Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy

Abstract

We report here the design, synthesis and application of pyrene binary oligonucleotide probes for selective detection of cellular mRNA. The detection strategy is based on the formation of a fluorescent excimer when two pyrene groups are brought into close proximity upon hybridization of the probes with the target mRNA. The pyrene excimer has a long fluorescence lifetime (>40 ns) compared with that of cellular extracts (approximately 7 ns), allowing selective detection of the excimer using time-resolved emission spectra (TRES). Optimized probes were used to target a specific region of sensorin mRNA yielding a strong excimer emission peak at 485 nm in the presence of the target and no excimer emission in the absence of the target in buffer solution. While direct fluorescence measurement of neuronal extracts showed a strong fluorescent background, obscuring the detection of the excimer signal, time-resolved emission measurements indicated that the emission decay of the cellular extracts is approximately 8 times faster than that of the pyrene excimer probes. Thus, using TRES of the pyrene probes, we are able to selectively detect mRNA in the presence of cellular extracts, demonstrating the potential for application of pyrene excimer probes for imaging mRNAs in cellular environments that have background fluorescence.

Figure 1

Binary pyrene probes in the absence of target (a) and after hybridizing with target (b).

Figure 2

Parameters for the optimization of excimer emission in Py-BP: probe sequences (a), target sequences (b) and pyrene linkers used to optimize Py-BP excimer emission (c). Monomer to excimer ratio for the different probes studied with (red) and without (blue) target (d); emission spectra of P₁P₂L₆ with (red) and without (blue) target (T₀) (e). [Probes] = 0.1 µM; Buffer: 20 mM Tris–HCl (pH 7.5), 500 mM NaCl, 60 mM MgCl₂; hybridization time = 30 min.

Figure 3

Pyrene probe pairs Py-BP-R2 (a), region of sensorin mRNA target sequence complementary to Py-BP-R2 (b) and Py-BP-R2 hybridized with target mRNA (c).

Figure 4

Steady-state fluorescence spectra of Py-BP-R2 in buffer solution (a) and cellular protein extract (b). [Py-BP-R2] = 0.1 µM; Buffer: 10 mM Tris (pH 7.5), 400 mM NaCl; hybridization time = 2 h; normalized at 400 nm.

Figure 5

Time-resolved fluorescence spectra of Py-BP-R2 with and without target in neuronal protein extract, gating from 0–300 ns (a) or from 30–150 ns (b). Fluorescence decay of Py-BP-R2 (λ_em = 398 nm) showing the signal gating for the spectra in (a) (c) and in (b) (d). [Py-BP-R2] = 0.1 µM; hybridization time = 30 min.