Tetranucleotides as a scaffold for diporphyrin arrays

The incorporation of porphyrin-substituted nucleosides into tetranucleotides using phosphoramidite chemistry on solid support is reported. Both diphenyl and tetraphenyl porphyrin nucleosides were used as building blocks. This method allows the synthesis of chiral homo- and heteroporphyrinic arrays, where the composition and thus the physical properties of the array can be modulated simply by reprogramming the DNA synthesizer. The porphyrin arrays are initially isolated in the free-base form. Remetallation to give the zinc-porphyrins can be achieved using standard procedures in solution. The UV-vis spectra of the arrays are reproducible by a superposition of the absorbance spectra of the individual porphyrins, indicating an undisturbed electronic ground state of the porphyrins in the arrays. The same is true for the steady-state emission spectra of the homoporphyrinic arrays, which are not influenced by the presence of the nucleotide strand. In the mixed porphyrin arrays, large differences in the excited-state properties compared to an equimolar mixture of the building blocks are observed by means that the emission of the diphenyl porphyrin moiety is quenched to a large extent, and the overall emission is dominated by the tetraphenyl porphyrin. The covalent connection of the porphyrins via the DNA-derived backbone therefore substantially alters the excited-state and energy-transfer properties of mixed porphyrin systems. The circular dichroism (CD) spectra show induced negative cotton effects in the region of the porphyrin B-band absorption, which is due to the attachment of the chromophores to the chiral oligonucleotide backbone. Addition of a complementary tetra-adenosine did not alter any of the spectroscopic properties, neither in chloroform nor in acetonitrile solutions. Therefore, it can be concluded that no duplex is formed, which is corroborated by 1H NMR spectroscop

Selektiver DNS-Strangbruch an fester Phase : eine neue Methode zur Sequenzerkennung

Introduction: In recent times, interest in genetic modification called for the development of important techniques in the analysis of modified oligonucleotide sequences. Many DNA sequence detection methods are known to date, including the use of DNA chips and real-time PCR methodology techniques. However, they all require target amplification and/or labelling steps during analysis. The method developed herein allows the qualitative and quantitative detection of DNA sequences without the need for target amplification or labelling steps prior to the analysis, but is based on the site specific DNA cleavage on solid-support. Method: Akin to other currently employed techniques, our innovation makes use of an immobilised DNA strand. However, we incorporate a photocleavable site X, in addition to a marker tag M at the terminus of the strand. Together with the target sequence, a double helix is formed and upon irradiation, X is cleaved and the marker containing fragments remain on the support through base pairing with the complementary target strand. In case of mismatches, located either in the upper or lower section, the stability of the double strand is attenuated, and releasing the fragment containing the marker tag M, which subsequently is detectable in solution (Figure A). Results: For the construction of the device we made use of the disulfide exchange mechanism which lead to quantitative yield for the immobilisation of the used 3’ modified oligonucleotides (Scheme A). Furthermore, hybridisation of counter strands to SP-B showed good yields. After heating to 85 °C and cooling for a minimum of 1.5 h with stirring, quantitative hybridisation yields were obtained. Irradiation of an X and M modified single strand SP-B affords quantitative yield of cleavage produkt ON-B (Scheme A). Identical irradiation experiments using a duplex with the complementary strand did not release the same amount of ON-B. The main part of ON-B (~80%) remaining on the support could be released upon heat treatment. This is the expected „counter-strand-effect“. Introduction of two mismatches into the counter strand lead to a destabilisation of the helix and therefore deliberation of ON-B indicating the mismatches. Incorporation of only one mismatch gives various results, but it could be shown that at selected positions single mismatches (SNPs) can be detected using our methodology. With the obtained “counter-strand-effect” the potential of the herein presented method for qualitative sequence detection could be shown. Furthermore, the effective strand cleavage on the solid support offers the opportunity for quantitative sequence detection (e.g. for GMO evidence)

Porphyrin-DNA: a supramolecular scaffold for functional molecules on the nanometre scale

We are pursuing the aim to use DNA as a supramolecular scaffold for the creation of electronically functional molecules on the nanometre scale. Here, we give a review on our results on porphyrin modified nucleotides used for this purpose. A general synthetic route to porphyrin-nucleotides has been devised, and the building blocks can be incorporated into oligonucleotides using standard solid phase synthesis methods. Up to 11 porphyrins were incorporated into DNA, reaching a length of approximately 4 nm in the array. The spectroscopic data are consistent with a porphyrin induced secondary structure stabilisation in the single strands

Tetranucleotides as a scaffold for diporphyrin arrays

The incorporation of porphyrin-substituted nucleosides into tetranucleotides using phosphoramidite chemistry on solid support is reported. Both diphenyl and tetraphenyl porphyrin nucleosides were used as building blocks. This method allows the synthesis of chiral homo- and heteroporphyrinic arrays, where the composition and thus the physical properties of the array can be modulated simply by reprogramming the DNA synthesizer. The porphyrin arrays are initially isolated in the free-base form. Remetallation to give the zinc-porphyrins can be achieved using standard procedures in solution. The UV-vis spectra of the arrays are reproducible by a superposition of the absorbance spectra of the individual porphyrins, indicating an undisturbed electronic ground state of the porphyrins in the arrays. The same is true for the steady-state emission spectra of the homoporphyrinic arrays, which are not influenced by the presence of the nucleotide strand. In the mixed porphyrin arrays, large differences in the excited-state properties compared to an equimolar mixture of the building blocks are observed by means that the emission of the diphenyl porphyrin moiety is quenched to a large extent, and the overall emission is dominated by the tetraphenyl porphyrin. The covalent connection of the porphyrins via the DNA-derived backbone therefore substantially alters the excited-state and energy-transfer properties of mixed porphyrin systems. The circular dichroism (CD) spectra show induced negative cotton effects in the region of the porphyrin B-band absorption, which is due to the attachment of the chromophores to the chiral oligonucleotide backbone. Addition of a complementary tetra-adenosine did not alter any of the spectroscopic properties, neither in chloroform nor in acetonitrile solutions. Therefore, it can be concluded that no duplex is formed, which is corroborated by H-1 NMR spectroscopy