No abstract

It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays.FigureHybridization with a nucleic acid target or affinity interactions with a nonnucleic acid target generate changes in the fluorescence characteristics of a nucleic acid-based fluorescent probe

Ion probes: A potassium‐sensing oligonucleotide with terminal pyrene moieties can be used as a fluorescent probe for the real‐time monitoring of the extracellular concentration of K+ ions under physiological conditions. The excimer emission intensity (b) of the chair‐type quadruplex structure formed depends on the K+ ion concentration (0–200 mm), and differs significantly from that in the absence of potassium (a).

The dual-labeled oligonucleotide derivative, FAT-0, carrying 6- carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) labels at the 5' and 3' termini of the thrombin-binding aptamer (TBA) sequence 5'-GGT TGG TGT GGT TGG-3', and its derivatives, FAT-n (n=3, 5, and 7) with a spacer at the 5'-end of a TBA sequence of T(m)A (m=2, 4, and 6) have been designed and synthesized. These fluorescent probes were developed for monitoring K(+) concentrations in living organisms. Circular dichroism, UV-visible absorption, and fluorescence studies revealed that all FAT-n probes could form intramolecular tetraplex structures after binding K(+). Fluorescence resonance energy transfer and quenching results are discussed taking into account dye-dye contact interactions. The relationship between the fluorescence behavior of the probes and the spacer length in FAT-n was studied in detail and is discussed.

Ionensonde: Ein Kalium‐empfindliches Oligonucleotid mit terminalen Pyreneinheiten kann als Fluoreszenzsonde für die Verfolgung der extrazellulären Konzentration von K+‐Ionen in Echtzeit unter physiologischen Bedingungen genutzt werden. Die Intensität der Excimer‐Emission (b) der gebildeten stuhlförmigen Quadruplexstruktur hängt von der K+‐Ionenkonzentration (0–200 mM) ab und unterscheidet sich deutlich von der ohne Kalium gemessenen Intensität (a).