Selection and characterization of DNA aptamers targeting the surface borrelial protein CspZ with high-throughput cross-over SELEX

Abstract

Lyme borreliosis (LB) is the most prevalent tick-borne illness, with an estimated 700 000 cases annually in the United States and Europe. LB diagnosis based on two-tiered serology remains controversial due to its indirect nature and low sensitivity during the early stage of the disease. Aptamers are single-stranded DNA or RNA oligonucleotides that exhibit high selectivity and specificity for their target due to their unique three-dimensional structure. By applying cross-over-SELEX process, an enrichment of DNA oligonucleotide sequences against a surface protein of Borrelia, named CspZ, has been performed and monitored using absorbance at 260 nm, melting curves and NGS analyses. Beyond sequence enrichment, oligonucleotides binding to CspZ were observed during the selection rounds by Dot Blot and beads assays. Thirteen unique and highly redundant oligonucleotide sequences have been further characterized using multiple approaches such as Dot Blot, BioLayer Interferometry and Surface Plasmon Resonance. The selected aptamers showed KD values from tens of nanomolar to the micromolar range by BLI and SPR. Two aptamers, characterized by flow cytometry and epifluorescence microscopy, were able to specifically recognize Borrelia burgdorferi sensu stricto. This novel strategy holds promise for the development of an improved diagnosis assay. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/632687v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@3a2697org.highwire.dtl.DTLVardef@1bba993org.highwire.dtl.DTLVardef@17a0a60org.highwire.dtl.DTLVardef@13f4a4f_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIDNA aptamers were selected by cross-over SELEX. C_LIO_LIAptamers KDs ranged from tens of nanomolar to the micromolar range. C_LIO_LIAptamer characterization was performed by Dot Blot, beads assay, BLI, and SPR. C_LIO_LIInteraction with Borrelia was tested by flow cytometry and epifluorescence microscopy. C_LIO_LIAptamers bind to both the recombinant and borrelial surface CspZ protein. C_LI