Fine Tuning of Pyrene Excimer Fluorescence in Molecular Beacons by Alteration of the Monomer Structure

Abstract

Oligonucleotide probes labeled with pyrene pairs that form excimers have a number of applications in hybridization analysis of nucleic acids. A long excited state lifetime, large Stokes shift, and chemical stability make pyrene excimer an attractive fluorescent label. Here we report synthesis of chiral phosphoramidite building blocks based on (R)-4-amino-2,2-dimethylbutane-1,3-diol, easily available from an inexpensive d-(-)-pantolactone. 1-Pyreneacetamide, 1-pyrenecarboxamide, and DABCYL derivatives have been used in preparation of molecular beacon (MB) probes labeled with one or two pyrenes/quenchers. We observed significant difference in the excimer emission maxima (475-510 nm; Stokes shifts 125-160 nm or 7520-8960 cm) and excimer/monomer ratio (from 0.5 to 5.9) in fluorescence spectra depending on the structure and position of monomers in the pyrene pair. The pyrene excimer formed by two rigid 1-pyrenecarboxamide residues showed the brightest emission. This is consistent with molecular dynamics data on excimer stability. Increase of the excimer fluorescence for MBs after hybridization with DNA was up to 24-fold.

Structure and piezochromism of pyrene-1-carbaldehyde at high pressure

“…Polycyclic aromatic hydrocarbons (PAHs), including derivatives of pyrene, have for some time been attracting attention due to their potential applications as optoelectronic materials (Anthony, 2008;Figueira-Duarte & Mü llen, 2011) or luminescent markers (Faggi et al, 2016;Aparin et al, 2017) as well as due to the relative ease of chemical modification. Their ability to form aggregates, ranging from -stacked dimers to more intricate arrangements (Hinoue et al, 2012) both in solution and in the solid state, is vital to many of these applications, together with their general sensitivity to environment (Petaccia et al, 2016;Niko et al, 2012), which manifests for instance in varying spectroscopic properties.…”

Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides

“…Due to the remarkable property of pyrene fluorophores to form excimers and exciplexes with pronounced changes in output fluorescence signals, pyrene-modified MBs have been well developed. To obtain MBs with improved emission characteristics and specificity, several interesting design decisions have been proposed (see, for instance, [ 29 , 30 , 66 , 85 , 92 , 94 , 95 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 ]; some of them are illustrated in Figure 9 ), among them are excimer-forming MBs containing a quencher (for instance, types MB1a [ 85 , 100 , 105 ], MB1b [ 105 ], MB1c and MB1d [ 100 ], and MB2 [ 30 ]) and numerous quencher-free MBs (for instance, types MB3 [ 66 ], MB4 [ 94 ], MB5 [ 29 , 101 ], MB6 [ 109 ], MB7 [ 106 , 108 ], and MB8 [ 107 ]), including systems that utilize γ-cyclodextrin or hybridization chain reaction-mediated signal amplification (types MB9 [ 95 , 111 ] and MB10 [ 104 ]).…”

“…Recently, Korshun and coauthors developed a number of new MBs (types MB1a and MB1b ) labeled with one or two non-nucleoside pyrene monomers P1 and/or P2 and one or two moieties of Dabcyl quencher (units Dabcyl and Double Dabcyl ) [ 105 ] ( Figure 10 ).…”

“…They have found that the use of Double Dabcyl quencher does not result in any benefit in fluorescence properties of the MBs; in contrast, in some cases the excimer fluorescence signal was even higher than for the single Dabcyl quencher. Excimer fluorescence maxima of the MBs with different compositions of pyrene units varied from 475 nm to 510 nm; at that, the probes displayed a high excimer/monomer emission ratio and a considerable increase in excimer fluorescence upon hybridization to a complementary DNA target [ 105 ].…”

“… Structures of non-nucleoside pyrene units P1 and P2 and quencher units Dabcyl and Double Dabcyl [ 105 ] . …”