Hans-Achim Wagenknecht

Perylene Bisimide Dimers as Fluorescent “Glue” for DNA and for Base-Mismatch Detection†

Perylene-3,4:9,10-tetracarboxylic acid bisimide (“PB”) and its derivatives are applied as fluorescent dyes in organic materials owing to their excellent photochemical stability as well as the high fluorescence quantum yields. The strong hydrophobic stacking interactions between the PB chromophores make this dye an important building block for functional supramolecular architectures. Based on these properties, PB in the dimeric form should be also of potential interest as a probe for fluorescent DNA/RNA analytics as well as for functionalized DNA-based architectures. Its noncovalent DNA-binding interactions have been studied with PB derivatives that had been modified with spermine or other amines. Moreover, an increasing number of publications about covalent modifications of oligonucleotides with PB have appeared over the last few years. Recently, we presented a facile route for the synthetic incorporation of PB as an artificial DNA base in order to study the stacking interactions of this dye at specific sites in duplex DNA. Herein, we present the evaluation of fluorescent PB dimers for the optical functionalization of DNA using three representative duplexes (DNA1, DNA3, and DNA4a). For the synthetic modification of the corresponding oligonucleotides with the PB chromophore (Scheme 1), the 2-deoxyribofuranoside moiety was replaced by an acyclic linker system which is tethered to one of the imide nitrogens of the PB dye. This linker allows the chromophore to intercalate in the base stack while providing high chemical stability during the automated phosphoramidite chemistry. DNA1 bears one interstrand PB dimer inside the duplex, whereas DNA3 contains a PB monomer outside the duplex at each 5’ end. Both duplexes contain palindromic sequences. When DNA1 and DNA3 are excited at 505 nm, the fluorescence spectra of both duplexes (Figure 1) are dominated by a broad band at 660 nm without fine structure. This band corresponds to the excimer-type emission of the PB dimer that has been observed in nanoaggregates of perylene bisimides. 15] The UV/Vis spectra of DNA1 and DNA3 (Figure 2) at low temperatures show two major bands that are hyposochromically (506 nm) or bathochromically (545 nm) shifted in comparison to the 0!1 and 0!0 vibronic transitions of the PB monomer. This result shows the strong p–p excitonic interactions of the two PB chromophores inside (DNA1) and outside (DNA3) of the duplex. Both the excimer-type fluorescence band and the shifted absorption bands of DNA1 vanish at higher temperatures. It is remarkable that this occurs cooperatively at a temperature (75.9 8C) that corresponds to the cooperative thermal dehybridization of the whole DNA duplex, which is typically measured at 260 nm (Tm = 78.6 8C). Apparently the intact helical duplex is required as a framework for the PB dimer formation. In comparison to the unmodified DNA2 (Tm = 76.2 8C) the duplex DNA1 is stabilized by 2.4 8C through the Scheme 1. PB-modified duplexes DNA1–DNA3 and DNA4a–DNA4e.

Electron transfer processes in DNA: mechanisms, biological relevance and applications in DNA analytics

In principle, DNA-mediated charge transfer processes can be categorized as oxidative hole transfer and reductive electron transfer. With respect to the routes of DNA damage most of the past research has been focused on the investigation of oxidative hole transfer or transport. On the other hand, the transport or transfer of excess electrons has a large potential for biomedical applications, mainly for DNA chip technology.

Fluorescent color readout of DNA hybridization with thiazole orange as an artificial DNA base.

A fluorescent chameleon: A single thiazole orange (TO) dye, when used as an artificial DNA base shows the typical green emission, whereas the interstrand TO dimer exhibits an orange excimer-type emission inside duplex DNA (see picture).

1-Ethynylpyrene as a Tunable and Versatile Molecular Beacon for DNA

Fluorescent or luminescent probes that are sensitive to the local environment within DNA duplexes represent important tools for DNA hybridisation and conformational changes caused by DNA±protein interactions, or for the detection of physiologically important DNA base mismatches or lesions on DNA chips or microarrays. As a consequence, there is a continuously increasing demand for new fluorophores that have a clear and specific range of spectral characteristics which are tunable to distinct excitation or emission wavelengths. One suitable and important way to create new emission properties is to attach chromophores covalently to natural DNA bases. Recently, we applied this modification strategy to the preparation of photoexcitable charge donors, which have been used for the investigation of DNA-mediated electron transport. 4] Herein, we report the properties of DNA duplexes bearing the 1-ethynylpyrene moiety (Py C C) covalently attached to the bases dX=dA, dC, dG, or dU. Three structural features of these Py C C dX-modified DNA duplexes are important: i) a clear steric separation of the pyrene moiety from the DNA base stack due to the rigid ethynyl group, ii) a strong electronic coupling between the pyrene and the base moiety provided by the acetylene bridge and iii) a partial stacking of the base moiety as part of the delocalised Py C C dX chromophore. Moreover, the incorporation of the Py C C dX moiety could influence only the local conformation, but should not perturb the overall BDNA duplex conformation. The Py C C dX-modified oligonucleotides were synthesised by a semiautomated synthetic strategy with solid-phase Sonogashira-type cross-coupling conditions (Scheme 1). 5] It is important to note that a time-consuming synthesis of Py C C modified phosphoramidites can be avoided because this modification protocol is based on commercially available DNA building blocks. First, the oligonucleotide was synthesised by following standard protocols on a DNA synthesiser up to the position of the Py C C dX unit. At this position, either 8bromo-2’-deoxyadenosine, 2’-deoxy-5-iodocytidine, 8-bromo2’-deoxyguanosine, or 2’-deoxy-5-iodouridine was inserted automatically without the final deprotection of the terminal 5’OH group. Subsequently, the CPG vials were removed from the synthesiser and a Sonogashira-coupling reagent mixture containing Pd(PPh3)4 (60 mm), 1-ethynylpyrene (120 mm) and CuI (60 mm) in DMF/Et3N (3.5:1.5) was added to the CPG vials under dry conditions with syringes. After a coupling time of 3 h at room temperature, the CPGs were washed with different solvents, dried and attached to the DNA synthesiser to finish the synthesis automatically. Modification of the standard procedures for deprotection and cleavage of the oligonucleotides from the solid phase, or during workup was not necessary. The Py C C dX-modified oligonucleotides were purified by semipreparative HPLC and identified by MALDI-TOF mass spectrometry. HPLC analysis of the unpurified oligonucleotides showed excellent coupling efficiencies of the Py C C unit to the oligonucleotides. In the present work, a representative range of Py C C dXmodified duplexes 1±4 was prepared, that differ only by the base dX to which the pyrene modification group has been attached (Scheme 1). The base sequences of all duplexes 1±4 are the same and are based on modified duplexes we have used previously in electron-transport experiments. 4,7] Due to this experimental design, all observed spectroscopic differences between 1±4 can be attributed selectively to the different base [a] Dipl.-Chem. E. Mayer, Dipl.-Chem. L. Valis, Dipl.-Chem. C. Wagner, Dr. M. Rist, Dipl.-Chem. N. Amann, Dr. H.-A. Wagenknecht Chemistry Department Technical University of Munich 85747 Garching (Germany) Fax: (+49)89-289-13210 E-mail : wagenknecht@ch.tum.de Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Scheme 1. Schematic representation for the synthesis of the Py C C dX-modified DNA duplexes 1±4. DMTO=dimethoxytrityl, B=DNA base, CPG=controlled pore glass, R=benzoyl or isobutyroyl, I= inosine.

