Efficient Quenching of Oligomeric Fluorophores on a DNA Backbone (original) (raw)
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Oligodeoxyfluorosides: strong sequence dependence of fluorescence emission
Tetrahedron, 2007
We describe the properties of a series of oligomeric polyfluorophores assembled on the DNA backbone. The eleven oligomers (oligodeoxyfluorosides, ODFs), 4 to 7 monomers in length, were composed of only two fluorescent monomers and a spacer in va ried sequences, and were designed to test how fluorescent nucleobases can interact electronically to yield complexity in fluorescence emission. The monomer fluorophores were deoxyribosides of pyrene and perylene, which emit light in violet and blue wavele ngths respectively. The experiments show that simple variation of sequence and spacing can dramatically change fluorescence, yielding emission maxima ranging from 380 to 557 nm and visible colors from violet to orange-red. Fluorescence lifetimes data, excitation spectra, and absorption data point to a number of multi-fluorophore electronic interactions, including pyrenepyrene and perylene-perylene excimers, pyrene-perylene exciplexes, as well as monomer dye emissions, contributing to the final spectral outcomes. Thus, two simple fluorophores can be readily combined to give emissions over much of the visible spectrum, all requiring only a single excitation. The results demonstrate that fluorescent nucleobases in oligomeric form can act cooperatively as electronic units, and that fluorophore sequence in such oligomers is as important as fluorophore composition in determining fluorescence properties.
Polyfluorophore Labels on DNA: Dramatic Sequence Dependence of Quenching
Chemistry - A European Journal, 2009
We describe studies carried out in the DNA context to test how a common fluorescence quencher, dabcyl, interacts with oligodeoxynu-cleoside fluorophores (ODFs)-a system of stacked, electronically interacting fluorophores built on a DNA scaffold. We tested twenty different tetrameric ODF sequences containing varied combinations and orderings of pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and spacer (S) monomers conjugated to the 3′ end of a DNA oligomer. Hybridization of this probe sequence to a dabcyl-labeled complementary strand resulted in strong quenching of fluorescence in 85% of the twenty ODF sequences. The high efficiency of quenching was also established by their large Stern-Volmer constants (K SV) of between 2.1 × 10 4 and 4.3 × 10 5 M −1 , measured with a free dabcyl quencher. Interestingly, quenching of ODFs displayed strong sequence dependence. This was particularly evident in anagrams of ODF sequences; for example, the sequence BYDS had a K SV that was approximately two orders of magnitude greater than that of BSDY, which has the same dye composition. Other anagrams, for example EDSY and ESYD, also displayed different responses upon quenching by dabcyl. Analysis of spectra showed that apparent excimer and exciplex emission bands were quenched with much greater efficiency compared to monomer emission bands by at least an order of magnitude. This suggests an important role played by delocalized excited states of the π stack of fluorophores in the amplified quenching of fluorescence.
Photophysical characterization of oligopyrene modules for DNA-based nanosystems
Photochemical & Photobiological Sciences, 2009
The photophysics of free pyrenedicarboxamide (Py-DCA) in solution as well as of single-stranded and double-stranded oligonucleotides (ss and ds ONs) containing 1-7 pyrene building blocks per strand were studied by steady-state and time-resolved fluorescence spectroscopy. It was found that the fluorescence quantum yield U F of free Py-DCA chromophore in solution is rather high (U F = 0.44). However, after incorporation of the chromophore into a ss ON the monomeric chromophore fluorescence is quenched more than 40-fold due to electron-transfer reactions with ON bases. An increase of the number n of neighboring pyrenes in an ON results in U F growth up to 0.25 at n = 6. Starting from n = 2, all fluorescence belongs mainly to excimer formed by pyrene chromophores. Sections composed of multiple pyrenes may be considered as robust functional entities that may serve as independent modules in DNA-based, functional nano-architectures.
