Surpassing Specificity Limits of Nucleic Acid Probes via Cooperativity

Abstract

The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions. These cooperative probe constructs (Tentacle probes) were then compared by modeling thermodynamic and hybridization characteristics to both Molecular Beacons (stem loop DNA probes) and Taqman probes (a linear oligonucleotide). The biophysical models reveal that cooperative probes compared with either Molecular beacons or Taqman probes have enhanced specificity. This was a result of increased melting temperature differentials and the concentration-independent hybridization revealed between wild-type and variant sequences. We believe these findings of order of magnitude enhanced melting temperature differentials with probes possessing concentration independence and more favorable binding kinetics have the potential to significantly improve molecular diagnostic assay functionality.