ABSTRACT
8-(Pyren-1-yl)-2'-deoxyguanosine (Py-G) was incorporated synthetically as a modified DNA base and optical probe into oligonucleotides. A variety of Py-G-modified DNA duplexes have been investigated by methods of optical spectroscopy. The DNA duplex hybridization can be observed by both fluorescence and absorption spectroscopy since the Py-G group exhibits altered properties in single strands versus double strands for both spectroscopy methods. The fluorescence enhancement upon DNA hybridization can be improved significantly by the presence of 7-deazaguanin as an additional modification and charge acceptor three bases away from the Py-G modification site. Moreover, Py-G in DNA can be applied as a photoinducable donor for charge transfer processes when indol is present as an artificial DNA base and charge acceptor. Correctly base-paired duplexes can be discriminated from mismatched ones by comparison of their fluorescence quenching.
Subject(s)
DNA/analysis , Fluorescent Dyes/chemistry , Guanosine/chemistry , Oligonucleotide Probes/chemical synthesis , Pyrenes , Base Pair Mismatch , Base Pairing , DNA/chemistry , Nucleic Acid Hybridization , Spectrum AnalysisABSTRACT
In this work, fluorescence anisotropy was used to study DNA binding of the DNA methyltransferase M.TaqI. For this purpose short DNA molecules labelled with three different fluorophores (Cy3, thiazole orange, and ethidium bromide) were prepared in various topologies and their suitability for detection of DNA-protein complex formation was investigated.
Subject(s)
DNA/chemistry , Fluorescence Polarization , Fluorescent Dyes/chemistry , Nucleic Acid Conformation , Site-Specific DNA-Methyltransferase (Adenine-Specific)/chemistry , Benzothiazoles/chemistry , Carbocyanines/chemistry , Ethidium/chemistry , Quinolines/chemistryABSTRACT
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 pi-stacked nucleic acids.
Subject(s)
Base Pairing , DNA/chemistry , DNA/metabolism , Movement , Base Sequence , Models, Molecular , Oxidation-ReductionABSTRACT
8-(Pyren-1-yl)-2'-deoxyguanosine (Py-G) was incorporated synthetically as an optical probe into oligonucleotides. The Py-G group in DNA does not discriminate between any of the four natural nucleosides as a counterbase and exhibits altered optical properties in single strands versus double strands. Thus, the duplex hybridization of Py-G-modified DNA can be observed by both fluorescence and absorption spectroscopy. Moreover, Py-G in DNA can be applied as photoinducable donor for charge transfer processes with small peptides.
Subject(s)
DNA/chemistry , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/chemistry , Nucleic Acid Probes/analysis , Nucleic Acid Probes/chemistry , Peptides/chemistry , Pyrenes/chemistry , Base Sequence , Nucleic Acid Hybridization , Spectrum Analysis , Titrimetry , Transition TemperatureABSTRACT
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.
