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We use machine learning for the selection and classification of single–molecule trajectories to replace commonly used user–dependent sorting algorithms. Measured fluorescence time series of labelled single molecules need to be sorted into ’good molecules’ and ’bad’ molecules before further kinetic and thermodynamic analysis.
Currently, processing, sorting and analysis of the data is mainly done with the help of laboratory specific programs.
Although there are freely available programs for processing smFRET data, they do not offer ’molecular sorting’ or it is purely empirical. Only recently, new approaches came up to solve this problem by means of machine learning. Here, we describe a sound terminology for molecular sorting of smFRET data and present an efficient workflow for manual annotation followed by the training of the ML algorithm. Descriptive statistics of our generated dataset are provided and will serve as the basis for supervised ML-based molecular sorting algorithms yet to be developed.
Long-range tertiary interactions between RNA tetraloops and their receptors stabilize the folding of ribosomal RNA and support the maturation of the ribosome. Here, we use FRET-assisted structure prediction to develop a structural model of the GAAA tetraloop receptor (TLR) interaction and its dynamics. We build the docked TLR de novo, label the RNA in silico and compute FRET histograms based on MD simulations. The predicted mean FRET efficiency is remarkably consistent with single-molecule experiments of the docked tetraloop. This hybrid approach of experiment and simulation will promote the elucidation of dynamic RNA tertiary contacts and accelerate the discovery of novel RNA and RNA-protein interactions as potential future drug targets.
RNA tertiary contact interactions between RNA tetraloops and their receptors stabilize the folding of ribosomal RNA and support the maturation of the ribosome. Here we use FRET assisted structure prediction to develop structural models of two ribosomal tertiary contacts, one consisting of a kissing loop and a GAAA tetraloop and one consisting of the tetraloop receptor (TLR) and a GAAA tetraloop. We build bound and unbound states of the ribosomal contacts de novo, label the RNA in silico and compute FRET histograms based on MD simulations and accessible contact volume (ACV) calculations. The predicted mean FRET efficiency from molecular dynamics (MD) simulations and ACV determination show agreement for the KL-TLGAAA construct. The KL construct revealed too high FRET efficiency and artificial dye behavior, which requires further investigation of the model. In the case of the TLR, the importance of the correct dye and construct parameters in the modeling was shown, which also leads to a renewed modeling. This hybrid approach of experiment and simulation will promote the elucidation of dynamic RNA tertiary contacts and accelerate the discovery of novel RNA interactions as potential future drug targets.