FRET can be used as a strategy to assign different simultaneous events in the same sample but {"}cross-talk{"} problems are a limitation. Here we present a contribution for the better understanding of the {"}cross-talk{"} in FRET experiments that include several pairs in the same sample. Using oligonucleotide probes labeled with fluorescent dyes which can be selectively excited at a specific wavelength, and using target oligonucleotides tagged with a fluorescent dye with specific characteristics that allow only it to emit light upon selective excitation of a specific probe by energy transfer (FRET), we aim to identify the exact probe-target hybridized pair. When using three donors to probe the presence of complementary targets, only 20% of possible donor/acceptor combinations give straightforward signals readily identifiable with the sample composition, while in the remaining cases severe cross-excitation prevents the direct identification of the sample composition. To correctly resolve the samples identity, we developed a theoretical model that enables the unequivocal attribution of a sample composition to a given set of fluorescence signals, in the presence of three donors.

O presente invento relaciona-se com um método para controlo de reac{\c c}ões enzimáticas de síntese de ácidos nucleicos, recorrendo a nucleótidos funcionalizados com derivados de 4-metilcumarinas (1) protectores e fotolábeis. Quando ligado aos nucleótidos (2), o grupo cumarinico (3) impede que estes sejam utilizados como substrato por parte das enzimas, impossibilitando a ocorrência de reac{\c c}ão. Através de irradia{\c c}ão com radia{\c c}ão electromagnética, o grupo cumarinico é libertado, ficando o nucleótido disponível para a reac{\c c}ão. Desta forma, as reac{\c c}ões enzimáticas de síntese de ácidos nucleicos podem ser controladas através da luz.