We developed an activator independent chemical DNA ligation or replication system combined with a fluorescence quenching analytic system suitable to monitor self-replication in microfluidic experiments. The ligation reaction is strongly pH-dependent and can therefore be manipulated. We successfully oppressed undesirable side reactions. In opposite to EDC activated replication systems the new ligation chemistry allows to analyze ligation reactions at low concentrations and in any suitable temperature range. To monitor the ligation reaction in online kinetics and microfluidics a FRET-quenching system was developed, containing a Dabcyl-modified derivative of Ellman´s reagent as a quencher-leaving group.
Therefore two buffered solutions (0,1 M MES, pH 6,7; 0,1 M NaCl, 35°C), containing the educt building blocks A+T or B+T, respectively, were mixed in a y-structure. The reaction was monitored in the outlet channel of an Y-shaped microfluidic structure in various positions, giving a spatial resolution of the emerging fluorescence signal. At a flow rate of 5µl/h with increasing displacement an increase of fluorescence was observed. Furthermore, “stop flow”-experiments revealed an increasing fluorescence due to a local replication (upper curve).