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.

Figure

Hybridization 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

Fig. 1

Schematic layout of a fluorescent biosensor

Fig. 2

Schematic representation of nucleic acid hybridization probes: quenching-type binary probe (a), FRET-type binary probe (b), quenching-type molecular beacon (c), and FRET-type molecular beacon (d)

Fig. 3

Schematic representation of TacMan hydrolysis probe (a) and fluorescent primers Scorpions (b) and Amplifluor (c) used for monitoring QPCR amplification progress

Fig. 4

Structure of G-tetrad showing hydrogen bonds between four guanines and the interactions with a cation (a) and schematic representation of G-quadruplex structures: an antiparallel “chair-type” G-quadruplex with all lateral loops (b), an antiparallel “basket-type” G-quadruplex with one diagonal and two lateral loops (c), a hybrid-type quadruplex with parallel-antiparallel loops orientation (d), and a parallel quadruplex with all loops positioned alongside the grooves (e)

Fig. 5

Schematic representation of generation of fluorescence with different aptamer affinity probes

Fig. 6

Schematic representation of generation of fluorescence with aptazyme sensors for ATP (a) and Pb²⁺ ion (b)

Fig. 7

Schematic illustration of strategies used to develop oligonucleotide sensors for potassium ion: a FRET-based probe (a), an excimer emission sensor based on pyrene-labeled thrombin binding aptamer (b), and a FRET probe sensitive to the electrostatic interactions between cationic conjugated polymer (CCP) and G-quadruplex labeled with fluorescein (c)