Computational Resources and Strategies to Construct Single-Molecule Models of FISH

Abstract

Currently, the interactions occurring between oligonucleotides and the cellular envelope of bacteria are not fully resolved at the molecular level. Understanding these interactions is essential to gain insights on how to improve the internalization of the tagged oligonucleotides during fluorescence in situ hybridization (FISH). Agent-based modelling (ABM) is a promising in silico tool to dynamically simulate FISH and bring forward new knowledge on this process. Notably, it is important to simulate the whole bacterial cell, including the different layers of the cell envelope, given that the oligonucleotide must cross the envelope to reach its target in the cytosol. In addition, it is also important to characterise other molecules in the cell to best emulate the cell and represent molecular crowding. Here, we review the main information that should be compiled to construct an ABM on FISH and provide a practical example of an oligonucleotide targeting the 23S rRNA of Escherichia coli.

Estimation of nucleic acid mimics (NAMs) diffusion in bacteria with fluorescence recovery after photobleaching (FRAP) and PyFRAP

“…The calculated DC values are also in good agreement with the theoretical DC reported in the study by Magalhães et al for an 18-mer FAM-NAM sequence and calculated using the Stokes-Einstein equation (2.67 x 10 -3 µm 2 •s -1 ) 42 .…”

“…For the NAMs diffusion assays, the coverslip was removed, and a 5 μL drop of the 2 μM FAM-NAMs in hybridization solution was added to the agarose pad with trapped bacteria. This concentration of NAMs allows for a large surplus of probes when compared to the number of ribosomes in the cell 42 . Cells in saline without the addition of NAMs served as negative control for auto uorescence.…”