvents at high concentrations, and the residual aromatic regions offer sites for interaction with nucleobases and aromatic fluorophores. By using these properties, we designed the QF-MB/GO by using GO as an external quencher. Our QF-MB/GO system exhibited a higher S/B ratio (31.0) relative to that (2.2) of the same system in the absence of GO, while retaining a high selectivity for fully matched over single-base-mismatched targets.
We also focused on the charge interaction between the oxidized surface of GO and the phosphate backbone of oligonucleotides. Even though these two components have a negative charge–charge repulsion, the π–π interaction between the residual aromatic surface of GO and the aromatic parts in fluorescent oligonucleotides, nucleobases, and fluorophores compensates for this repulsion. We thought accurate pH changes could control the association and dissociation of the fluorescent oligonucleotides from GO without needing complementary strands. We have designed and performed a molecular switch based on self-assembly of multi-PyA-substituted oligodeoxyadenylate that can operate by pH regulation in a GO solution. This molecular switch signal was visualized in a high pH environment (pH 9.0) with the dissociation and self-assembly of multi-PyA-substituted oligodeoxyadenylate in GO solution, which results in an increase in fluorescence signal at 570 nm. The binding and response of the multi-PyA-substituted oligodeoxyadenylate to the GO surface was rapid and capable of discriminating the off-state at a low pH (pH 4.0). Moreover, the multi-PyA-substituted oligodeoxyadenylate/GO system showed a high reproducibility of fluorescence signal through multiple cycles and displayed the real on/off state via the different interactions between GO and pyrenyl-modified oligodeoxyadenylate through different pH conditions.
By using π–π interaction between arene ruthenium metalla-cage between pyrene-linked nucleoside, we synthesized the anticancer nucleoside-ruthernium metalla-cages to overcome low cellular uptake of anticancer nucleosides. The water-soluble arene ruthenium metalla-cages were used to deliver pyrenyl-nucleosides to cancer cells. Due to high π–π interaction between arene ruthenium metalla-cage between pyrene-linked nucleoside, the complexes were synthesized with good yield. Moreover these complexes showed the proper stability to determine the anticancer acitivity. Consequently, the different floxuridine-metalla-cage combinations exhibit excellent anti-proliferative effects on both A2780 ovarian cancer cells and their cisplatin resistant strains. Hence these molecules could be a good therapeutic alternative to floxuridine which suffers from poor cellular uptake and bioavailability.
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