Fluorescence Quenching of Xanthene Dyes during Amide Bond Formation Using DMTMM

Fluorophore bioconjugation to proteins, nucleic acids, and other important molecules can provide a powerful approach to sensing, imaging, and quantifying chemical and biological processes. One of the most prevalent methods for fluorophore attachment is through the formation of amide bonds, which are often facilitated by coupling agents to activate carboxylic acid moieties for subsequent nucleophilic attack by amines. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) is among the most popular of these coupling agents for bioconjugation due to its ability to facilitate amide bond formation in water. After observing quenching of 5-fluoresceinamine (5-FAM)-conjugated oligonucleotides in the presence of DMTMM, we sought to evaluate the magnitude and scope of this challenge by surveying the effect of DMTMM on a range of fluorescent dyes. A higher quenching effect was consistently observed for xanthene dyes compared to that for cyanine dyes. Further analysis of the impact of DMTMM on FAM shows that quenching occurs independently of whether the dye is free in solution or attached to an oligonucleotide or antibody. Furthermore, we found that FAM-conjugated DNA was unable to recover its fluorescence after the removal of DMTMM, and UV–vis and NMR analyses suggest the formation of new products, such as an adduct formed between FAM and the dimethoxytriazine of DMTMM. As such, DMTMM at high concentrations is not recommended for coupling reactions where targets are fluorescently labeled. This research serves as a word of caution to those utilizing xanthene-containing fluorophores in bioconjugation reactions involving DMTMM.


Table Of Contents
Materials……………………………………………………………………………………………………………... 1 μM dye in 1% DMSO was exposed to DMTMM concentrations of 1 M, 500 mM, 250 mM, 100 mM, 10 mM, 1 mM, 100 μM, 10 μM and 0 μM in a 96-well plate. Solutions ranging between 0-2.0 μM dye were made from each dye solution in triplicate to generate a calibration curve on the same plate as the reaction mixtures. Raw fluorescence values were compared to the calibration curve and normalized to fluorescence intensity at t=0 h, and formulas for calibration curves as well as concentrations were quantitatively determined using Excel. All fluorescence experiments were performed at N=6 and error bars represent standard deviation. All fluorescence measurements were taken using a Biotek Cytation 5 imaging reader. In between measurements, the 96-well plate was covered with an opaque lid and placed under a box to minimize photobleaching. Each well in the perimeter of the 96-plate was filled with 200 μL water to minimize evaporation over the 24-hour observation period.
Scheme S1. General Mechanism of Reactivity for DMTMM S3 a. b.
c.  Spectral Scan 1 μM dye in 1% DMSO was exposed to DMTMM concentrations of 1 M, 500 mM, 250 mM, 100 mM, 10 mM, 1 mM, 100 μM, 10 μM and 0 μM in a 96-well plate. Solutions ranging between 0-2.0 μM dye were made from each dye solution in triplicate to generate a calibration curve on the same plate as the reaction mixtures. Raw fluorescence values were compared to the calibration curve and normalized to fluorescence intensity at t=0 h, and formulas for calibration curves as well as concentrations were quantitatively determined using Excel. All fluorescence experiments were performed at N=6 and error bars represent standard deviation. All fluorescence measurements were taken using a Biotek Cytation 5 imaging reader with the following parameters; 5-FAM -Fluorescence Spectra -Scale to peak: Scaled to three wells containing 2 uM dye in calibration curve, 80,000 RFU, expected peak: 518 nm -Excitation: 462 nm -Emission Start: 490 nm, Emission Stop: 700 nm -Steps: 5 -Read Height: 7.00 mm Cy3 Azide -Fluorescence Spectra -Scale to peak: Scaled to three wells containing 2 uM dye in calibration curve, 80,000 RFU, expected peak: 590 nm -Excitation: 500 nm -Emission Start: 540, Emission Stop 700 -Steps: 5 -Read Height: 7.00 mm . In between measurements, the 96-well plate was covered with an opaque lid and placed under a box to minimize photobleaching. Each well in the perimeter of the 96-plate was filled with 200 μL water to minimize evaporation over the 24-hour observation period.

Characterization of reactivity.
50 μL of FAM stock was added to 50 μL of DMTMM such that the reaction between DMTMM and FAM in 10:20 mM, 10:10 mM, 1:2 μM and 1:1 μM ratios were made in triplicate in a 96 well plate. Standards of 10 mM and 1 μM DMTMM as well as 10 mM, 20 mM, 1 μM and 2 μM FAM were added to well plates in triplicate, and fluorescence was observed over the course of 24 hours. As two well plates were run per dye, in total N=6. With values normalized to fluorescence at t=0, fluorescence intensity tends decrease with higher concentrations of both FAM and DMTMM which supports concentration being a more significant factor than molar ratio in determining depletion. Following this, reactivity was explored in MOPS buffer at pH 8.0, phosphate buffer at pH 7.4, and MilliQ water at pH 6.9. The first two of these conditions were chosen, as they represent typical buffers used for bioconjugation reactions with DMTMM Figure S3. Fluorescence of FAM after exposure to various concentrations of DMTMM in a) MilliQ Water, pH=6.9 b) phosphate buffer, pH=7.4, and c) MOPS buffer, pH=8.0. All experiments were performed at N=3. Points below zero removed from plot

Fluorescence quenching experiments in biomolecules
Kanamycin A binding aptamer (Ky2) modified with a 5' FAM (Table S1) and monitored fluorescence quenching in MOPS buffer at pH 8 in the presence of 30 μL of 300 mM DMTMM for 24 hours. DNA concentration (via absorbance at 260 nm) and fluorescence intensity at Ex/λEm=498/518 nm were quantified using a Biotek Cytation 5 imaging reader prior to and 24 hours following DMTMM exposure. Similarly, Goat Anti-Dog IgA antibody labeled with FITC was incubated in PBS buffer at pH=7.4 with 30 μL of 300 mM DMTMM for 24 hours. DNA concentration (via absorbance at 280 nm) and fluorescence intensity at λEx/λEm=491/516 nm were quantified using a Biotek Cytation 5 imaging reader prior to and 24 hours following DMTMM exposure. Experiments were performed at N=2 and error bars represent standard deviation. Buffer Recipes are as follows;          Figure S17. 1 H NMR comparison of Pyronin Y and DMTMM at t=0 (green) and t=24 (purple), 6.7-8.9 ppm.