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.

Combating small molecule environmental contaminants: detection and sequestration using functional nucleic acids.

Small molecule contaminants pose a significant threat to the environment and human health. While regulations are in place for allowed limits in many countries, detection and remediation of contaminants in more resource-limited settings and everyday environmental sources remains a challenge. Functional nucleic acids, including aptamers and DNA enzymes, have emerged as powerful options for addressing this challenge due to their ability to non-covalently interact with small molecule targets. The goal of this perspective is to outline recent efforts toward the selection of aptamers for small molecules and describe their subsequent implementation for environmental applications. Finally, we provide an outlook that addresses barriers that hinder these technologies from being widely adopted in field friendly settings and propose a path forward toward addressing these challenges.

Sequestration and Removal of Multiple Small-Molecule Contaminants Using an Optimized Aptamer-Based Ultrafiltration System.

Small-molecule toxins pose a significant threat to human health and the environment, and their removal is made challenging by their low molecular weight. Aptamers show promise as affinity reagents for binding these toxins, and recently, aptamers have been utilized for both sensing and remediation applications. We found that functionalization of ultrafiltration membranes with aptamers provides a convenient scaffold for toxin sequestration, but our initial efforts in this area were limited by low functionalization efficiencies and the ability to only capture a single target molecule. Herein, we describe detailed optimization of our aptamer-functionalized ultrafiltration membrane system and subsequent use for simultaneous removal of multiple small-molecule toxins. We examine multiple critical components involved in fabricating and functionalizing the membranes, including PEG polymer molecular weight for membrane fabrication, grafting conditions for pMAA attachment, and coupling reagents for aptamer functionalization. This screening enabled us to identify a set of unique conditions in which we were able to achieve high flux, near quantitative yield for DNA attachment, and effective overall depletion of both toxins and bacterial cells. Furthermore, we demonstrate the attachment of multiple aptamers and subsequent parallel removal of atrazine, bisphenol A, and microcystin-LR in a complex lake water matrix. Our rigorous evaluation resulted in depletion of multiple small-molecule toxins, contaminants, and microorganisms, demonstrating the potential of aptamer-functionalized membranes as point-of-use decontamination systems.