Multi-path quenchers: efficient quenching of common fluorophores

Abstract

Fluorescence quenching groups are widely employed in biological detection, sensing, and imaging. To date, a relatively small number of such groups are in common use. Perhaps the most commonly used quencher, dabcyl, has limited efficiency with a broad range of fluorophores. Here, we describe a molecular approach to improve the efficiency of quenchers by increasing their electronic complexity. Multi-Path Quenchers (MPQ) are designed to have multiple donor or acceptor groups in their structure, allowing for a multiplicity of conjugation pathways of varied length. This has the effect of broadening the absorption spectrum, which in turn can increase quenching efficiency and versatility. Six such MPQ derivatives are synthesized and tested for quenching efficiency in a DNA hybridization context. Duplexes placing quenchers and fluorophores within contact distance or beyond this distance are used to measure quenching via contact or FRET mechanisms. Results show that several of the quenchers are considerably more efficient than dabcyl at quenching a wider range of common fluorophores, and two quench fluorescein and TAMRA as well as or better than a Black Hole Quencher.

Figure 1

Structures of Multiple Pathway Quenchers (MPQs) and commercially available quenchers used in this study.

Figure 2

Absorption spectra (normalized to absorbance at 260nm) of quenchers on (a) 20mer and (b) 15mer oligonucleotide sequences. MPQ1 (), MPQ2 (), MPQ3 (), MPQ4 (), MPQ5 (), MPQ6 (), Dabcyl (), BHQ2 ().

Figure 3

DNA hybridization formats used for determining (a) contact quenching and (b) mixed mechanism quenching and (c) FRET quenching efficiencies of MP quenchers.

Figure 3

Fluorescence quenching ratios for contact () and mixed mechanism () quenching with (a) AlexaFluor 350, (b) Fluorescein, (c) Cy 3, (d) TAMRA, (e) ATTO 590 and (f) Quasar 670.

Scheme 1

Synthesis of Multiple Pathway Quenchers. a) β-alanine, EtOH, (73%). b) SnCl₂, MeOH, HCl, (71%). c) i) NaNO₂, H₂SO₄ ii) aniline derivative iii) NaOAc (20-62%). d) KOTMS, THF (36-87%). e) i) NaNO₂, HCl ii) 1H-perimidine, NaOAc. f) 5-hydroxy-2-pentanone, pTsOH (43%). g) Fast Corinth V, 1:1 THF:acetone, 0°C (14%). h) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIPEA.