Next, we incorporated the modified thymine into a molecular beacon comprising a Cy3 dye on the 5' end and a black hole quencher (BHQ-2) on the 3' end of a 30-base oligomer (Fig. ) and measured its melting temperature by fluorescence spectroscopy (see Fig ). The molecular beacon we chose is based on a hairpin that, without any artificial modification, has a melting temperature (Tm) of 40.4°C under the applied conditions (H-0, Fig. ). We anticipated that incorporating one modified thymine would decrease the Tm due to steric hindrance and electrostatic repulsion of the modified thymine compared to regular thymine. Thus, we designed H-1, which is H-0 with a point-mutation of one thymine for modified thymine in the stem region of the molecular beacon. As expected, we found that its melting temperature was 10.5°C lower compared to H-0. This decrease in Tm was independent of where the mutation was performed (H-2), provided it was in the stem region, i.e., H-3 was modified in the loop region, and its Tm was almost equal to the unmodified H-0. If we added two mutations in the stem region, Tm decreased by 20.6 °C (H-4). Taken together, the incorporation of modified thymines in the stem region destabilizes the duplex. The destabilizing effect of the modified thymine can be attributed to electrostatic repulsion of the carboxylates and /or steric hindrance of the phthalic acid and triazole in the crowded environment of the duplex. To test which of these forces was the dominant one, we synthesized the methyl-ester of modified thymine which lacks the anionic nature compared to modified thymine but is similar in size (Fig. ). When incorporated in the molecular beacon (H-5), we found a small drop in Tm (4.4°C) compared to non-modified H-0. This observation suggests that both a steric hindrance and the presence of the negative charges are responsible for the change in the Tm upon incorporating modified thymine.