Fluorescence detection of potassium ion using the G-quadruplex structure

Oligonucleotides with sequences of human telomere DNA or thrombin binding aptamer (TBA) are known to form tetraplex structures upon binding the K(+) ion. Structural changes associated with the formation of tetraplex assemblies led to the development of potassium-sensing oligonucleotide (PSO) probes, in which two fluorescent dyes were attached to both termini of particular oligonucleotide. The combination of dyes included fluorescence resonance energy transfer (FRET) and excimer emission approaches, and the structural changes upon binding K(+) ion could be monitored by a fluorescence technique. These systems showed a very high preference for K(+) over Na(+) ion, which was suitable for fluorescence imaging of the potassium concentration gradient in a living cell. In the case of human telomere DNA, it was also possible to follow the polymorphism of its tetraplex structures.     

The Fluorescence Properties and Lifetime Study of G-quadruplexes Single- and Double-labeled with Pyrene

We report steady state fluorescence and lifetime emission studies of d(GGTTGGTGTGGTTGG) (TBA) and d(GGGTTAGGGTTAGGGTTAGGG) (Htelom) oligonucleotides labeled with pyrene through a 3-aminopropyl linker. Such G-rich sequences are able to self-assemble into G-quadruplexes, especially in the presence of specific cations like potassium. A comparative studies with single- and double-labeled G-quadruplexes were carried out. For each probe we have measured fluorescence decays for emission wavelength of 390 and 480 nm in the varying concentration of potassium ion. We have calculated average lifetimes <τ> for every system as well as the fractional distribution α_i of emitting species.     

Photoisomerizable DNA Ligands. A Simple Method to Probe the Regions of AT-Rich Sequences

The dramatic effect of the microenvironment on the mechanism and quantum yield of photoisomerization have been widely evidenced; therefore, one can expect that the DNA matrix can modulate photoisomerization of properly designed ligands, and it may have an analytical importance.     

Fluorescence imaging of potassium ions in living cells using a fluorescent probe based on a thrombin binding aptamer-peptide conjugate

When a biotinylated FRET probe based on a peptide-thrombin binding aptamer conjugate was introduced together with streptavidin and biotinylated nuclear export signal peptide into HeLa cells, the resulting ternary complex enabled visualization of K(+) concentration changes in the cell.     

Extraction-spectrophotometric determination of lead in heavy metal salts with cryptand (2.2.2) and eosin

A rapid, extraction-spectrophotometric determination of trace amounts of lead in heavy metal salts is proposed. Owing to application of ammonia as masking agent, the extraction system cryptand (2.2.2)-eosin-chlorobenzene exhibits very high selectivity towards transition metal cations. Using standard addition method, lead is determined without matrix separation in nitrates, chlorides, acetates and sulphates of such metals as Ag, Cd, Ni, Cu and Zn. The effect of matrix on lead extraction efficiency and blank value is also discussed. The high sensitivity and selectivity of the proposed method allow to determine of lead at the level 10^–3–10^–4%.     

Synthesis and spectroscopic characterisation of (E)-2-(2-(9-(4-(1H-1,2,4-triazol-1-yl)butyl)-9H-carbazol-3-yl)vinyl)-3-ethylbenzo[d]thiazol-3-ium, a new ligand and potential DNA intercalator

Three new compounds based on carbazole planar skeleton were synthesised. Among them there is a new ligand and a potential DNA intercalator which contains a benzothiazolium moiety connected to the carbazole ring by a vinyl bridge. The absorption and emission spectral properties of this new ligand have been studied by spectroscopic methods.     

Differences in affinity of arylstilbazolium derivatives to tetraplex structures

Research into the interactions of small molecules (ligands) with DNA is a very important field of biochemistry. A ligand interacts with a DNA structure in many ways, depending on the structural features of the ligand (the presence of rings, substituent groups, length of bonds, etc.) or nucleic acid (number and association of strands, base sequence etc.). This study reports on an investigation of the preferential binding of arylstilbazolium ligands to a four-stranded DNA. For this purpose, an equilibrium dialysis was used. Equilibrium dialysis is a versatile method which enables many DNA structures to be investigated at the same time. A dozen different DNA structures of (single-stranded, double-stranded (duplex), triple-stranded (triplex), and four-stranded (tetraplex)) were involved in experiments with each ligand. Following the dissociation of DNA-ligand complexes by SDS, the concentration of the ligand bound was calculated from fluorescence and absorbance calibration curves. As a result, the amount of the ligand bound was directly related to the ligand-binding affinity. Equilibrium dialysis was used as a powerful tool to indicate which of the arylstilbazolium ligands investigated was the best therapeutic agent targeting G-quadruplex. Arylstilbazolium derivatives demonstrated strong interactions with the DNA samples used in the assay. The most interesting finding was a selective, preferential binding of anthryl derivative to c-MYC DNA (c-MYC is a DNA sequence that appears in an oncogene). Furthermore, as this derivative binds preferentially to one of the triplexes investigated, it can find an application in the TFO-triplex forming oligonucleotides which are used in gene therapy.     

Fluorogenic peptide sequences--transformation of short peptides into fluorophores under ambient photooxidative conditions

Long-lived proteins are susceptible to nonenzymatic chemical reactions and the evolution of fluorescence; however, little is known about the sequence-dependence of fluorogenesis. We synthesized a library of over half a million octapeptides and exposed it to light and air in pH 7.4 buffer to identify fluorogenic peptides that evolve under mild oxidative conditions. The bead-based peptide library was composed of the general sequence H(2)N-Ala-(Xxx)(6)-Ala-resin, where Xxx was one of nine representative amino acids: Asp, Gly, His, Leu, Lys, Pro, Ser, Trp, and Tyr. Next, we selected five highly fluorescent beads from the library and subjected them to microsequencing, revealing the sequence of the unreacted peptide. All five of the fluorogenic sequences were ionic; lacked Tyr, His, and Leu; and most of the sequences contained only one Trp. We then synthesized the five soluble peptides corresponding to the fluorogenic peptide sequences and exposed them to photooxidative conditions. In general, the soluble peptides reacted slowly, generating nonfluorescent monooxygenated and dioxygenated products. However, one peptide (H(2)N-AlaLysProTrpGlyGlyAspAla-CONH(2)) evolved into a highly fluorescent photoproduct as well as a nonfluorescent monooxygenated photoproduct. The fluorescent photoproduct consisted of a 2-carboxy-quinolin-4-yl moiety fused to the N-terminus of GlyGlyAspAla. The formation of this photoproduct requires cleavage of the peptide backbone and a dramatic reorganization of tryptophan. This work demonstrates that sequencing unreacted peptide on beads can reveal sequences with unique nonenzymatic reactivity. The study also confirms that peptide fluorogenesis is dependent on sequence and not merely on the presence of tryptophan. The potential importance of fluorogenic peptide sequences is two-fold. First, fluorogenic sequences that arise through mutation could prove to be hot spots for human aging. Second, fluorogenic sequences, particularly those compatible with intracellular conditions, may serve as fluorescent tags for proteins or as fluorescent biomaterials.