Stepwise and site-selective enzymatic introduction of multiple functional groups to turn-on multiple fluorescence in long DNA strands

We have developed a stepwise and site-specific method, employing DNA polymerase, for the introduction of many functional groups into long DNA strands. With this approach, we developed three novel fluorescence turn-on systems that respond specifically to amino, alkynyl, and hydrazino groups in oligonucleotide strands. These systems operate when adding fluorescence precursors (i.e., ylidenmalonitrile enamine, azidopyrene, and diethylsalicylaldehyde), each of which exists in its turned-off state in the absence of the specific reactions. MALDI-TOF mass spectrometry confirmed the site-specific introduction of these functional groups and their post-synthetic reactions with the fluorescence precursors. We also applied this method to incorporate several functional groups into long DNA strands; for example, we demonstrate the stepwise and site-specific introduction, mediated by several enzymes, of multiple functional groups at 18 different positions in a 144mer DNA. Multiple fluorescence turn-on was possible when this long DNA containing 18 functional groups encountered the fluorescence precursors. In a cellular study, we observed three different fluorescence turn-on processes, exhibiting blue, green, and red fluorescence signals, when using this post-synthetic approach. •We developed a step-wise and site-specific method for the multiple labeling into long DNA strands.•We also performed a cellular study using the post synthetic method, observing three different fluorescence turn-on emissions.•Such novel three-color fluorescence turn-on systems would be useful in various bioimaging studies.