Hybridization of the InflA probe with targets containing 74% mismatching 70-mers with one perfectly matching end of various lengths (74%xnt) was compared with hybridization of the InflA probe with short, perfectly matching targets (12-22 nt_free; Figure 3A , B and D; nt sequences in Supplementary Table S2B ) to analyze the effect of long mismatching ends. Utilizing the InflA probe / InflA target as reference, the 12nt_free target did not hybridize at 45 C (1.8% of the total MFI, 106 MFI) but the 15-, 18-and 22-nt_free targets gave successively stronger MFI signals (62, 98 and 123% of the total MFI; 3669, 5770 and 7269 MFI, respectively) than the 74%15nt, 74%18nt and 74%22nt targets (17.4, 55 and 62% of the total MFI; 1029, 3222 and 3637 MFI, respectively). Thus, the MFI is higher when the short, perfectly matching targets (xnt_free) hybridize with the InflA probe with only one long end protruding from the hybridized portion of the probe. Previous reports have demonstrated that 1-5 nt single dangling ends tend to stabilize duplex formation (23) (24) (25) (26) . This study shows that two long mismatching ends destabilize the hybridization of the matching part of the duplex. The long free mismatching ends could form intramolecular secondary structures that could have an effect on the hybridizing duplex. 