Mixed labeling as a FRET detection method for oligonucleotide replicators
Rapid online techniques to monitor chemical kinetics in a parallel
fashion are a major prerequisite to study chemical self-replication. As
such, HPLC- and NMR-kinetics, by which most of the systems so far
described have been studied are in a sense either laborious or
expensive.
We developed a scheme for the online monitoring of chemical
replication of self-complementary oligonucleotides by means of FRET.
The building blocks and basic reactions (1.4.1)
of the system studied point to the rationale behind "mixed labelling" (1.4.2),
viz. the use of a donor and an acceptor for one and same precursor
sequence.
The replicator reaction network embedding mixed labelling (1.4.3) looks a bit complicated at the first glance. Its analysis was possible however because the kinetics of its metabolic core could be derived independantly from NMR based kinetics (1.4.4). FRET-based kinetics (1.4.5) enabled both, the direct observation of sigmoidal growth profiles for mixed labeled template molecules, as well as the observation of non sigmoidal profiles expected for the synthesis of acceptor labelled templates in the presence of fixed concentrations of donor labelled templates. All profiles were taken together as input data for a SimFit analysis based on a reaction model (1.4.6), which embedded several simplifications. The main approximation was that structurally similar labels such as Cy3 and Cy5 should exhibit neligible differences with respect to the kinetics. This allowed us to reduce the complexity of the network in such a way that three composite however meaningful rate parameters could be extracted.