Using double-stranded DNA probes to promote specificity in target capture

[Display omitted] ► Double-stranded probes were immobilized on microspheres for displacement studies. ► 15 base-long targets differed in sequence by one base. ► Probes possessing a single-stranded toehold region exhibited target discrimination. ► Displacement rates were measured using flow cytometry. The most prevalent nucleic acid detection schemes employ single-stranded sequences as probes for capture and detection of oligonucleotide targets in solution. In these systems elevated temperature conditions are generally used to enhance specificity and limit false positives from occurring with mismatched targets. In contrast, the current study uses a strand displacement approach between soluble targets and double-stranded DNA probes (dsProbes) immobilized on microspheres. In our approach the displacement of reporter strands from the dsProbes by the target of interest is promoted by the affinity differences between the reporter strand and the soluble DNA or RNA targets for the immobilized sequences. While displacement activity occurred readily in center mismatched dsProbes with a weaker intrinsic affinity, incorporating a two base-long single-stranded segment at the free end of the immobilized dsProbes resulted in target discrimination not observed for dsProbes possessing only a center mismatch.