Effective Antisense Design Using An Ensemble of Energetically Sub-Optimal Secondary mRNA Structures

There is a current deficit of effective therapies against bacterial infection. Many strategies seek using small molecules to target the infectious pathogen. One approach involves direct manipulation of the pathogen at the RNA level. Messenger RNA (mRNA) is a genetic transcript that encodes the fundamental instruction for protein production. Inhibiting mRNA translation effectively prevents protein synthesis. The therapeutic agent must physically access mRNA to effectively block its message from being read. A technique has arisen where a complementary nucleic acid binding strand, called antisense, is generated to impede protein synthesis. An issue in creating effective antisense is finding mRNA target sites for inhibition. This problem is largely due to fluctuating secondary structure blocking target sites. In fact, the kinetics of physical accession are suggested to be the rate-limiting factor and thus the inefficiency of antisense. However, these secondary structure fluctuations are energetically inherent to the nucleic acid sequence and are predictable. A program, GenAVERT, has been developed to thermodynamically determine effective mRNA target sites. It identifies the ensemble of the most probable energetically suboptimal states and determines which regions are therefore most accessible. With proper identification, a successful, effective antisense can be synthesized. This work has proven GenAVERT’s capacity by constructing antisense to downregulate green fluorescent protein expression in Escherichia coli. The fluorescence reduction affirms this model’s efficacy. Supported by fundamental energetic principles, this method of producing an antisense sequence complement for the most accessible mRNA target region has the potential to greatly reduce the strife of pathogenic infection