By using (S)-2-amino-1,3-propanediol as a linker, thiazole orange (TO) was incorporated in a dimeric form into DNA. The green fluorescence (λ=530 nm) of the intrastrand TO dimer is quenched, whereas the interstrand TO dimer shows a characteristic redshifted orange emission (λ=585 nm). Steady-state optical spectroscopic methods reveal that the TO dimer fluorescence is independent of the sequential base contexts. Time-resolved pump–probe measurements and excitation spectra reveal the coexistence of conformations, including mainly stacked TO dimers and partially unstacked ones, which yield exciton and excimer contributions to the fluorescence, respectively. The helicity of the DNA framework distorts the excitonic coupling. In particular, the interstrand TO dimer could be regarded as an excitonically interacting base pair with fluorescence readout for DNA hybridization. Finally, the use of this fluorescent readout was representatively demonstrated in molecular beacons.

A fullerene was covalently attached to a (dA)₂₀ template that serves as structural scaffold to self-assemble an ordered and mixed array of ethynyl-pyrene- and ethynyl-Nile-red-nucleoside conjugates. Fluorescence spectroscopy revealed evidence for energy transfer between the two different chromophores. Moreover, fluorescence quenching is significantly enhanced by the attached fullerene in mixed assemblies of different chromophore ratios. This indicates exciton dissociation by electron transfer from the photo-generated exciton on the chromophore stack to the fullerene. The fullerene–DNA-conjugate was integrated as a photo-active layer in solar cells that showed charge-carrier generation in the spectral regime of all three components of the conjugate. This work clearly demonstrates that DNA is suitable as structural element for chromophore assemblies in future organic optoelectronic devices, such as solar cells.

Fulleren wurde kovalent mit einem (dA)₂₀-Templat verknüpft, das als Strukturgerüst für den Aufbau eines selbst-assoziierenden, geordneten und gemischten Stapels von Ethinylpyren- und Ethinylnilrot-Nucleosid-Konjugaten diente. Der Energietransfer zwischen den beiden verschiedenen Chromophoren wurde durch Fluoreszenzspektroskopie nachgewiesen. Darüber hinaus wurde die Fluoreszenzlöschung der Stapel verschiedener Chromophormischungen durch das angeknüpfte Fulleren deutlich verstärkt. Das deutet darauf hin, dass das photogenerierte Exciton vom Chromophorstapel durch Elektronentransfer auf das Fulleren dissoziiert. Das Fulleren-DNA-Konjugat wurde als photoaktive Schicht von Solarzellen verwendet, die daraufhin Ladungsträger im spektralen Bereich aller drei Komponenten der Konjugate zeigten. Diese Arbeit macht deutlich, dass DNA als strukturgebendes Gerüst für die Assoziation von Chromophoren in zukünftigen organischen optoelektronischen Bauelementen, wie Solarzellen, gut geeignet ist.

Four benzophenone nucleosides that are para-substituted (–NH₂, –NMe₂, –OMe, and –Me) in relation to the carbonyl group were synthesized and characterized by their optical properties. The electron-donating character of the substituents influenced the optical properties of these nucleosides, especially the bathochromic shift of the charge-transfer band in their UV/Vis absorption spectra. The solubility of the synthetic nucleosides in aqueous solution allowed for a photocatalytic intramolecular [2+2] cycloaddition of a quinolone substrate to take place in H₂O/MeCN by irradiating the mixture with 365 nm light-emitting diode (LED) lamps. The MeO- and Me-substituted benzophenone nucleosides were subjected to these reaction conditions in substoichiometric amounts. After prolonged irradiation times, substrate conversions competed with product decomposition. On the basis of our results, we determined that the Me-substituted benzophenone nucleoside has potential applications in the development of photocatalytically active DNAzymes for both in vivo applications in chemical biology and enantioselective photocatalysis in aqueous solutions.

The chromophores ethynyl pyrene as blue, ethynyl perylene as green and ethynyl Nile red as red emitter were conjugated to the 5-position of 2′-deoxyuridine via an acetylene bridge. Using phosphoramidite chemistry on solid phase labelled DNA duplexes were prepared that bear single chromophore modifications, and binary and ternary combinations of these chromophore modifications. The steady-state and time-resolved fluorescence spectra of all three chromophores were studied in these modified DNA duplexes. An energy-transfer cascade occurs from ethynyl pyrene over ethynyl perylene to ethynyl Nile red and subsequently an electron-transfer cascade in the opposite direction (from ethynyl Nile red to ethynyl perylene or ethynyl pyrene, but not from ethynyl perylene to ethynyl pyrene). The electron-transfer processes finally provide charge separation. The efficiencies by these energy and electron-transfer processes can be tuned by the distances between the chromophores and the sequences. Most importantly, excitation at any wavelength between 350 and 700 nm finally leads to charge separated states which make these DNA samples promising candidates for light-harvesting systems.

