Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania which affects an estimated 12 million people worldwide. The work presented herein describes characterization of several DNA aptamers which were developed against two different 39-amino acid synthetic peptides derived from the highly conserved repetitive regions of a 230 kD flagellar Leishmania kinesin for their potential use in Leishmania diagnostics. Following ten rounds of aptamer affinity selection against the kinesin peptides and PCR amplification (i.e., SELEX), several candidate aptamers demonstrated strong binding to the kinesin peptides in an enzyme-linked aptamer sorbent assay (ELASA). Confocal fluorescence microscopy using... More
Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania which affects an estimated 12 million people worldwide. The work presented herein describes characterization of several DNA aptamers which were developed against two different 39-amino acid synthetic peptides derived from the highly conserved repetitive regions of a 230 kD flagellar Leishmania kinesin for their potential use in Leishmania diagnostics. Following ten rounds of aptamer affinity selection against the kinesin peptides and PCR amplification (i.e., SELEX), several candidate aptamers demonstrated strong binding to the kinesin peptides in an enzyme-linked aptamer sorbent assay (ELASA). Confocal fluorescence microscopy using several aptamers with the highest affinities and the microtubule marker paclitaxel revealed localization patterns characterized by diffuse, but often punctate, staining within the cytoplasm and in some cases along the flagella of L. major promastigotes. Aptamer association with microtubules was further evidenced by aptamer-colloidal gold staining and electron microscopy supporting the binding and specificity of these aptamers for Leishmania kinesins. In addition, Southwestern blots confirmed that some of the top aptamers were specific for their respective ∼4 kD peptides. One aptamer in particular (K2-13R) also demonstrated clear detection of a 100 kD protein band and >250 kD bands in L. major promastigote lysate. Given that the presence of antibodies recognizing the 39 kD kinesin (K39) or repetitive region of the 230 kD kinesin in patient serum is useful for the serodiagnosis of visceral leishmaniasis, but that few commercial antibodies exist for detection of K39 itself, these kinesin aptamers may have value as novel cellular and molecular probes for Leishmania diagnosis.
