Thymine-based molecular beacon for sensing adenosine based on the inhibition of S-adenosylhomocysteine hydrolase activity

This study presents a thymine (T)-based molecular beacon (MB) used for probing S-adenosylhomocysteine hydrolase (SAHH)-catalyzed hydrolysis of S-adenosylhomocysteine (SAH) and for sensing adenosine based on the inhibition of SAHH activity. The designed MB (T8-MB-T8) contained a 15-mer loop and a stem that consisted of a pair of 8-mer T bases, a fluorophore unit at the 5′-end, and a quencher unit at the 3′-end. In the presence of Hg2+, a change in the conformation of T8-MB-T8 placed the fluorophore unit and the quencher in proximity to each other and caused collisional quenching of fluorescence between them. The Hg2+-induced fluorescence quenching of T8-MB-T8 occurred because the Hg2+ induced T-T mismatches to form stable T-Hg2+-T coordination in the MB stem. SAHH catalyzed the hydrolysis of SAH to produce homocysteine. The generated homocysteine enabled the Hg2+ to be removed from a hairpin-shaped T8-MB-T8 through the formation of a strong Hg2+-S bond, leading to the restoration of its fluorescence. The T8-MB-T8·Hg2+ probe showed a limit of detection for SAHH of 4 units L−1 (approximately 0.24 nM) and was reusable for detecting the SAHH/SAH system. Because adenosine was an effective SAHH activity inhibitor, the T8-MB-T8·Hg2+ probe combining the SAHH and SAH systems was used for sensitive and selective detection of adenosine in urine without the interference of other adenosine analogs.