Anti-Francisella tularensis DNA aptamers detect tularemia antigen from different subspecies by Aptamer-Linked Immobilized Sorbent Assay

Abstract

Aptamers are powerful candidates for molecular detection of targets due to their unique recognition properties. These affinity probes can be used to recognize and bind to their targets in the various types of assays that are currently used to detect and capture molecules of interest. They are short single-stranded (ss) oligonucleotides composed of DNA or RNA sequences that are selected in vitro based on their affinity and specificity for the target. Using combinatorial oligonucleotide libraries, we have selected ssDNA aptamers that bind to Francisella tularensis subspecies (subsp) japonica bacterial antigen. F. tularensis is an intracellular, nonmotile, nonsporulating, Gram-negative bacterial pathogen that causes tularemia in man and animals. Just as antibodies have been used to detect specific targets in varying formats, it is possible that nucleic acid-binding species or aptamers could be used to specifically detect biomolecules. Aptamers offer advantages over antibody-based affinity molecules in production, regeneration and stability due to their unique chemical properties. We have successfully isolated a set of 25 unique DNA sequences that specifically bind to F. tularensis subspecies japonica. When tested in a sandwich Aptamer-Linked Immobilized Sorbent Assay (ALISA) and dot blot analysis, the aptamer cocktail exhibited specificity in its ability to bind only to tularemia bacterial antigen from subspecies japonica, holarctica (also known as palaearctica) and tularensis but not to Bartonella henselae. Moreover, there is no binding observed either to pure chicken albumin or chicken lysozyme. Thus, it appears that this novel antitularemia aptamer cocktail may find application as a detection reagent for a potential biological warfare agent like F. tularensis.