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Sondy fluorescencyjne typu „light up” do detekcji i obrazowania G-kwadrupleksów in vitro i in vivo. Cz. 1 i 2

Baranowski Daniel

Wiadomości Chemiczne

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2020

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[Z] 74, 11-12

883--901

EN

G-quadruplexes are non-canonical guanosine rich four stranded nucleic acids structures consisting of at least two or more G-tetrads stabilized by an array of Hoogsteen hydrogend bonds and monovalent cations. The distinguishing feature of the G-quadruplexes is their high thermal stability and structural polymorphism in aqueous media. In parallel, a great number of GQ structures have been extensively surveyed ex vivo by means of biophysical techniques such as nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and X-ray crystallography. Accumulating evidence suggesting that G-quadruplexes play essential role in a numerous biological processes in vivo, including DNA replication and transcription, RNA translation as well as genomic maintenance. Consequently, G-quadruplexes has attracted attention as therapeutic targets in cancer or hereditary diseases as well as molecular target in cellular biology. Study on G-quadruplexes:ligand interaction by NMR, CD, UV and fluorescence spectroscopy in vitro or in vivo has become an intensive research work area of many groups in recent years. Nowadays, there are available large amount of organic compounds that selectively bind to G-quadruplexes and their photophysical and kinetic properties were comprehensively characterized but only few of them are endowed with fluorescence properties that could be applicable as fluorescent probes in cellular biology or in vitro detection. Interestingly, the group of these fluorescent probes is characterized by a vast structural diverisity resulting from the different way of interaction with G-quadruplexes as well as G-quadruplex polymorphism. This review focuses on the G-quadruplex-selective light up fluorescent probes that have been employed for in vitro detection as well as cellular imaging along with a summary of the key photophysical, biophysical, and biological properties of reported examples.

2

Dializa równowagowa. Metoda badania selektywności oddziaływań ligand.DNA

Czerwińska I., Głuszyńska A., Juskowiak B.

Wiadomości Chemiczne

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2010

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[Z] 64, 1-2

105-122

EN

Equilibrium or competition dialysis is a powerful tool for binding study of ligands that are expected to bind to nucleic acids with selectivity related to their structure or sequence. In the equilibrium dialysis experiment, a set of nucleic acid samples that differ in structure and sequence is dialyzed against a test ligand solution. After equilibration, the concentration of ligand bound to each structure or sequence is determined by UV-Vis absorption or fluorescence spectroscopy in each dialysis unit. Since all nucleic acid samples are in equilibrium with the same free ligand concentration, the amount of bound ligand is directly related to the ligand binding affinity. Thus, equilibrium provides a direct measure of selectivity and identifies the nucleic acid sample, which is preferred by a particular ligand. We describe here the principles and practice of the method. Examples of an application of the method are limited to the discovery of small molecules that selectively recognize the unique structural features of G-quadruplexes. There are proofs for important functional roles of G-quadruplex structures in biology (maintenance of telomeres, transcriptional regulation, and modulation of mRNA translation). G-quadruplex DNA can exist in a variety of structural forms that may possess numerous potential binding sites for small molecules. Therefore equilibrium dialysis provides a useful tool for discovery of new mall-molecule therapeutic agents targeting G-quadruplexes.

3

Procesy przenoszenia energii (FRET) w badaniach czteroniciowych form DNA

Juskowiak B., Gałęzowska E., Paczesny J.

Wiadomości Chemiczne

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2006

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[Z] 60, 7-8

547-566

EN

DNA sequences with stretches of multiple guanines can form four-stranded tetraplex DNA structures called guanine-quadruplexes or G4 DNA. They contain stacked arrays of planar guanine quartets linked by connecting loops. Evidence supports the occurrence of quadruplexes in the cell nucleus and a number of biological functions have been attributed to quadruplexes. In eukaryotic systems guanine-rich sequences are positioned at the ends of chromosomes and are called tclomcric DNA. The study of telomeric DNA has acquired importance through the discovery of the relationship between telomerase activity in most cancer cells and telomere folding into tetraplex structure. Coordination of certain metal cations stabilizes G-quadruplex as do some promising small organic molecules, which are regarded as potential anticancer drugs. Among many techniques employed to explore properties of G-quadrupIexes, fluorescence resonance energy transfer (FRET) has been recognized as a powerful tool to explore tetraplex formation due to extreme sensitivity and the distance dependency of the FRET process. This review shows how FRET contributes to G-quadruplex research and focuses on the FRET application, describing briefly also the underlying principles. Information about molecular structure, binding events, and motion are considered to be potentially available from FRET measurements. In a typical FRET experiment a guanine-rich oligonucleotide labeled with proper fluorophores (FRET donor and acceptor) undergoes structural transformations (folding or unfolding), which are monitored by spectral changes in the fluorescence spectra of FRET partners. We tried to summarize the current developments in the field of the various applications of FRET measurements for the fundamental structural and kinetic investigations of G-quadru-plexes and their complexes with metal cations and organic ligands. Fundamental applications include studies of quadruplex unfolding kinetics with the use of complementary DNA or PNA (Peptide Nucleic Acid) strands as a duplex trap or determination of thermodynamic parameters. Practical applications are illustrated by the FRET-based selection of quadruplex-binding ligands, construction of the quadruplex-based nanomotor, design of molecular probes for protein recognition, and development of sensors for the detection of potassium ions in aqueous media. The presented examples of FRET studies showed that FRET is particularly use ful in structural studies of oligonucleotides capable of folding into tetraplex structure.