Development of inhibitory RNA aptamers using ultrahigh-throughput screening to fight antibiotic resistance.

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The increase of antibiotic resistance is becoming a serious health threat worldwide, but the number of newly released antibiotics remains low. One way to address this problem is to target resistance proteins to restore the efficacy of antibiotics. β-lactams are the most successful class of antibiotic drugs but they are vulnerable to inactivation by a growing class of β-lactamases. For a long time, medically relevant β-lactamases encompassed only serine β-lactamases (SBLs) for which several inhibitors have now been developed. However, more recently metallo-β-lactamases (MBLs) emerged as a global threat for which no inhibitor has been developed yet. However, their increasing importance in drug failure is a strong incentive to target them. Here, we propose to develop RNase resistant inhibitory aptamers targeting both types of β-lactamases using an innovative pipeline combining the use of in vitro selection (SELEX) in tandem with microfluidic-assisted ultrahigh-throughput screening and next generation sequencing (NGS). Indeed, whereas SELEX will allow to enrich aptamers from very large libraries in sequence displaying the potential to bind the target protein, microfluidic screening will allow refining this pre-selection by searching for those aptamers really able to inhibit enzyme activity. Finally, using NGS and bioinformatics will allow analyzing the whole process at once to rapidly identify most promising sequences. Most key elements of this pipeline have already been validated during preliminary experiments, allowing the majority of the project to be focused on the actual development of new aptamers able to inhibit three medically relevant extracellular enzymes: the metallo-β-lactamase NDM-1 from Klebsiella pneumoniae and the serine-β-lactamases BlaZ and the protease Aureolysin, both from Staphylococcus aureus. By the end of this project we will not only have validated this new technology through the actual development of efficient aptamers, but we will also have developed new prototypes of drugs that could later be optimized and enter into the arsenal required to fight bacterial infection that are foreseen to be a major thread surpassing every other disease in the up-coming decades. Moreover, the potential application spectrum of the DIRA pipeline refined and used here will be much wider than antibiotic discovery as discussed in the present proposal.