Concentration-Dependent Study of Nucleic Acid Blockers Used for Sequence-Specificity Enhancement in Nucleic Acids Detection

Smart probes (SP) are fluorescent oligonucleotide hairpin probes that are used for the sequence-specific detection of nucleic acids. These probes are able to discriminate between a perfect target and mismatch sequences, which may have sequences very similar to the perfect target sequence. For mismatch sequences very similar to the target of interest, the SP may be unable to discriminate between the perfect target and mismatch sequences due to non-specific hybridization between the SP and mismatch sequences. To prevent such non-specific hybridization, nucleic acid blockers (NABs) are being used to block possible non-specific interactions. However, the concentration dependence of NAB when used for this application has not been clearly established and the appropriate NAB concentration is largely unknown. In this work, we have performed concentration-dependent experiments with NABs in order to establish the appropriate concentration for blocking non-specific SP-mismatch interactions. We mixed the SP with its complementary target in the presence of mismatch sequences and varying concentrations of NABs that are complementary to the mismatch sequences and monitored the resulting fluorescence signal. The fluorescence signal reported by the SP decreases with increasing NAB concentration in an exponential fashion, reaching a limiting NAB concentration of about 500 nM. When used in a complex mixture consisting of SP, target and excess mismatch sequences and NABs, the fluorescence signals slightly decreased, suggesting target–NAB interaction and/or masking effect by the excess NABs. Thus, our results show optimal NAB/SP molar ratio of 10:1, while higher NAB/SP molar ratio can lead to unintended target–NABs interactions and masking effect.