DNA aptamers specific for Legionella pneumophila: systematic evolution of ligands by exponential enrichment in whole bacterial cells

Abstract

Legionella pneumophila is the major causative agent of Legionnaires' disease and Pontiac fever, which pose major public health problems. Rapid detection of L. pneumophila is important for global control of these diseases. Aptamers, short oligonucleotides that bind to targets with high a nity and speci city, have great potential for use in pathogenic bacterium detection, diagnostics, and therapy. Here, we used a whole-cell SELEX (systematic evolution of ligands by exponential enrichment) method to isolate and characterize single-stranded DNA (ssDNA) aptamers against L. pneumophila. A total of 60 ssDNA sequences were identi ed after 17 rounds of selection. Other bacterial species (Escherichia coli, Bacillus subtilis, Pseudomonas syringae, Staphylococcus aureus, Legionella quateirensis, and Legionella adelaidensis) were used for counterselection to enhance the speci city of ssDNA aptamers against L. pneumophila. Four ssDNA aptamers showed strong a nity and high selectivity for L. pneumophila, with K d values in the nanomolar range. Bioinformatic analysis of the most speci c aptamers revealed predicted conserved secondary structures that might bind to L. pneumophila cell walls. In addition, the binding of these four uorescently labeled aptamers to the surface of L. pneumophila was observed directly by uorescence microscopy. This is the rst study to use SELEX to target L. pneumophila whole cells. The aptamers identi ed in this study could be used in the future to develop medical diagnostic tools and public environmental detection assays for L. pneumophila.