Research On Bacteria-sensing RNA-cleaving Fluorescent DNA Probe

Bacterial infection represents a serious global crisis and results in serious economic loss. The detection of bacteria is the firstline of defence to keep them from threatening human health. Through in vitro selection(IVS), an RNA-cleaving fluorogenic DNA probe, named RFD-EC1, for rapid and specific E. coli detection was created, which is promised to provide the probe model for next-generation bacteria-detecting assays. In this dissertation, the structure, target and further applications of RFD-EC1 are explored to provide experience for the future selections of bacteria-sensing RFDs.By our study, the RFD-EC1 is capable of determining the minimal inhibitory concentration(MIC) of antibiotics much faster than the conventional optical density(OD) method. In addition, since the target for RFD-EC1 is an extracellular protein molecule from E. coli, RFD-EC1 is able to identify pore-forming antibiotics or compounds that can cause membrane leakage. Finally, RFD-EC1 can be used to analyse the competition of cohabitatingbacteria, specifically the inhibition of growth of E. coli by Bacillus subtilis.Based on the nature of the target for RFD-EC1 in E. coli cell is protein, the target was successfully identified as RNase I through screening of the reactions between RFD-EC1 and the mutanted E. coli cells in the Keio Collection(a single gene mutanted E. coli cells library). Then by the single amino acid mutations at the catalytic site, the RNase I was proved to play an important role in the cleavage reaction with RFD-EC1. On the other hand, the fluorescent substarte RS28-cleaving activity of RNase I would be inhibited in presence of 30 m M Ba2+; but through the addition of the ADZ3(the activ e part of RFD-EC1), the RNase I recovered its capability to cleave RS28 at 30 m M Ba2+. RFD-EC1 not only specifically binds with RNase I, but also is pivotal in the catalytic reaction.Through reselction and truncation experiments, the sequence of RFD-EC1 was successfully optimized. The length of effective RFD-EC1 part is shortened to 29 nucleotide(nt) from 99 nt; meantime its cleavage acitivity is improved. The second structure of the optimized sequence is defined, which provides convenience for its futher modification as well as other applications for microbiological area.The RFD probe for Staphylococcus aureus(SA) named RFD-SA was obtained through the reasonable selection design. It is the first RFD probe for pathogen indication. After the optimization of the reaction conditions, the RFD-SA successfully detected the SA from milk; among the cultures of bacterial samples from hospital, the RFD-SA specifically responsed to SA one.