The rapid increase in occurrence of carbapenemase producing multi drug resistant Entero-bacteriaceae represents a world wide health risk. Carbapenem antibiotics have long been used as a treatment of last resort for infections with multi resistant bacteria. Now that bacteria develop enzymes that render these agents useless, treatment options are limited, and the health care system is left with a fast-growing challenge. Easy to use and cost-effective detection of carbapenem resistance and discrimination between different carbapenemases are important to prevent spread and to facilitate appropriate treatment. The development of reliable detection systems strongly depends on the availability of highly affine and specific molecular recognition molecules. Aptamers are promising molecular recognition molecules. They are small and chemically synthesized, single stranded (ss) DNA or RNA molecules that bind to their targets with high affinity and specificity. Aptamers are selected by an in vitro selection process that can be tailored to suit the desired application. Chemical synthesis of aptamers minimizes batch-to-batch variations and facilitates easy and cost-effective modifications. Yet, an effective aptamer selection process is often hampered by non-specific binding to side targets. Thus, negative selections against potential side targets are recommended. In this work, selections were carried out against N-terminally polyhistidine (His)-tagged New Delhi metallo-beta-lactamase 1 (His-NDM-1) and Klebsiella pneumoniae carbapenemase-2 (His-KPC-2). Eleven selection rounds (SRs), partially conducted with negative selections against His-tagged carbapenemases did not result in the identification of aptamers. Three consecutive SRs without negative selections against His-tagged proteins resulted in the identification of high-affinity His-tag aptamers, only one His-KPC-2 and no His-NDM-1 aptamer. To facilitate carbapenemase binding while preventing His-tag binding, the selection against His-KPC-2 was restarted from SR eleven and three different selection strategies were designed. The strategies included masking of the His-tag and competitive elution of His-tag-binding sequences using a truncated version of the previously selected His-tag aptamer as well as immobilization of the protein via its His-tag. After three SRs, sequencing data was analyzed for the enrichment of a motif that is likely involved in His-tag binding. Based on this analysis, the masking approach was identified as the most promising strategy. Binding to His-KPC-2 was demonstrated for several aptamers. Two of these aptamers were further characterized for binding properties. Both very likely did not bind to the His-tag. Consequently, an aptamer-based lateral flow assay was developed for His-KPC-2. The masking approach was also applied to selection against His-NDM-1. Here, an aptamer with a binding affinity in the high nanomolar to low micromolar range, without cross reactivity to another His-tagged carbapenemase and a synthetic hexa-His peptide was identified. This aptamer may serve as a valuable tool for the detection of the very prevalent and harmful carbapenemase NDM-1.