Development of a Metal‐Ion‐Mediated Base Pair for Electron Transfer in DNA

A new C-nucleoside structurally based on the hydroxyquinoline ligand was synthesized that is able to form stable pairs in DNA in both the absence and the presence of metal ions. The interactions between the metal centers in adjacent Cu(II)-mediated base pairs in DNA were probed by electron paramagnetic resonance (EPR) spectroscopy. The metal-metal distance falls into the range of previously reported values. Fluorescence studies with a donor-DNA-acceptor system indicate that photoinduced charge-transfer processes across these metal-ion-mediated base pairs in DNA occur more efficiently than over natural base pairs.

Thiazole orange and Cy3: Improvement of fluorescent DNA probes with use of short range electron transfer

Thiazole orange was synthetically incorporated into oligonucleotides by using the corresponding phosphoramidite as the building block for automated DNA synthesis. Due to the covalent fixation of the TO dye as a DNA base surrogate, the TO-modified oligonucleotides do not exhibit a significant increase of fluorescence upon hybridization with the counterstrand. However, if 5-nitroindole (NI) is present as a second artificial DNA base (two base pairs away from the TO dye) a fluorescence increase upon DNA hybridization can be observed. That suggests that a short-range photoinduced electron transfer causes the fluorescence quenching in the single strand. The latter result represents a concept that can be transferred to the commercially available Cy3 label. It enables the Cy3 fluorophore to display the DNA hybridization by a fluorescence increase that is normally not observed with this dye.

DNA as a supramolecular scaffold for the helical arrangement of a stack of 1-ethynylpyrene chromophores

A highly organized helical pi-stacked arrangement of 1-ethynylpyrene moieties along the major groove of duplex DNA can only be achieved if more than three chromophores have been synthetically incorporated adjacent to each other.

Detection of single base mismatches and abasic sites using phenanthridinium as an artificial DNA base and charge donor

Combining the fluorescence properties of phenanthridinium as an artificial DNA base together with DNA-mediated charge transfer processes allows the homogeneous detection of DNA base mismatches and abasic sites.

Helical Arrangement of Porphyrins along DNA: Towards Photoactive DNA-Based Nanoarchitectures†

Stack them helically: A self-assembled helically stacked array of up to 11 porphyrin (Po)-modified uridines (red and blue in the double strand shown) is based on the supramolecular scaffold of duplex DNA and shows promising optical properties. Such architectures could find application as functional molecules for photoactive nanomaterials and photonic nanostructures.

Base pair motions control the rates and distance dependencies of reductive and oxidative DNA charge transfer.

In 1999, Wan et al. [Proc. Natl. Acad. Sci. USA 96, 6014–6019] published a pioneering paper that established the entanglement between DNA base pair motions and the transfer time of the charge carrier. The DNA assemblies contained an ethidium covalently bound via a flexible alkyl chain to the 5′ hydroxyl group of the DNA backbone. Although covalently attached, the loose way in which the ethidium was linked to DNA allowed for large degrees of conformational freedom and thus raised some concern with respect to conformational inhomogeneity. In this letter, we report studies on a different set of ethidium DNA conjugates. In contrast to the “Caltech systems,” these conjugates contain ethidium tightly incorporated (as a base pair surrogate) into the DNA base stack, opposite to an abasic site analog. Despite the tight binding, we found that charge transfer from the photoexcited ethidium base pair surrogate across two or more base pairs is several orders of magnitude slower than in case of the DNA systems bearing the tethered ethidium. To further broaden the scope of this account, we compared (oxidative) electron hole transfer and (reductive) electron transfer using the same ethidium chromophore as a charge donor in combination with two different charge acceptors. We found that both electron and hole transfer are characterized by similar rates and distance dependencies. The results demonstrate the importance of nuclear motions and conformational flexibility and underline the presence of a base gating mechanism, which appears to be generic to electronic transfer processes through π-stacked nucleic acids.