Fluorescence-Quenching Phenomenon by Photoinduced Electron Transfer between a Fluorescent Dye and a Nucleotide Base

Takasu, Masaki; Kurata, Shinya; Yamada, Kazutaka; Yokomaku, Toyokazu; Kamagata, Yoichi; Kanagawa, Takahiro; Kurane, Ryuichiro

doi:10.2116/analsci.17.155

Cited by 329 publications

(294 citation statements)

References 43 publications

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“…This quenching of fluorescence could be attributed to partial stacking of the chromophore and alteration of its microenvironment, or by neighboring bases via a photoinduced electron transfer mechanism, or both. 22 Similar reduction in emission has also been seen with other fluorescent nucleoside analogs (e.g., 2-aminopurine, pyrroloC). 23 Remarkably, the emission of the duplex (7•10) containing the fluorescent nucleoside 4 opposite to C, was enhanced over 7-fold as compared to the perfect duplex 7•9 (Fig.…”

supporting

confidence: 54%

A highly fluorescent nucleoside analog based on thieno[3,4-d]pyrimidine senses mismatched pairing

2008

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A highly emissive nucleobase analog, based on a thieno [3,4-d]pyrimidine core, is enzymatically incorporated into RNA oilgonucleotides that function as base discriminating fluorescent probes.A fundamentally simple yet practically challenging task in nucleic acid diagnostics is the detection of single nucleotide polymorphism (SNP). 1 Such mutated base pairs, while in many cases associated with benign biological outcomes, could also be linked to certain diseases or reflect susceptibility to specific therapeutics. 2 Whereas several approaches for the detection of SNPs have been developed and commercialized, 3,4 efforts have recently been made to advance base-discriminating fluorescent (BDF) nucleosides, a term coined by Saito et al. 5 Upon hybridization to a given target, such emissive modified nucleobases can, in principle, identify the paired base on the opposite strand by eliciting a specific change in their photophysical characteristics. 6 Such fluorescence-based tools can facilitate the future development of enzyme-free and homogenous SNP detection protocols. They present, however, a considerable challenge in nucleobase probe design, as our ability to correlate structure and microenvironment with photophysical properties is currently limited. 7 Our program aims at the development of simple and minimally perturbing emissive nucleobases. The primary design principle entails maintaining the highest possible structural similarity to the natural nucleobases, while bestowing useful photophysical properties upon the probes. Of particular significance is their ability to respond to and report on changes in their microenvironment. Several useful nucleobase analogs have been obtained by either conjugating or fusing five-membered aromatic heterocyclic rings onto the pyrimidines at their 5 or 5,6 positions, respectively. 8 Conjugating furan to a dU core, for example, has generated an emissive analog that responds to the presence of abasic sites in DNA via enhanced emission. 9 The analogous furan containing ribonucleoside has been shown to serve as a useful probe for monitoring RNA-ligand binding. 10 Here we report on a new motif for highly emissive nucleobase analogs based on a thieno [3,4-d]pyrimidine core. We report the synthesis of the ribonucleoside, its basic photophysical characteristics, and its conversion into a triphosphate, as well as its enzymatic incorporation into RNA † Electronic supplementary information (ESI) available: Details of synthesis and characterization of compounds, transcription reactions and gel shift experiments, and additional fluorescence and thermal melting data. See DOI: 10.1039/b801054dCorrespondence to: Yitzhak Tor. Short RNA constructs were selected in this case as fluorescent hybridization probes as such oligonucleotides tend to form stable heteroduplexes with DNA, frequently of higher stability than the corresponding DNA-DNA duplexes. 11 Although solid-phase synthesis is a powerful tool for the site specific modification of nucleic acids, we have chosen transcription reactio...

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supporting

confidence: 54%

A highly fluorescent nucleoside analog based on thieno[3,4-d]pyrimidine senses mismatched pairing

2008

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“…S1). The increase in fluorescence lifetime and the enhancement of fluorescence intensity are consistent with PET mechanism reported by others and our previous studies [63,66,70,71,75]. These results suggest that the intramolecular interaction between the labeled TMR and adjacent guanine bases in the aptamer probe gives rise to PET, which is eliminated upon binding to Pb 2+ , resulting in the formation of a G-quadruplex.…”

Section: Principle Of Sensing Pb 2+ By Fasupporting

confidence: 91%

“…The TBA has a random-coil structure in the absence of Pb 2+ . Because the internally labeled TMR can intramolecularly interact with the adjacent guanine bases by photo-induced electron transfer (PET) mechanism due to its good electron donating property [67][68][69][70][71], the rotation of TMR is firmly restricted, which results in the aptamer probe having large FA value. In the presence of Pb 2+ , because Pb 2+ can interact with aptamer probe and induce conformational change from a random-coil structure into a highly-ordered G-quadruplex [65,72], this would weaken or eliminate the intramolecular interaction between the TMR and the adjacent guanine bases involved in formation of the G-quadruplex.…”

Section: Principle Of Sensing Pb 2+ By Famentioning

confidence: 99%

A sensitive fluorescence anisotropy method for detection of lead (II) ion by a G-quadruplex-inducible DNA aptamer

