Nature employs nanoscale machines that self-assemble into dynamic structures of complex architecture and functionality. Single molecule fluorescence microscopy offers a non-invasive tool to probe and ultimately dissect and control these nanoassemblies in real-time, often with the aid of computational tools to interpret and complement the experimental data. In particular, single molecule fluorescence resonance energy transfer (smFRET) allows us to measure biologically relevant distances and changes thereof at the 2-8 nm scale, whereas complementary super-resolution localization techniques based on Gaussian fitting of imaged point spread functions measure distances in the 10 nm and longer range. In this talk, I will describe how we have developed Single Molecule Cluster Analysis (SiMCAn) based on a vast smFRET dataset to dissect the complex conformational dynamics of a pre-mRNA as it is spliced by the spliceosomal processing machinery. In addition, I will demonstrate how a single molecule systems biology can be implemented that feeds super-resolved single particle tracking data into lattice-based Monte-Carlo simulations to generate novel hypotheses on the gene regulation of mRNAs by microRNAs during RNA silencing.