Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes

Abstract

An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is the efficiency of energy transfer between the fluorophore and quencher used to label the probes. We have determined the efficiency of energy transfer for a large number of combinations of commonly used fluorophores and quenchers. We have also measured the quenching effect of nucleotides on the fluorescence of each fluorophore. Quenching efficiencies were measured for both the resonance energy transfer and the static modes of quenching. We found that, in addition to their photochemical characteristics, the tendency of the fluorophore and the quencher to bind to each other has a strong influence on quenching efficiency. The availability of these measurements should facilitate the design of oligonucleotide probes that contain interactive fluorophores and quenchers, including competitive hybridization probes, adjacent probes, TaqMan probes and molecular beacons.

Figure 1

Structure of oligodeoxyribonucleotide hybrids used to measure the efficiencies of quenching. (A) Blunt-ended hybrids that place the fluorophore (F) and the quencher (Q) so close together that contact quenching occurs. (B) Staggered hybrids that place the fluorophore and quencher at a distance at which FRET is the predominant mode of quenching. (C) Hybrids used to study the quenching effect of nucleotides (N).

Figure 2

Spectral characteristics of hybrids with an overhang of 10, 5 or 0 nt. (A) Emission spectra of the TET-labeled oligodeoxyribonucleotides alone (dashed lines) and emission spectra of the hybrids they form with the TMR-labeled oligodeoxyribonucleotide (solid line), when they are both stimulated by 522 nm light, which is the optimal excitation wavelength for TET. A dotted line shows the emission spectrum of the TMR-labeled oligodeoxyribonucleotide alone, when stimulated by 555 nm light, which is the optimal excitation for TMR. Dashed arrows indicate the magnitude of the decrease in TET fluorescence that occurs when the TET-labeled strands are hybridized to the TMR-labeled strand, and solid arrows indicate the magnitude and direction of the change in TMR fluorescence that occurs when the TET-labeled strands are hybridized to the TMR-labeled strand. (B) Solid lines show the absorption spectra of the hybrids and dotted lines show the absorption spectra obtained by combining the absorption spectra of the individual oligodeoxyribonucleotides.

Figure 3

Absorption spectra of each of the five quencher-labeled oligodeoxyribonucleotides normalized for absorption at 260 nm (which is due to the nucleotides), in order to compare the absorption spectra of the quenchers.

Figure 4

Determination of the melting temperature of a hybrid possessing a 5 nt overhang that was labeled with FAM and dabcyl. The fluorescence of the hybrid (open circles) was divided by the fluorescence of the FAM-labeled oligodeoxyribonucleotide alone (open squares), which decreases as the temperature is increased, in order to obtain the corrected fluorescence of the hybrid as a function of temperature (filled circles). The corrected values were used to calculate the melting temperature of the hybrid.

Figure 5

Correlation between quenching efficiency and melting temperature for blunt-ended hybrids containing different fluorophore–quencher pairs. Even though all of the hybrids were formed from the same complementary oligodeoxyribonucleotides, they exhibited different melting temperatures, due to the degree of attraction or repulsion of the fluorophore and the quencher.