Conformational analysis of the natural iron chelator myo-inositol 1,2,3-trisphosphate using a pyrene-based fluorescent mimic.

myo-Inositol phosphates possessing the 1,2,3-trisphosphate motif share the remarkable ability to completely inhibit iron-catalysed hydroxyl radical formation. The simplest derivative, myo-inositol 1,2,3-trisphosphate [Ins(1,2,3)P(3)], has been proposed as an intracellular iron chelator involved in iron transport. The binding conformation of Ins(1,2,3)P(3) is considered to be important to complex Fe(3+) in a 'safe' manner. Here, a pyrene-based fluorescent probe, 4,6-bispyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-bispyrenoyl Ins(1,2,3,5)P(4)], has been synthesised and used to monitor the conformation of the 1,2,3-trisphosphate motif using excimer fluorescence emission. Ring-flip of the cyclohexane chair to the penta-axial conformation occurs upon association with Fe(3+), evident from excimer fluorescence induced by pi-pi stacking of the pyrene reporter groups, accompanied by excimer formation by excitation at 351 nm. This effect is unique amongst biologically relevant metal cations, except for Ca(2+) cations exceeding a 1 : 1 molar ratio. In addition, the thermodynamic constants for the interaction of the fluorescent probe with Fe(3+) have been determined. The complexes formed between Fe(3+) and 4,6-bispyrenoyl Ins(1,2,3,5)P(4) display similar stability to those formed with Ins(1,2,3)P(3), indicating that the fluorescent probe acts as a good model for the 1,2,3-trisphosphate motif. This is further supported by the antioxidant properties of 4,6-bispyrenoyl Ins(1,2,3,5)P(4), which closely resemble those obtained for Ins(1,2,3)P(3). The data presented confirms that Fe(3+) binds tightly to the unstable penta-axial conformation of myo-inositol phosphates possessing the 1,2,3-trisphosphate motif.

Fluorescent probe: complexation of Fe3+ with the myo-inositol 1,2,3-trisphosphate motif.

Natural myo-inositol phosphate antioxidants containing the 1,2,3-trisphosphate motif bind Fe(3+) in the unstable penta-axial conformation.

SNP detection for cytochrome P450 alleles by target-assembled tandem oligonucleotide systems based on exciplexes.

We report the first use of exciplex-based split-probes for detection of the wild type and *3 mutant alleles of human cytochrome P450 2C9. A tandem 8-mer split DNA oligonucleotide probe system was designed that allows detection of the complementary target DNA sequence. This exciplex-based fluorescence detector system operates by means of a contiguous hybridization of two oligonucleotide exciplex split-probes to a complementary target nucleic acid target. Each probe oligonucleotide is chemically modified at one of its termini by a potential exciplex-forming partner, each of which is fluorescently silent at the wavelength of detection. Under conditions that ensure correct three-dimensional assembly, the chemical moieties on suitable photoexcitation form an exciplex that fluoresces with a large Stokes shift (in this case 130 nm). Preliminary proof-of-concept studies used two 8-mer probe oligonucleotides, but in order to give better specificity for genomic applications, probe length was extended to give coverage of 24 bases. Eight pairs of tandem 12-mer oligonucleotide probes spanning the 2C9*3 region were designed and tested to find the best set of probes. Target sequences tested were in the form of (i) synthetic oligonucleotides, (ii) embedded in short PCR products (150 bp), or (iii) inserted into plasmid DNA (approximately 3 Kbp). The exciplex system was able to differentiate wild type and human cytochrome P450 2C9 *3 SNP (1075 A-->C) alleles, based on fluorescence emission spectra and DNA melting curves, indicating promise for future applications in genetic testing and molecular diagnostics.

Target-assembled exciplexes based on Scorpion oligonucleotides.

Scorpion probes, specific DNA probe sequences maintained in a hairpin-loop, can be modified to carry the components of an exciplex for use as a novel fluorescence-based method for specific detection of DNA. The exciplex partners (5'-pyrenyl and 3'-naphthalenyl) were attached to oligonucleotides via phosphoramidate links to terminal phosphate groups. Hybridization of the probe to a complementary target in a buffer containing trifluoroethanol produced an obvious fluorescence change from blue (pyrene locally excited state emission) to green (exciplex emission).

Detection of nucleic acids in situ: novel oligonucleotide analogues for target-assembled DNA-mounted exciplexes.

This research describes the effects of structural variation and medium effects for the novel split-oligonucleotide (tandem) probe systems for exciplex-based fluorescence detection of DNA. In this approach the detection system is split at a molecular level into signal-silent components, which must be assembled correctly into a specific 3-dimensional structure to ensure close proximity of the exciplex partners and the consequent exciplex fluorescence emission on excitation. The model system consists of two 8-mer oligonucleotides, complementary to adjacent sites of a 16-mer DNA target. Each probe oligonucleotide is equipped with functions able to form an exciplex on correct, contiguous hybridization. This study investigates the influence of a number of structural aspects (i.e. chemical structure and composition of exciplex partners, length and structure of linker groups, locations of exciplex partner attachment, as well as effects of media) on the performance of DNA-mounted exciplex systems. The extremely rigorous structural demands for exciplex formation and emission required careful structural design of linkers and partners for exciplex formation, which are here described. Certain organic solvents (especially trifluoroethanol) specifically favour emission of the DNA-mounted exciplexes, probably the net result of the particular duplex structure and specific solvation of the exciplex partners. The exciplexes formed emitted at approximately 480 nm with large Stokes shifts ( approximately 130-140 nm). Comparative studies with pyrene excimer systems were also carried out.

Synthetic analogues of the manzamenones and plakoridines which inhibit DNA polymerase.

An array of novel analogues of the marine oxylipins, the manzamenones and plakoridines, have been prepared in divergent fashion using an approach modelled on a biogenetic theory. Many of the target compounds show potent inhibition of DNA polymerases alpha and beta and human terminal deoxynucleotidyl transferase (TdT).