Zhang

Meng

et al. 2014

Analytica Chimica Acta

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h i g h l i g h t s• A fluorescence anisotropy approach for detection of Pb 2+ was developed.• The strategy was based on bindinginduced allosteric conformational change of aptamer probe.• The sensing mechanism was established by testing the photoinduced electron transfer interaction. in homogeneous solution by a G-rich thrombin binding aptamer (TBA). The TBA labeled with 6-carboxytetramethylrhodamine (TMR) at the seventh thymine nucleotide was used as a fluorescent probe for signaling Pb 2+ . It was found that the aptamer probe had a high FA in the absence of Pb 2+ . This is because the rotation of TMR is restricted by intramolecular interaction with the adjacent guanine bases, which results in photoinduced electron transfer (PET). When the aptamer probe binds to Pb 2+ to form G-quadruplex, the intramolecular interaction should be eliminated, resulting in faster rotation of the fluorophore TMR in solution. Therefore, FA of aptamer probe is expected to decrease significantly upon binding to Pb 2+ . Indeed, we observed a decrease in FA of aptamer probe upon Pb 2+ binding. Circular dichroism, fluorescence spectra, and fluorescence lifetime measurement were used to verify the reliability and reasonability of the sensing mechanism. By monitoring the FA change of the aptamer probe, we were able to real-time detect binding between the TBA probe and Pb 2+ . Moreover, the aptamer probe was exploited as a recognition element for quantification of Pb 2+ in homogeneous solution. The change in FA showed a linear response to Pb 2+ from 10 nM to 2.0 M, with 1.0 nM limit of detection. In addition, this sensing system exhibited good selectivity for Pb 2+ over other metal ions. The method is simple, quick and inherits the advantages of aptamer and FA.

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“…The dramatic FA decrease accompanied significant increase of fluorescence intensity indicates that the target-binding induced change of the intramolecular interaction leads to the alteration of FA of these TMR-labeled aptamers. The low fluorescence intensity of TMR-labeled aptamer suggests that fluorescence quenching of TMR occurs due to the intramolecular interactions between TMR and the adjacent G bases of the aptamer prior to OTA binding, 37 causing a low local rotation of TMR and a high FA. In contrast, the high fluorescence intensity of TMR-labeled aptamers means that the intramolecular interaction between TMR and G bases of the aptamer upon OTA binding is eliminated, 37 resulting in a large local rotation of TMR and a low FA.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Identification of Allosteric Nucleotide Sites of Tetramethylrhodamine-Labeled Aptamer for Noncompetitive Aptamer-Based Fluorescence Anisotropy Detection of a Small Molecule, Ochratoxin A

Zhao

Wang

2013

Anal. Chem.

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Aptamer-based fluorescence anisotropy (FA) assay combines the advantages of affinity aptamers in good stability, easy generation, and facile labeling and the benefits of FA in homogeneous analysis, such as robustness, simplicity, and high reproducibility. By using a fluorophore-labeled aptamer, FA detection of a small molecule is not as easy as detection of protein because the binding of a small molecule cannot cause significant increase of molecular weight of the dye-labeled aptamer. The intramolecular interaction between labeled tetramethylrhodamine (TMR) and DNA aptamer bases dramatically affects the local rotation and FA of TMR. This intramolecular interaction can be altered by aptamer conformation change upon target binding, leading to a significant change of FA of TMR. Taking this unique feature of a TMR-labeled aptamer, we described a noncompetitive aptamer-based fluorescence anisotropy assay for detection of small molecules by using ochratoxin A (OTA) as a model. We successfully identified the specific TMR-labeling sites of aptamers with sensitive FA response to OTA from the 5′-end, 3′-end and the internal thymine (T) bases. The aptamer with a TMR labeled on the 10th T base exhibited a remarkable FA reduction response to OTA (Δr = 0.078), without requiring any proteins or nanomaterials as FA signal enhancers. This FA approach for OTA showed high sensitivity with a detection limit of 3 nM, a dynamic range from 3 nM to 3 μM, and good selectivity over the tested compounds with similar structures to OTA. The new strategy allowed the detection of OTA in diluted red wine and urine samples. F luorescence anisotropy (FA) is a powerful and unique analytical technique for molecular interactions study and assay developments, which relies on the rotational diffusion of fluorescent tracers or probes under polarized excitation light. 1−5Fluorescence anisotropy shows strength in real-time analysis, simplicity, robustness, and sensitivity. FA assays have been applied to the studies about protein−protein interaction and protein−DNA interaction and the immunoassays for drug discovery, diagnostics, environmental monitoring, and food analysis. 1−7The emergence of aptamers as affinity ligands makes the aptamer-based FA assay attractive due to their advantages over antibodies in ease of synthesis and labeling, great stability, good selectivity, and high binding affinity. 8−12 Target-dependent allosteric structural change is one unique feature of an aptamer, and it allows the construction of an aptamer switch for target analysis through the adaptive transition converting a binding event into a detectable signal. 8,13−17 The applications of fluorophore-labeled aptamers to direct FA analysis of proteins are relatively straightforward as aptamers are usually smaller than proteins in size. 5,8,11,12 The binding of the fluorophorelabeled aptamers to proteins can cause enhanced FA signals by restricting the rotational diffusion of the labeled fluorophore as a result of increased molecular size. 18−23 In contrast, the ...

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Fluorescence-Quenching Phenomenon by Photoinduced Electron Transfer between a Fluorescent Dye and a Nucleotide Base

Cited by 329 publications

References 43 publications

A highly fluorescent nucleoside analog based on thieno[3,4-d]pyrimidine senses mismatched pairing

A highly fluorescent nucleoside analog based on thieno[3,4-d]pyrimidine senses mismatched pairing

A sensitive fluorescence anisotropy method for detection of lead (II) ion by a G-quadruplex-inducible DNA aptamer

Identification of Allosteric Nucleotide Sites of Tetramethylrhodamine-Labeled Aptamer for Noncompetitive Aptamer-Based Fluorescence Anisotropy Detection of a Small Molecule, Ochratoxin A

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