Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer.

Abstract

Three approaches were used to study hybridization of complementary oligodeoxynucleotides by nonradiative fluorescence resonance energy transfer. (i) Fluorescein (donor) and rhodamine (acceptor) were covalently attached to the 5' ends of complementary oligodeoxynucleotides of various lengths. Upon hybridization of the complementary oligodeoxynucleotides, energy transfer was detected by both a decrease in fluorescein emission intensity and an enhancement in rhodamine emission intensity. In all cases, fluorescein emission intensity was quenched by about 26% in the presence of unlabeled complement. Transfer efficiency at 50C decreased from 0.50 to 0.22 to 0.04 as the distance between donor and acceptor fluorophores in the hybrid increased from 8 to 12 to 16 nucleotides. Modeling of these hybrids as double helices showed that transfer efficiency decreased as the reciprocal of the sixth power of the donor-acceptor separation R, as predicted by theory with a corresponding Ro of 49 A. (u) Fluorescence resonance energy transfer was used to study hybridization of two fluorophore-labeled oligonucleotides to a longer, unlabeled oligodeoxynucleotide. Two 12-mers were prepared that were complementary to two adjacent sequences separated by four bases on a 29-mer. The adjacent 5' and 3' ends of the two 12-mers labeled with fluorescein and rhodamine exhibited a transfer efficiency of -0.60 at 50C when they both hybridized to the unlabeled 29-mer. (Wi) An intercalating dye, acridine orange, was used as the donor fluorophore to a single rhodamine covalently attached to the 5' end of one oligodeoxynucleotide in a 12-base-pair hybrid. Under these conditions, the transfer efficiency was =0.47 at 50C. These results establish that fluorescence modulation and nonradiative fluorescence resonance energy transfer can detect nucleic acid hybridization in solution. These techniques, with further development, may also prove useful for detecting and quantifying nucleic acid hybridization in living cells.In this paper we describe how fluorescently labeled oligodeoxynucleotides (ODNTs) and the process of nonradiative fluorescence resonance energy transfer (FRET) can be used to study nucleic acid hybridization. When two fluorophores whose excitation and emission spectra overlap are in sufficiently close proximity, the excited-state energy of the donor molecule is transferred by a resonance dipole-induced dipole interaction to the neighboring acceptor fluorophore. The results are a decrease in donor lifetime, a quenching of donor fluorescence, an enhancement of acceptor fluorescence intensity, and a depolarization of fluorescence intensity. The efficiency of energy transfer, Et, falls off rapidly with the distance between donor and acceptor molecule, R, and is expressed as Et = 1/[1 + (R/R0)6], [1] where Ro is a value that depends upon the overlap integral of the donor emission spectrum and the acceptor excitation spectrum, the index of refraction, the quantum yield of the donor, and the orientation of the donor emission and the acce...

Single-Cell Gene Expression Analysis: Implications for Neurodegenerative and Neuropsychiatric Disorders

“…First, DNA polymerases including Taq polymerase possess 5Ј to 3Ј exonuclease activity (48). Second, the principle of fluorescence resonance energy transfer (FRET) (49) can be applied to double-labeled oligonucleotide probes (e.g., PCR and/or RT-PCR primers). In a double-labeled oligonucleotide probe system (ideal for qRT-PCR), FRET occurs when fluorescence energy of a reporter dye is quenched by close proximity of the quencher dye.…”

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

“…However, this achievement also poses a challenge for the development of new and improved methods for the detection and monitoring of DNA or RNA either in vitro or in vivo. Different approaches have been taken in the design of oligonucleotide probes for DNA and RNA detection including molecular beacons (MBs), [5][6][7][8] binary probes (BPs), [9][10][11][12][13] and quenched autoligation probe pairs (QUAL) [14][15][16] . Other probes like scorpion primers, 17 5′-nuclease probes, 18 and cyclicons 19 have been used in vitro to detect amplification products in polymerase chain reactions (PCR).…”

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“…9 BPs rely mostly on FRET for target detection. [9][10][11][12] In general, one of the sequences in the BP contains a donor fluorophore, while the second sequence should have an acceptor fluorophore, as shown schematically in Figure 1d. In the absence of the target, the probes are dispersed in solution and no FRET is observed.…”

Triple-FRET Technique for Energy Transfer Between Conjugated Polymer and TAMRA Dye with Possible Applications in Medical Diagnostics

“…In order that FRET may be efficient, it is necessary that there should be maximum overlap between the PL spectrum of the donor and absorption spectrum of the acceptor [3][4][5]. Also, the distance between the acceptor and the donor must be within the Förster radius.…”