In this paper, a new fluorescence polarization (FP) assay strategy is described reporting the first demonstration of a noncompetitive FP technique dedicated to the small molecule sensing. This approach was based on the unique induced-fit binding mechanism of nucleic acid aptamers which was exploited to convert the small target binding event into a detectable fluorescence anisotropy signal. An anti-L-tyrosinamide DNA aptamer, labeled by a single fluorescent dye at its extremity, was employed as a model functional nucleic acid probe. The DNA conformational change generated by the L-tyrosinamide binding was able to induce a significant increase in the fluorescence anisotropy signal. The method allowed enantioselective sensing of tyrosinamide and analysis in practical samples. The methodology was also applied to the L-argininamide detection, suggesting the potential generalizability of the direct FP-based strategy. Such aptamer-based assay appeared to be a sensitive analytical system of remarkable simplicity and ease of use.

Formation of DNA-protein cross-links involving the initial formation of a guanine radical cation was investigated. For this purpose, riboflavin-mediated photosensitization of a TGT oligonucleotide in aerated aqueous solution in the presence of the KKK tripeptide was performed. We have shown that the nucleophilic addition of the epsilon-amino group of the central lysine residue of KKK to the C8 atom of either the guanine radical cation or its deprotonated form gives rise to the efficient formation of a Nepsilon-(guanin-8-yl)-lysine cross-link. Interestingly, the time course of formation of the above-mentioned cross-link was found to be not linear with the time of irradiation, and its formation rapidly reached a plateau. This is explained by secondary decomposition of the initially generated cross-link which could be further oxidized more efficiently than starting TGT oligonucleotide. One-electron oxidation of the initially generated cross-link was found to produce mainly two diastereomeric cross-links exhibiting a spiroimino-trilysine-dihydantoin structure as inferred from enzymatic digestion, CD, UV, NMR and mass spectrometry measurements. In addition, other minor cross-links, for which formation was favored at acidic pH, were assigned as lysine-guanine adducts in which the modified guanine base exhibits a guanidino-trilysine-iminohydantoin structure. A proposed mechanism for the formation of the different detected oligonucleotide-peptide cross-links is given. The high yield of formation of the detected cross-links strongly suggests that a DNA-protein cross-link involving a lysine residue linked to the C8 position of guanine could be generated in cellular systems if a lysine is located in the close vicinity of a guanine radical cation.

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Here, we describe a new fluorescence polarization aptamer assay (FPAA) strategy which is based on the use of the single-stranded DNA binding (SSB) protein from Escherichia coli as a strong FP signal enhancer tool. This approach relied on the unique ability of the SSB protein to bind the nucleic acid aptamer in its free state but not in its target-bound folded one. Such a feature was exploited by using the antiadenosine (Ade)−DNA aptamer (Apt-A) as a model functional nucleic acid. Two fluorophores (fluorescein and Texas Red) were introduced into different sites of Apt-A to design a dozen fluorescent tracers. In the absence of the Ade target, the binding of the labeled aptamers to SSB governed a very high fluorescence anisotropy increase (in the 0.130−0.200 range) as the consequence of (i) the large global diffusion difference between the free and SSB-bound tracers and (ii) the restricted movement of the dye in the SSB-bound state. When the analyte was introduced into the reaction system, the formation of the folded tertiary structure of the Ade−Apt-A complex triggered the release of the labeled nucleic acids from the protein, leading to a strong decrease in the fluorescence anisotropy. The key factors involved in the fluorescence anisotropy change were considered through the development of a competitive displacement model, and the optimal tracer candidate was selected for the Ade assay under buffer and realistic (diluted human serum) conditions. The SSB-assisted principle was found to operate also with another aptamer system, i.e., the antiargininamide DNA aptamer, and a different biosensing configuration, i.e., the sandwich-like design, suggesting the broad usefulness of the present approach. This sensing platform allowed generation of a fluorescence anisotropy signal for aptamer probes which did not operate under the direct format and greatly improved the assay response relative to that of the most previously reported small target FPAA.

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Steady-state (1)H photo-chemically induced dynamic nuclear polarization (CIDNP) experiments were conducted at 14.1 T on deoxygenated (buffered pH 7) aqueous solutions of [Ru(phen)(3)](2+), [Ru(tap)(2)(phen)](2+), and [Ru(tap)(3)](2+) (tap = 1,4,5,8-tetraazaphenanthrene; phen = 1,10-phenanthroline) in the presence of guanosine-5'-monophosphate or N-acetyltyrosine. For the first time, CIDNP arising from photo-oxidation by polyazaaromatic Ru(II) complexes is reported. In agreement with the occurrence of a photo-electron-transfer process, photo-CIDNP effects are observed with [Ru(tap)(3)](2+) and [Ru(tap)(2)(phen)](2+) but not with [Ru(phen)(3)](2+). With [Ru(tap)(2)(phen)](2+), no significant photo-CIDNP is observed for the (1)H nuclei of the phen ligand, consistent with the fact that the metal-to-ligand charge-transfer triplet excited states responsible for the photo-oxidation involve a tap ligand. Successive experiments with [Ru(tap)(3)](2+) highlight the accumulation of long-lived radical species in solution that cause (1)H NMR signal broadening and photo-CIDNP extinction. The (1)H photo-CIDNP observed for the biomolecules is rather weak, less than about 30% of the equilibrium magnetization. However, up to 60% polarization enhancement is observed for H-2 and H-7 of the tap ligands, which indicates high unpaired electron density in the vicinity of these atoms in the transient radical pair. This is consistent with the structure of known photoadducts formed, for instance, between the metallic compounds and the guanine base of mono- and polynucleotides. Indeed, in these adducts the covalent bond involves carbon C-2 or C-7 of a tap ligand. The occurrence of photo-CIDNP with polyazaaromatic Ru(II) complexes opens new perspectives for the study of this type of compound.

A novel fluorescence polarization (FP) aptasensing platform based on target-induced aptamer enzymatic cleavage protection is reported. The method relies on the FP analysis of the phosphodiesterase I mediated size variation of a dye-labeled aptamer. The tyrosinamide/antityrosinamide DNA aptamer couple was firstly tested as a model system to establish the proof-of-concept. In the absence of the target, the labeled aptamer was enzymatically cleaved into small DNA fragments, leading to a low FP signal. Upon tyrosinamide binding, the DNA substrate was partially protected against the enzymatic attack, leading to an increase in the fluorescence anisotropy response as a result of the higher average molecular volume of the weakly digested probe. The method was subsequently applied to two other systems, i.e., for the detection of ochratoxin A and adenosine. Such an approach was found to combine simplicity and general applicability features.

Fluorescence polarization/anisotropy is a very popular technique that is widely used in homogeneous-phase immunoassays for the small molecule quantification. In the present Feature, we discuss how the potential of this signaling approach considerably expanded during the last 2 decades through the implementation of a myriad of original transducing strategies that use functional nucleic acid recognition elements as a promising alternative to antibodies.