Fingerloop sRNA Nanotechnology for Metabolic Engineering and Synthetic Biology
Functional "fingerloop" RNA antisense motifs for metabolic engineering and RNA nanotechnology applications.
Synthetic biology holds tremendous promise to revolutionize how numerous products are made. For example, microorganisms can be harnessed to produce useful products such as biofuels, chemicals, therapeutics, fragrances, and antibodies, just to name a few examples. However, metabolic engineering of these microorganisms is often a prerequisite for commercially viable levels of product generation. Current metabolomic engineering strategies exist, such as siRNA and genome editing, but suffer from serious drawbacks. For example, expression of a metabolic pathway protein can be reduced using siRNA, but typically more than one protein must be targeted to effectively control metabolism, necessitating introduction of multiple siRNAs. Furthermore, siRNA target specificity is often poor. Permanent genome editing, another strategy, is difficult to reliably perform, not tractable in all microorganisms, and often results in off-target mutations that must be filtered. Moreover, the complete knockout of multiple metabolism genes is often too harsh, with more subtle modulation necessary for optimal results.
Researchers at The Ohio State University led by Dr. Richard Lease have created a novel bacterial sRNA-based genetic manipulation tool capable of transient, tunable, and highly specific control of the expression of multiple proteins using a single engineered sRNA. A single sRNA molecule can contain multiple fingerloop motifs, each motif capable of base pairing with a distinct mRNA promoter, thus down–regulating expression of the corresponding protein. Consequently, the introduction of a single sRNA vector can comprehensively reprogram a metabolic pathway by altering the expression of multiple proteins in that pathway to maximize product yield. Furthermore, by modulating the degree of base complementarity, the level of protein expression can be precisely tuned to optimize product formation without over-taxing the cell. Finally, due to the fingerloop motif structure, sRNAs display superior mRNA target specificity relative to traditional siRNAs or non-structured antisense sRNAs.
Dr. Lease’s team has demonstrated one embodiment of this technology by downregulating both hydrogen-generating hydrogenase (hydA) and butyrate kinase (buk), proteins involved in Clostridium acetobutylicum butanol biosynthesis, using a single sRNA. The invention involves using a plasmid reporter system to optimize the sRNA sequence required to yield the desired degree of target mRNA/protein repression. This optimized sRNA can then be introduced into the microorganism to alter its metabolism. sRNA technology is broadly compatible with bacteria and could be used to target any set of proteins that a user wishes to modulate.