Journal of Organic Chemistry, 2008
The DNA probes (ODNs) containing a 2′-N-(pyren-1-yl)-group on the conformationally locked nucleosides [2′-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2′-N-(pyren-1-yl)carbonyl-aza-ENA thymidine, Aza-ENA-pyr (Y)], show that they can bind to complementary RNA more strongly than to the DNA. The Aze-pyr (X) containing ODNs with the complementary DNA and RNA duplexes showed an increase in the fluorescence intensity (measured at λ em ≈ 376 nm) depending upon the nearest neighbor at the 3′-end to X [dA (∼12-20-fold) > dG (∼9-20-fold) > dT (∼2.5-20-fold) > dC (∼6-13-fold)]. They give high fluorescence quantum yields (Φ F ) 0.13-0.89) as compared to those of the single-stranded ODNs. The Aza-ENA-pyr (Y)-modified ODNs, on the other hand, showed an enhancement of the fluorescence intensity only with the complementary DNA (1.4-3.9-fold, Φ F ) 0.16-0.47); a very small increase in fluorescence is also observed with the complementary RNA (1.1-1.7-fold, Φ F ) 0.17-0.22), depending both upon the site of the Y modification introduced as well as on the chemical nature of the nucleobase adjacent to the modification site into the ODN. The fluorescence properties, thermal denaturation experiments, absorption, and circular dichroism (CD) studies with the X-and Y-modified ODNs in the form of matched homo-and heteroduplexes consistently suggested (i) that the orientation of the pyrene moiety is outside the helix of the nucleic acid duplexes containing a dT-d/rA base pair at the 3′-end of the modification site for both X and Y types of modifications, and (ii) that the microenvironment around the pyrene moiety in the ODN/DNA and ODN/RNA duplexes is dictated by the chemical nature of the conformational constraint in the sugar moiety, as well as by the nature of neighboring nucleobases. The pyrene fluorescence emission in both X and Y types of the conformationally restricted nucleotides is found to be sensitive to a mismatched base present in the target RNA: (i) The X-modified ODN showed a decrease (∼37-fold) in the fluorescence intensity (measured at λ em ≈ 376 nm) upon duplex formation with RNA containing a G nucleobase mismatch (dT-rG pair instead of dT-rA) opposite to the modification site. (ii) In contrast, the Y-modified ODN in the heteroduplex resulted in a ∼3-fold increase in the fluorescence intensity upon dT-rG mismatch, instead of matched dT-rA pair, in the RNA strand. Our data corroborate that the pyrene moiety is intercalated in the X-modified mismatched ODN/RNA (G mismatch) heteroduplex as compared to that of the Y-modified ODN/RNA (G mismatch) heteroduplex, in which it is located outside the helix.
Polyfluorophores on a DNA Backbone: A Multicolor Set of Labels Excited at One Wavelength
Journal of the American Chemical Society, 2009
We recently described the assembly of fluorescent deoxyriboside monomers ("fluorosides") into DNA-like phosphodiester oligomers (oligodeoxyfluorosides, or ODFs) in which hydrocarbon and heterocyclic aromatic fluorophores interact both physically and electronically. Here we report the identification of a multicolor set of water-soluble ODF dyes that display emission colors across the visible spectrum, and all of which can be simultaneously excited by long-wavelength UV light at 340−380 nm. Multispectral dye candidates were chosen from a library of 4096 tetramer ODFs constructed on PEG-polystyrene beads using a simple long-pass filter to observe all visible colors at the same time. We re-synthesized and characterized a set of 23 ODFs containing one to four individual chromophores, and included 2−3 spacer monomers to increase aqueous solubility and minimize aggregation. Emission maxima of this set range from 376 nm to 633 nm, yielding apparent colors from violet to red, all of which can be visualized directly. The spectra of virtually all ODFs in this set varied considerably from the simple combination of monomer components, revealing extensive electronic interactions between the presumably stacked monomers. In addition, comparisons of anagrams in the set (isomers having the same components in a different sequence) reveal the importance of nearest-neighbor interactions in the emissive behavior. Preliminary experiments with human tumor (HeLa) cells, observing two ODFs by laser confocal microscopy, showed that they can penetrate the outer cellular membrane, yielding cytoplasmic localization. In addition, a set of four distinctly-colored ODFs was incubated with live zebrafish embryos, showing tissue penetration, apparent biostability, and no apparent toxicity. The results suggest that ODF dyes may be broadly useful as labels in biological systems, allowing the simultaneous tracking of multiple species by color, and allowing visualization in moving systems where classical fluorophores fail.
Journal of The American Chemical Society, 2002
Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5′ end with a fluorophore and at the 3′ end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through Fö rster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5′Cy3.5--actin-3′BHQ1 and 5′FAM--actin-3′BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5-BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.
Chemical Society Reviews, 2011
Pyrene-functionalized oligonucleotides (PFOs) are increasingly explored as tools in fundamental research, diagnostics and materials science. Their popularity is linked to the ability of pyrenes to function as polarity-sensitive and quenchable fluorophores, excimer-generating units, aromatic stacking moieties and nucleic acid duplex intercalators. These characteristics have motivated development of PFOs for detection of complementary DNA/RNA targets, single nucleotide polymorphisms (SNPs), and generation of π-arrays on nucleic acid scaffolds. This Review will highlight the physical properties and applications of PFOs that are likely to provide high degree of positional control of the chromophore in nucleic acid complexes. Particular emphasis will be placed on pyrene-functionalized Locked Nucleic Acids (LNAs) since these materials display distinctive properties such as large fluorescence quantum yields, efficient discrimination of SNPs, and recognition of mixed-sequence double stranded DNA. † Part of a themed issue on the advances in DNA-based nanotechnology This journal is The Review is structured to provide: a) a physicochemical background to assist interpretation of key experimental observations with PFOs, b) an overview of pyrenefunctionalized nucleotide monomers and their hybridization properties, and c) an introduction to LNA and overview of pyrene-functionalized LNA monomers. This then sets the stage for a survey of recent PFO-based applications.