The hybridization of a long probe containing dInosines is stronger than that of a long probe containing the same amount of 5-Nitroindole, at either high or low temperatures 5-Nitroindole is a second-generation universal base nt analog that was chosen for comparison with the first-generation dInosine with respect to hybridization properties in 3M TMAC. According to Loakes and Brown (1994) , 5-nitroindole is less destabilizing than its 4-and 6-isomers (27) and than 3-nitropyrrole (9). A probe with 18 5-nitroindole residues (5-NitroInd_18) was designed; the nt analogs were distributed to match the pattern of the dInosines in the Ino18 probe ( Figure 3D ). The probes were allowed to hybridize with the InflA target and the set of targets with sm, 33%_xF and 33%_xnt ( Figure 3C ). At 45 C, hybridization of 5-NitroInd_18 with the InflA (44% of the total MFI), 33%_xF (23-37% of the total MFI), and 33%_xnt (4-7% of the total MFI) targets resulted in hybridization signals that were much lower than those seen with the Ino18/InflA (73% of the total MFI), Ino18/33%_xF (54-78% of the total MFI), and Ino18/33%_xnt (47-59% of the total MFI) probes ( Figure 3C, 45 C) . Increasing the temperature to 55 C destabilized the 5-NitroInd_18 probe even more, resulting in hybridization of only 1-8% of the total MFI. In conclusion, dInosine functions much better than 5-nitroindole as a universal nt analog, under 3 M TMAC buffer conditions.