Molecular Beacon DNA Probes with Fluorescein Bifluorophore

Abstract

An azido-derivative of a fluorescein bifluorophore was obtained and used for the synthesis of "molecular beacon"-type oligonucleotide fluorogenic probes for RT-PCR. Eight probe variants were synthesized based on an optimized sequence: with one or two quencher residues at the 3'-end, with a single or bifluorophore fluorescein label attached to 5'-end using modifying phosphoramidites (short linker) or "click reaction" (long linker). Comparison of probes in RT-PCR showed that probes with a doubled quencher (single fluorescein on a short linker) and doubled dye on a short linker (single dye) are somewhat superior in sensitivity to a standard probe (single quencher, single dye on a short linker) by the value of ΔC_t = 1-2.

Supplementary information: The online version contains supplementary material available at 10.1134/S1068162021030055.

Keywords: 3,5-diaminobenzoic acid; 5-carboxyfluorescein; fluorescence quenching; fluorogenic DNA probes; real-time qPCR.

Fig. 1.

The principle of operation of a fluorogenic molecular beacon DNA probe. F, fluorescent dye; Q, quencher.

Fig. 2.

Synthesis of oligonucleotide probes with 5-FAM-bifluorophore. Pv, pivaloyl; Pfp, pentafluorophenyl; FAM, 5-FAM fluorophore. (a) Probe synthesis schemes MB3 and MB4 using the amidophosphite method; (b) synthesis of the azido derivative (V) and probes MB7 and MB8; (c) the main components of the probe, Q, quencher; L, linker; B, the branching fragment based on 3,5-diaminobenzoic acid. The sequence of the probe complementary to the target is underlined; the fragments that form the stem of the hairpin are highlighted in bold; (d) detected sequence of a 290-bp fragment of the gene encoding translation elongation factor 1α of Fusarium avenaceum. Areas complementary to primers Fat65R and Fat65F are underlined; the section of the gene that binds to probes MB1–MB8 is highlighted in bold.

Fig. 3.

Fluorescence enhancement profiles in quantitative RT-PCR; fluorescence detection at 55°C. (a) Detection of different numbers of target molecules using probe MB1; (b) comparison of probes MB1–MB8 in RT-PCR with 2.7 × 10⁷ (left) and 2.7 × 10⁸ target molecules (right).