Protein-Induced Fluorescence Enhancement and Quenching in a Homogeneous DNA-Based Assay for Rapid Detection of Small-Molecule Drugs in Human Plasma

Abstract

Homogeneous assays for determining the concentration of small molecules in biological fluids are of importance for monitoring blood levels of critical drugs in patients. We have developed a strand displacement competition assay for the drugs dabigatran, methotrexate, and linezolid, which allows detection and determination of the concentration of the drugs in plasma; however, a surprising kinetic behavior of the assay was observed with an initial rapid change in apparent FRET values. We found that protein-induced fluorescent enhancement or quenching (PIFE/Q) caused the initial change in fluorescence within the first minute after addition of protein, which could be exploited to construct assays for concentration determination within minutes in the low nanomolar range in plasma. A kinetic model for the assay was established, and when taking the new finding into account, the in silico simulations were in good agreement with the experimentally observed results. Utilizing these findings, a simpler assay was constructed for detection of dabigatran, which allowed for detection within minutes without any time dependencies.

A new twist on PIFE: photoisomerisation-related fluorescence enhancement

“…From an applicative perspective, the utilisation of PIFE in various emerging platforms to tackle previously unexplored problems is experiencing a clear surge. These platforms encompass a wide range of innovative techniques, such as smPIFE with continuous repositioning of single molecules within an anti-Brownian electrokinetic trap [111], the integration of smPIFE with force spectroscopy [112], the application of PIFE for investigating liquid-liquid phase separation of the RNA-binding protein FUsed in Sarcoma (FUS) [113], as well as bio-detection and sensing applications [114,115], including the introduction of aptamer-based PIFE [116,117]. Undoubtedly, this surge in the adoption of smPIFE points towards a promising future of PIFE in the realms of biomolecular science and biomedical application development.…”

Fluorescent Investigation of Proteins Using DNA-Synthetic Ligand Conjugates

“…Intriguingly, a recent study from the same group challenged the mechanistic hypothesis of SDC, revealing that the increase in FRET due to displacement is a long-term effect, while a surprising kinetic effect can be detected in the initial stages of the assay. 83 A different sensing platform exploits the advantageous hybridization chain reaction, 84 facilitating the sensing of SM-Ags in human plasma. Detection was achieved using Cy3and Cy5-appended hybridizing strands and monitoring the FRET signal resulting from strand elongation.…”

“…Following the same scheme and in the presence of free SM Ags, the Ab will be precaptured; therefore, the equilibrium will not be shifted toward AS duplex formation, resulting in low FRET. Intriguingly, a recent study from the same group challenged the mechanistic hypothesis of SDC, revealing that the increase in FRET due to displacement is a long-term effect, while a surprising kinetic effect can be detected in the initial stages of the assay . A different sensing platform exploits the advantageous hybridization chain reaction, facilitating the sensing of SM-Ags in human plasma.…”

Insertion of Chemical Handles into the Backbone of DNA during Solid‐Phase Synthesis by Oxidative Coupling of Amines to Phosphites

“…Modified oligomers of nucleic acids are extensively used as a tool for research and diagnostics [1, 2] . More recently, conjugates of oligonucleotides (ON) have also been used clinically as therapeutics, especially antisense oligonucleotides (ASOs) and small interfering RNAs (siRNA) [3–6] .…”

“…Modified oligomers of nucleic acids are extensively used as a tool for research and diagnostics. [1,2] More recently, conjugates of oligonucleotides (ON) have also been used clinically as therapeutics, especially antisense oligonucleotides (ASOs) and small interfering RNAs (siRNA). [3][4][5][6] Both natural and artificial ribose, or backbone modified variants are routinely prepared by the means of phosphoramidite chemistry using solid-phase oligonucleotide synthesis (SPOS).…”