Phosphate-perylene modified G-quadruplex probes for the detection of Pb²⁺ using fluorescence anisotropy

Abstract

A simple and universal phosphate-perylene modification strategy was applied in the development of G-quadruplex probes with thrombin binding aptamer (TBA) and [d(TGGGT) 4 ] (G4) sequences. A perylene moiety was inserted at different phosphate positions of oligonucleotides without a significant effect on the G-quadruplex structures. Upon binding with K + or Pb 2+ , these probes showed different perylene fluorescence anisotropy responses due to the different labeling positions and G-quadruplex structures.Two probes (G4-9 and TBA-9) were successfully used in Pb 2+ detection through fluorescence anisotropy. Once the complexes of Pb 2+ with G4-9 or TBA-9 were formed, the rotational diffusion of the perylene moiety was limited, resulting in a significant increase in fluorescence anisotropy. Both probes showed good sensitivity to Pb 2+ and their fluorescence anisotropy signals demonstrated good linear responses to the logarithm of Pb 2+ concentrations and the detection limits were 24.5 nM and 30.0 nM for TBA-9 and G4-9, respectively.

On-Strand Knoevenagel Insertion of a Hemicyanine Molecular Rotor Loop Residue for Turn-On Fluorescence Detection of Pb-Induced G-Quadruplex Rigidity

“…Under similar conditions, Smirnov and Shafer reported a K d for Pb 2+ -binding by native TBA ∼100 nM or lower from a CD titration in the absence of coexisting cations in 10 mM Tris-acetate pH 6.8, 10 which is comparable with our K d value for Pb 2+ -binding by TBA-4PI[T3]. Using fluorescence anisotropy measurements in 10 mM Tris-acetate pH 7.5, Wang and co-workers obtained an LOD of 24.5 nM for TBA phosphate linked to perylene at T9, 22 which is also comparable to our LOD value of 19 nM using the molecular rotor 4PI surrogate at T3 of TBA. The US EPA has set a maximum contaminant level (MCL) of 70 nM for lead in drinking water.…”

“…It is well established that Pb 2+ templates have a smaller, more compact, and rigid antiparallel GQ structure than does K + . Wang and co-workers have taken advantage of this property and covalently attached a perylene group to the phosphates of GQ structures for monitoring Pb 2+ -binding using fluorescence anisotropy due to the restricted rotation of the perylene moiety upon Pb 2+ -induced GQ-folding . A lead ion also more strongly stabilizes H-bonds through polarization enhancement (Figure B), with a diagnostic long-wavelength circular dichroism (CD) maximum at ∼310 nm rather than ∼290 nm for monocationic antiparallel GQs .…”

“…21 Wang and co-workers have taken advantage of this property and covalently attached a perylene group to the phosphates of GQ structures for monitoring Pb 2+ -binding using fluorescence anisotropy due to the restricted rotation of the perylene moiety upon Pb 2+ -induced GQ-folding. 22 A lead ion also more strongly stabilizes H-bonds through polarization enhancement (Figure 1B), 21 with a diagnostic long-wavelength circular dichroism (CD) maximum at ∼310 nm rather than ∼290 nm for monocationic antiparallel GQs. 10 This feature has been employed by Kim using the 15mer thrombin binding aptamer (TBA, 5′-G 2 T 2 G 3 TGTG 2 T 2 G 2 -3′) as a CD-specific label-free sensor.…”

In What Ways Do Synthetic Nucleotides and Natural Base Lesions Alter the Structural Stability of G-Quadruplex Nucleic Acids?

“…The TBA and its analogs displayed positive peaks near 245 and 295 nm and a negative one near 265 nm, typical of the antiparallel GQ structures. In the presence of Pb ++ , the positive peaks of 265 nm and 295 nm of the structures were red-shifted, indicating the Pb ++ -stabilized antiparallel GQs in both scaffolds [ 60 ].…”

Recent advances in fluorescence anisotropy/polarization signal amplification

“…3,9 Third, because of its simplicity and speed, FA/FP assays are especially suitable for an automated high throughput format assay. 10–13…”