Biomolecules such as proteins and nucleic acids sometimes exhibit structural diversity in aqueous environment. Such conformational heterogeneity is closely related to the mechanisms of the structure formation and the function of the biomolecule. Therefore, it is desirable to develop an experimental method which enables us to resolve the heterogeneity for determining the relative population and the spectroscopic signature of each substate.<br/>We have developed a new method in which independent component analysis (ICA) is applied to time-resolved smFRET data for elucidating conformational heterogeneity of biomolecules. ICA is a data processing method used in combination with multichannel signal detection to resolve a mixed signal into independent components by examining the high-order correlations, or non-gaussianity, of the fluctuations of the signal. The developed method, which we name independent fluorescence component analysis (IFCA), has advantages over existing analysis methods of smFRET photon data. First, it is model-free, i.e., it does not rely on fitting to an arbitrary theoretical model. Second, it can be applied to a relatively highly concentrated sample. Lastly, the analysis result does not depend on the temporal binning width of the photon data, so that IFCA can be applied to study transient species with a lifetime of microseconds.<br/>In order to realize IFCA, we developed a numerical procedure for analyzing the third-order correlation tensor of multichannel photon signals. The photon signal was obtained with a time-resolved smFRET setup which utilizes the time-correlated single photon counting and the pulsed interleaved excitation scheme. We will demonstrate the potential of IFCA by using the developed procedure for analyzing synthetic photon data generated by numerical simulation assuming five independent components as well as experimental data from FRET-labeled DNA oligonucleotides.

Independent component analysis of smFRET photon data for resolving conformational heterogeneity of biomolecules

2019 Spring National Meeting

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