Nucleic acid-based fluorescent probes and their analytical potential

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

Fluorescent sensing ochratoxin A with single fluorophore-labeled aptamer

“…Combining the aptamer affinity and easy signal transduction aptamer-based sensors have attracted wide and increasing interest [2][3][4]. In the aptamer-based sensors, the socalled signaling aptamers rely on the specific recognition of the targets by the aptamers and the binding-induced signal generations [5][6][7][8]. The signal responses of the signaling aptamers are unique properties of the aptamers.…”

“…The signal responses of the signaling aptamers are unique properties of the aptamers. The generated signals can be electrochemical, colorimetric, or fluorescent output [3][4][5][6][7][8]. By using fluorophores on aptamers, the fluorescent signaling aptamers exhibited benefits in sensitivity, simplicity, convenience of detection, diverse detection approaches, and usual homogeneous binding process [5][6][7][9][10][11][12][13][14].…”

“…The generated signals can be electrochemical, colorimetric, or fluorescent output [3][4][5][6][7][8]. By using fluorophores on aptamers, the fluorescent signaling aptamers exhibited benefits in sensitivity, simplicity, convenience of detection, diverse detection approaches, and usual homogeneous binding process [5][6][7][9][10][11][12][13][14]. Usually, fluorescent dyes can be readily introduced on the ends of the aptamers through chemical synthesis to fabricate the aptamer switch [5][6][7][9][10][11][12][13][14].…”

“…By using fluorophores on aptamers, the fluorescent signaling aptamers exhibited benefits in sensitivity, simplicity, convenience of detection, diverse detection approaches, and usual homogeneous binding process [5][6][7][9][10][11][12][13][14]. Usually, fluorescent dyes can be readily introduced on the ends of the aptamers through chemical synthesis to fabricate the aptamer switch [5][6][7][9][10][11][12][13][14]. It is also feasible to conjugate fluorophores on the internal bases or to use the fluorescent analogue of the bases in oligonucleotides for sensing [5][6][7][15][16][17][18].…”

“…Usually, fluorescent dyes can be readily introduced on the ends of the aptamers through chemical synthesis to fabricate the aptamer switch [5][6][7][9][10][11][12][13][14]. It is also feasible to conjugate fluorophores on the internal bases or to use the fluorescent analogue of the bases in oligonucleotides for sensing [5][6][7][15][16][17][18].…”