Postsynthetic modification of nucleic acids has the advantage that the chemical development of only a few building blocks is necessary, each bearing a chosen reactive functional group that is applicable to its reactive counterpart for a variety of different labeling types. The reactive group is either linked to phosphoramidites for chemical synthesis on solid phase or attached to nucleoside triphosphates for application in primer extension experiments and PCR. Chemoselectivity is required for this strategy, together with bioorthogonality to perform these labelings in living cells or even organisms. Currently, the copper-free reactions include strain-promoted 1,3-dipolar cycloadditions, “photoclick” reactions, Diels–Alder reactions with inverse electron demand, and nucleophilic additions. The majority of these modification strategies show good to excellent reaction kinetics, an important prerequisite for labeling inside cells and in vivo in order to keep the concentrations of the reacting partners as low as possible.

DNA-based aptamers are commonly used recognition elements in biosensors for a range of target molecules. Here, the development of a wavelength-shifting optical module for a DNA-based adenosine-binding aptamer is described. It applies the combination of two photostable cyanine-styryl dyes as covalent modifications. This energy-transfer pair is postsynthetically attached to oligonucleotides via a copper(I)-catalyzed azide–alkyne cycloaddition by two structurally different approaches: 1) as nucleotide modifications at the 2′-position of uridines and 2) as nucleotide substitutions using (S)-amino-1,2-propanediol as acyclic linker between the phosphodiester bridges. Both dyes exhibit a remarkable photostability. A library of DNA aptamers consisting of different combinations of the two dyes in diagonal orientations were evaluated by their emission color contrast as readout. Further optimization led to aptasensors with improved fluorescent readout as compared with previously reported aptasensors. This approach described is synthetically facile using simple propargylated phosphoramidites as DNA building blocks. As such, this approach could be applied for other dyes and other chemical/biological applications.

A new cyano-substituted thiazole red derivative as a red emitter and a novel green fluorescent donor dye of the cyanine styryl type were synthesized in good yields. Characterization of their optical properties revealed excellent photostabilities and large apparent Stokes' shifts. Both dyes can be incorporated into oligonucleotides through postsynthetic “click”-type chemistry and combined in a diagonal arrangement in double-stranded DNA. As a result, both dyes combine to an energy-transfer pair in DNA that shows remarkable optical properties such as significant emission wavelength shift from green to red upon hybridization owing to high energy-transfer efficiency between the two dyes and remarkable emission red/green color contrast ratio. The combination of these dyes as an energy-transfer pair according to our concept of “DNA traffic lights” has high potential for applications in molecular imaging.

Ein neu synthetisierter DNA-Baustein auf Basis von 2′-Desoxyuridin trägt in der 5-Position ein über eine Aminopropinylgruppe verknüpftes, push-pull-substituiertes Diaryltetrazol. Das damit modifizierte Oligonucleotid ermöglicht die postsynthetische Markierung mit einem Maleinimid tragenden Sulfo-Cy3-Farbstoff, N-Methylmaleinimid und Methylmethacrylat als Dipolarophile jeweils durch Bestrahlung mit Licht bei λ_exc=365 nm (LED). Die experimentell bestimmte Geschwindigkeitskonstante beträgt (23±7) M⁻¹ s⁻¹ und ist bemerkenswert groß gegenüber denen anderer kupferfreier, bioorthogonaler Reaktionen und ähnlich groß wie jene der kupferkatalysierten Cycloaddition von Aziden mit Acetylenen.

A new DNA building block bearing a push–pull-substituted diaryltetrazole linked to the 5-position of 2′-deoxyuridine through an aminopropynyl group was synthesized. The accordingly modified oligonucleotide allows postsynthetic labeling with a maleimide-modified sulfo-Cy3 dye, N-methylmaleimide, and methylmethacrylate as dipolarophiles by irradiation at 365 nm (LED). The determined rate constant of (23±7) M⁻¹ s⁻¹ is remarkably high with respect to other copper-free bioorthogonal reactions and comparable with the copper-catalyzed cycloaddition between azides and acetylenes.

A new type of a bifunctional DNA architecture based on a three way junction is developed that combines the structural motif of sticky perylene bisimide caps with a tris-bipyridyl metal ion lock in the center part. A clear stabilizing effect was observed in the presence of Fe³⁺, Ni²⁺ and Zn²⁺ by the formation of corresponding bipyridyl complexes in the branching part of the DNA three way junctions. The dimerization of the 5′-terminally attached perylene diimides (PDI) chromophores by hydrophobic interactions can be followed by significant changes in the UV/Vis absorption and steady-state fluorescence. The PDI-mediated DNA assembly occurs at temperatures below the melting temperature and is not influenced by the metal-ion bipyridyl locks in the central part. The corresponding AFM images revealed the formation of higher-ordered structures as the result of DNA assemblies mediated by the PDI interactions.

