Safety of Delivery System

Many turn off strategies have been developed, most of these are the inducible suicide system that can be activated at certain condition. For instance, in our project, we plan to use temperature and small molecule as activating signals( following picture). When the course of treatment ends, administration of small amount of tetracycline agonist will induce bacterial to commit suicide, leaving human body also cause suicide gene activation thereby avoid recombinant strain/gene polluting. And splitting suicide system to provide repression in trans can prevent plasmid transfering to wild type strains. There have been many off-the-rack parts can be used.

general scheme of suicide system

However, these design cannot totally eliminate the risk of horizontal gene transfer(HGT), which recombinant genes can move to other organisms independent of suicide system. So besides suicide system, we have a new idea to deal with these kinds of HGT risks by RNA interaction.

Although bacteria lack for RNAi pathway, expressing well designed antisense RNAs have been shown to have inhibitory effect on target RNAs through competitive inhibition, and recent study showed that peptide nucleic acid (PNA) that antisense to antitoxin RNA 5' sequence can cause bacteria death. Putting appropriate antisense RNAs on untranslated region of transcripts may interfere target RNA function or translation. This property might be used to prevent HGT. For instance, HGT is more likely to occur between related species like lab E.coli & O157, laboratory E.coli have inactivated all its hok/sok toxin-antitoxin system by mutation, but wild bacteria especially pathogenic bacteria usually have more active TA locus on its chromosome like E.coli O157. we plan to put a stem loop from hok mRNA which can pair with sok RNA 5’sequence on UTR of antibiotic resistance genes we used in pepdEX system. IF wild bacteria steal our antibiotic resistance genes and express it, its antitoxin will be competitive inhibited and its toxin will express and kill the thief thus preventing HGT between lab & wild coli. This idea can have wide extension. Besides targeting antitoxin (functional RNA) of type I TA, designing antisense sequences that target RBS to down regulate targeted protein is also possible. Targeting antitoxin of type II TA, essential genes for metabolism, housekeeping genes and any sequences exist in potential HGT receivers but not our coli can be used. Even if the design cannot kill thieves, it can weaken receivers and reduce advantages antibiotic genes bring about thus reduce possibility and danger of HGT.

In the past the repression efficiencies of antisense RNA in bacteria are low, but after invention of the paired termini antisense RNA(PTasRNA) method and incorporate U turn/YUNR motif etc., this idea will become more and more feasible.

Is it possible to use RNA interaction to avoid HGT?