Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes

Abstract

Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore, rapid, cost-effective and reliable detection of mutations is an object of extensive research. Today, Förster resonance energy transfer (FRET) probes are among the most often used tools for the detection of nucleic acids and in particular, for the detection of mutations. However, multiple parameters must be taken into account in order to create efficient FRET probes that are sensitive to nucleic acid mutations. In this review; we focus on the design principles for such probes and available computational methods that allow for their rational design. Applications of advanced, rationally designed FRET probes range from new insights into cellular heterogeneity to gaining new knowledge of nucleic acid structures directly in living cells.

Keywords: FRET; binary probe; computational strategies; fluorescence; in vitro hybridization; molecular beacon; mutation; nucleic acid.

Figure 1

Main parameters of FRET: (a) Spectral overlap, J, of donor emission and acceptor absorption (necessary for FRET); (b) Transition dipole orientation of the donor D and the acceptor A.

Figure 2

Design of FRET probes for nucleic acid detection: molecular beacons (a); binary probes (b); RNA aptamer labelled with fluorophore and quencher (shown as a star and cloud, respectively) (c).

Figure 3

Mismatch sensitive FRET probe designs [61,62,63,64,65]: Invader probes (a); fluorescently labelled triphosphate terminators (b) and duplex probes (c). WT = wild type; MUT = mutant.

Figure 4

Chemical structures of synthetic oligonucleotides containing modified backbones.

Figure 5

SSchematic representation of allele-specific PCR using FRET probes. WT = wild-type (a) and MUT = mutant (b) targets; Pol = polymerase. Signal is increased upon amplification of the specific allele.

Figure 6

Computational design of new FRET dyes: (a) Rhodamine undergoes a very characteristic absorption change between the ring opening and the ring-closing conformation. Yuan et al. improve this mechanism for imagining of small molecular targets such as Cu²⁺, NO, HOCl by separation of the interaction site (denoted x) and the energy donor [103]; (b) Quadracyclic adenine analogues with the different substituents, R, was introduced by Larsen et al. Adenine analogues substituted at position 1 and 2 with cyanogroups showed a stable fluorescence quantum yield and environment-sensitive emission. Both properties make them suitable for monitoring nucleic acids systems [104]. R: fluorine-, methoxy- and cyanogroups; (c) Molecular structure of flavin mononucleotide (FMN) [105].