A new donor-DNA-acceptor system has been synthesized containing Nile red-modified 2′-deoxyuridine as charge donor and 6-N,N-dimethylaminopyrene-modified 2′-deoxyuridine as acceptor to investigate the charge transfer in DNA duplexes using fluorescence spectroscopy and time-resolved femtosecond pump-probe techniques. Fluorescence quenching experiments revealed that the quenching efficiency of Nile red depends on two components: 1) the presence of a charge acceptor and 2) the number of intervening CG and AT base pairs between donor and acceptor. Surprisingly, the quenching efficiency of two base pairs (73 % for CG and the same for AT) is higher than that for one base pair (68 % for CG and 37 % for AT), while at a separation of three base pairs less than 10 % quenching is observed. A comparison with the results of time-resolved measurements revealed a correlation between quenching efficiency and the first ultrafast time constant suggesting that quenching proceeds via a charge transfer from the donor to the acceptor. All transients are satisfactorily described with two decays: a rapid charge transfer with 600 fs (∼10¹² s⁻¹) that depends strongly and in a non-linear fashion on the distance between donor and acceptor, and a slower time constant of a few picoseconds (∼10¹¹ s⁻¹) with weak distance dependence. A third time constant on a nanosecond time scale represents the fluorescence lifetime of the donor molecule. According to these results and time-dependent density functional theory (TDDFT) calculations a combination of single-step superexchange and multistep hopping mechanisms can be proposed for this short-range charge transfer. Furthermore, significantly less quenching efficiency and slower charge transfer rates at very short distances indicate that the direct interaction between donor and acceptor leads to a local structural distortion of DNA duplexes which may provide some uncertainty in identifying the charge transfer rates in short-range systems.

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.

Upconverting nanoparticles (UCNPs) are a highly attractive tool owing to their unique property of showing visible luminescence when excited in the near-infrared (NIR) region. Plain UCNPs have no biorecognition capabilities, but functionalization of their surface with azido groups renders them conjugatable to ethynyl-modified oligonucleotides in a bioorthogonal fashion. Single-stranded DNA was covalently attached to the surface of UCNPs by click chemistry and purified by size exclusion chromatography (SEC) at elevated temperature. Covalent attachment was evidenced by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. DNA conjugation makes the particle soluble in water and enables it to recognize its counter strand. Such UCNPs are capable of nonspecifically crossing cell membranes. Confocal microscopy reveals the high potential of the bright UCNPs for live cell imaging in the NIR, where the UCNP–DNA conjugates can be considered to act as a kind of nano-sized lamp. Furthermore, cross-linking of those DNA nanolamps yields highly emissive aggregates.

Perylene-3,4:9,10-tetracarboxylic acid bisimide represents a particularly attractive building block for DNA-based multichromophores and nanoassemblies due to its unique combination of optical and electronic properties. Despite the broad applicability of the perylene bisimide dye for DNA-based assemblies, significant fluorescence quenching by photoinduced charge-transfer processes to guanines in DNA represents a major disadvantage. In order to develop a redox actively and optically tuned perylene bisimide derivative with fluorescence that does not photooxidize guanines in DNA, we chose to attach two N-pyrrolidinyl substituents as strongly electron-donating substituents at the 1- and 7-positions of the bay area (“APBI”). The corresponding phosphoramidite 1 was synthesized and incorporated synthetically into oligonucleotides by using the automated DNA building block chemistry. The 2′-deoxyribofuranoside of natural nucleosides was replaced by (S)-1-aminopropane-2,3-diol as an acyclic linker between the phosphodiester bridges that is tethered to one of the imide nitrogen atoms of the APBI dye. Electrochemical and optical characterization of the isolated APBI dye shows that photoinduced charge transfer with guanines is indeed very unlikely. Single APBI DNA base substitutions show an exciplex-type emission with pH sensitivity. The interstrand APBI dimer in DNA could be regarded as a hydrophobically interacting base pair that stabilizes the thermal stability of the double strand. The APBI dimer behaves like an H aggregate, and the fluorescence is quenched quantitatively.

Perylene bisimides (PBI) have been synthetically incorporated as caps onto a Y-shaped DNA triple strand. These PBI caps serve as “sticky” ends in the spontaneous assembly of larger DNA ensembles, linking the triangular DNA through stacking interactions. This, in turn, yields a hypsochromic shift in the absorption and a red shift in the fluorescence as characteristic optical readouts. This assembly occurs spontaneously without any enzymatic ligation process and without the use of overhanging DNA as sticky ends. Instead, dimerizations of the PBI chromophores in the assembly are controlled by the DNA as a structural scaffold. Thereby, the PBI-driven assembly is fully reversible. Due to the fact that PBI dimerization does not occur in the single strand, the aggregates can be destroyed by thermal dehybridization of the DNA scaffold and reassembled by reannealing of the DNA construct. In view of the fact that PBI forms stable radical anions, the presented DNA architectures are not only interesting optical biomaterials, but are also promising materials for molecular electronics with DNA.