Developing a method for tracking RNA in live e. coli cells using fluorogenic DNA oligonucleotide probes

Thesis

Super-resolution microscopy and the development of single molecule Fluorescence In Situ Hybridisation (smFISH) allows detection and localisation of individual molecules inside cells, deciphering intricate mechanistic details of various biological processes. FISH is well established in fixed cells, but there has been limited success in live cells, particularly in bacteria owing to their smaller size and lack of well-defined partitions. We aim to develop an in vivo smFISH technique which uses short single stranded fluorogenic DNA probes which are electroporated into live Escherichia coli. The spatial information from our study can potentially lead to visualising mRNA molecules throughout their lifetime, from transcription to translation to degradation.

Our proof of principle target consists of a short plasmid-expressed RNA, that contains six binding sites for the fluorogenic probe. The observed foci can be tracked and exhibit a wide range of behaviour; from immobility to rapidly diffusing spots, with many transitions between these states. A range of fluorogenic probes were tried with their in vivo behaviour characterised.

Despite the large proportion of non-specific interactions, it was observed that on a population level the mobility distributions of the tracks vary between the control and test samples. For this reason, it was concluded that the method has some degree of success and improvements to the design are suggested.

Authors: Wheeler, E

• DOI: 10.5287/ora-bpqxvyonk
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English