Dye to nucleic acid conjugation is a chemical strategy to couple one or more fluorescent dyes onto an oligonucleotide molecule, endowing nucleic acids with a detectable component while retaining its ability to base pair. The attachment of a small dye to an oligonucleotide forms the basis for constructing a sensitive reagent. Fluorescent oligonucleotides have now been extensively used in fluorescent fluorimetry, fluorescence microscopy, fluorescence polarization spectroscopy, time-resolved fluorescence (TRF) and fluorescence energy transfer (FRET).
Since the invention of phosphoramidite chemistry and the successful adaption of solid-phase synthesis, oligonucleotide synthesis and modification have become widely applicable. There are two main approaches to incorporate dye molecules in oligonucleotides: 1) incorporation during solid-phase synthesis; 2) modifying oligonucleotides through post-synthesis conjugation.
Directly incorporating dyes into oligonucleotides during solid-phase synthesis makes it easy and convenient for researchers to develop various fluorescent DNA probes for the applications in biochemical and cellular studies. In general, dye modified phosphoramidites are used for 5’- modification, and modified solid supports (e.g. CPGs) are for 3’- modification. Both phosphoramidites and modified nucleobases can be used to achieve internal modifications, while modified nucleobases are better at maintaining Watson-Crick base paring.
However, sometimes in-synthesis approach could not provide satisfactory modification, for example, when the corresponding phorphoramidites are not commercially available or the labeling dye is not compatible with DNA synthesis. In these cases, one or more chemical handles are introduced in the DNA strand in the solid-phase synthesis process and the desired dye is subsequently coupled to the DNA strand through the reaction between the chemical handle on the DNA and the complementary functional group on dye. The most common handles in oligonucleotide modifications are amino groups, thiols, and alkynes. Similar strategies being used for preparing dye-protein conjugates, such as through NHS ester-amine reaction or maleimide-thiol reaction, thus could be used to couple dye molecules to oligonucleotides.
AAT Bioquest offers a variety of fluorescent labeling dyes, compatible for either in-synthesis or post-synthesis modification, facilitating the conjugation of dyes to oligonucleotides for various biological studies