Safety of Delivery System

Many turn-off strategies have been developed, and 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 . When the course of treatment ends, administration of small amount of tetracycline agonist will induce bacteria to commit suicide. Leaving human body also cause suicide gene activation thereby avoids recombinant strain/gene polluting. And splitting suicide system to provide repression in trans can prevent plasmid transferring to wild type strains. There have been many usable off-the-rack parts.

general scheme of suicide system

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

Although bacteria lack of RNA interference pathway, expressing well-designed antisense RNAs has been shown to have inhibitory effect on target RNAs through competitive inhibition[13]. Recent study also showed that peptide nucleic acid (PNA) that antisense to antitoxin RNA 5' sequence can cause death of bacteria [14]. Putting appropriate antisense RNAs on untranslated region of transcripts may interfere with functions of target RNA or it translation. This property might be used to prevent HGT. For instance, HGT is more likely to occur between related species like laboratory E. coli and O157. Laboratory E.coli have inactivated all its hok/sok toxin-antitoxin system by mutation, but wild bacteria, especially pathogenic bacteria such as E.coli O157, usually have more active TA locI on its chromosome. 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 type bacteria steal our antibiotic resistance genes and express it, the antitoxin will be competitive inhibited and the toxin will be expressed to kill the thief, thus preventing HGT between laboratory and wild type bacteria. 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 that exist in potential HGT receivers but not in our E. coli strains can be used. Even if the design cannot kill the thieves, it can weaken receivers and reduce the advantages that antibiotic genes bring about thus reduce the 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[13, 15, 16].

Is it possible to use RNA interaction to avoid HGT?