Double displacement: An improved bioorthogonal reaction strategy for templated nucleic acid detection

Abstract

Quenched autoligation probes have been employed previously in a target-templated nonenzymatic ligation strategy for detecting nucleic acids in cells by fluorescence. A common source of background signal in such probes is the undesired reaction with water and other cellular nucleophiles. Here, we describe a new class of self-ligating probes, double displacement (DD) probes, that rely on two displacement reactions to fully unquench a nearby fluorophore. Three potential double displacement architectures, all possessing two fluorescence quencher/leaving groups (dabsylate groups), were synthesized and evaluated for templated reaction with nucleophile (phosphorothioate) probes both in vitro and in intact bacterial cells. All three DD probe designs provided substantially better initial quenching than a single-Dabsyl control. In isothermal templated reactions in vitro, double displacement probes yielded considerably lower background signal than previous single displacement probes; investigation into the mechanism revealed that one dabsylate acts as a sacrificial leaving group, reacting nonspecifically with water, but yielding little signal because another quencher group remains. Templated reaction with the specific nucleophile probe is required to activate a signal. The double displacement probes provided a ca. 80-fold turn-on signal and yielded a 2-4-fold improvement in signal/background over single Dabsyl probes. The best-performing probe architecture was demonstrated in a two-color, FRET-based two-allele discrimination system in vitro and was shown to be capable of discriminating between two closely related species of bacteria differing by a single nucleotide at an rRNA target site.

Figure 1

Structures of the reactive parts of double displacement and control (single displacement) probes.

Figure 2. Fluorescence Timecourse of Double Displacement and Single-Dabsyl Ligation Reactions at 37°C

(A) Sequences of DNA probes and targets (T^f = fluorescein dT; Q = single-Dabsyl or Double Displacement linker; Nuc = phosphorothioate) (B) 100 nM of 1,3-Pentyl (blue), Isobutyl (green), 1,5-Pentyl (yellow) or single-Dabsyl (red) probe and 120 nM phosphorothioate probe were incubated with or without 100 nM template DNA at 37°C in pH 7.0 buffer containing 70 mM PIPES, 10 mM MgCl₂, 50 µM dithiothreitol. The background (no template) reactions for each probe are shown as broken lines. The fluorescence was measured every 120 s with 494 nm excitation and 525 nm emission. Reactions were repeated in triplicate; representative traces are shown.

Figure 3. Change in Signal:Background for Double Displacement and Single Dabsyl Probes Over Time

The signal/background ratios for the 1,3-Pentyl (blue), Isobutyl (green), 1,5-Pentyl (yellow) and single-Dabsyl (red) reactions over an eight hour time period. The reactions were run at 37°C. 100 nM quencher probe and 120 nM phosphorothioate probe were incubated with or without 100 nM template DNA at 25°C in pH 7.0 buffer containing 70 mM PIPES, 10 mM MgCl₂, 50 µM dithiothreitol. The fluorescence was measured every 120 s with 494 nm excitation and 525 nm emission. The data was smoothed using a 7-point running average.

Figure 4. Fold Change in Fluorescence Intensity Over Time

100 nM of 1,3-Pentyl (blue), Isobutyl (green), 1,5-Pentyl (yellow) or single-Dabsyl (red) probe and 120 nM phosphorothioate probe were incubated with 100 nM template DNA at 37°C in pH 7.0 buffer containing 70 mM PIPES, 10 mM MgCl₂, 50 µM dithiothreitol. The initial quenching was calculated before the phosphorothioate DNA was added to initiate the reaction. The fluorescence was measured every 120 s with 494 nm excitation and 525 nm emission. Reactions were repeated in triplicate and the traces shown represent the average fold change in fluorescence at each time point.

Figure 5. Monitoring Dabsylate Release in Double Displacement Reaction

The amount of free dabsylate released in solution, as monitored by HPLC from reactions containing 1 µM DD probe, 1 µM template strand and 1.2 µM phosphorothioate probe (blue) at 25°C. The DD probe and template with no nucleophile probe present is shown in pink.

Figure 6. Comparison of Double Displacement and Single Dabsyl Probes for Detecting Cellular 16S rRNA

The indicated double displacement or single-Dabsyl probe (200 nM), helper DNAs (3 µM) and 3’-phosphorothioate probes (2 µM) were incubated with E. coli cells in 6× SSC buffer with 0.05% SDS at 37°C for three hours. Images were taken with a black/white camera and false-colored green.

Figure 7. FRET probes for Two-color Detection Scheme

(A) Sequences of DNA probes and targets (T^f = fluorescein dT; DD = 1,3-pentyl linker; Nuc = phosphorothioate); (B) Normalized fluorescence emission spectra of a mixture of EC and SE probes incubated with 3’-phosphorothioate probe and either EC DNA or SE DNA. Conditions: 200 nM EC probe, 200 nM SE probe, 600 nM phosphorothioate DNA and 200 nM EC (green) or SE DNA (red). The reactions were incubated for 1 h at 37°C in pH 7.0 buffer containing 70 mM PIPES, 10 mM MgCl₂, 50 µM dithiothreitol. The excitation wavelength was 494 nm.

Figure 8. Two-color Discrimination of E. coli and S. enterica Based on a Single Nucleotide Polymorphism in the 16S rRNA

EC and SE double displacement probes (200 nM each), helper probes (3 µM) and 3’-phosphorothioate probes (2 µM) in 6× SSC + 0.05% SDS buffer were incubated in the indicated cell type or mixture of cells at 37°C for five hours. A B-2A filter was used for imaging. (a) E. Coli cells (b) E. Coli and S. enterica cells (c) S. enterica cells