Rationally designing antisense therapy to keep up with evolving bacterial resistance - Fig 1

Kinetic Diagram of Subspecies mutations: a. Mechanism of action for antisense molecule. To alleviate its effects, there are two kinds of mutations: 1) specific mutations, nucleotide change in the target sequence, these mutations would only affect a “specific” antisense sequence. 2) nonspecific mutation, disruption in the entry mechanisms of antisense molecules, these mutations would nullify any antisense in “nonspecific” manner. b. Simulated system is bacteria grown in liquid culture. Computational experiments started with inoculated bacteria to a fresh medium containing antisense molecules. Growth continued until early stationary phase. Grown culture is used to transition to another medium. Serially passaged bacteria ensure continued evolution. After sufficient time, bacteria gain resistance to the therapy (growth rate surpassed a set threshold), at that time antisense therapy is redesigned. This action may or may not restore therapy effectiveness. If not, then therapy finalized. c. The kinetic diagram represents the mutational pathways. First, mutations in the targeted sequence (specific mutations, horizontal direction). Second, mutations that prevent entry of antisense into the bacterial cell (nonspecific mutations vertical direction). The direction of horizontal change is from left to right for acquiring specific resistance mutations. The direction of vertical change is from top to bottom for loss of delivery mechanisms. d. shows an example of a constructed fitness landscape. The height of the surface represents the growth rates associated with a kinetic diagram. The black edge represents the growth rates for specific mutations with all entry mechanisms kept intact. The red edge represents the growth rates associated with failing of entry mechanisms with 0 specific mutants. These two edges are used to derive all other growth rates, see methods for details.