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

There is a deficit of effective therapies in the current research against bacterial infection. Many therapeutic strategies seek to use small molecules to target the infectious pathogen. One approach to disease inhibition involves direct manipulation of the pathogen at the RNA level. Part of the central dogma, messenger RNA (mRNA) is a transcript of genetic information that encodes the fundamental instruction for protein production. Inhibiting translation of mRNA effectively prevents synthesis of proteins.

In order to effectively block the RNA’s message from being read, it must be physically accessed by the therapeutic agent. A field of study has arisen in which a complementary nucleic acid binding strand, called antisense[1], is generated to impede protein synthesis.  One of the issues that arises in creating effective antisense RNA is the ability to find appropriate target sites on the mRNA to be inhibited.  This problem is in part due to fluctuating secondary structure sequestering target sites.  In fact, the kinetics of physical accession of the antisense to the target strand are suggested to be the rate-limiting factor[2] and thus the cause of inefficiency of this method. However, the fluctuations among suboptimal conformations are energetically inherent to the nucleic acid sequence and can be predicted.  A software package, GenAVERT, has been developed to thermodynamically determine effective target sites by identifying the ensemble of the most probable energetically suboptimal states and determine which regions of mRNA are most accessible[3].  Once properly identified, a successful and effective antisense can, in theory, be synthesized.

In this work, a proof of principle will be demonstrated in E. coli with the downregulation of a short-lived variant of the green fluorescent protein through construction of an inducible antisense molecule. Statistical reduction in the fluorescence upon antisense induction will affirm the efficacy of this model.

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.