Construction of Aptamer-Based Molecular Beacons with Varied Blocked Structures and Targeted Detection of Thrombin

A kind of blocked aptamer-functionalized molecular beacon (MB) was designed as fluorescence sensors to detect thrombins by binding-induced turn on structural transformation. Three MBs named MB(8 + 8), MB(15 + 8), and MB(15 + 6) consisted of two single-stranded oligonucleotides. One long single-stranded oligonucleotide (abbreviated as SS) contained a thrombin aptamer sequence and was modified with a fluorescence group and quenching group on each end side. Another short single-stranded oligonucleotide (written as cDNA) was partially complementary to the long SS. It was interesting to find that the complementary sequence length of cDNA greatly influenced the structure of the MBs. The construction of MB experiments proved that MB(8 + 8) and MB(15 + 8) could form the quenching MBs but MB(15 + 6) could not. MB(8 + 8) was composed of a SS strand paired with a complementary cDNA(8 + 8), which was called one-to-one combination, while MB(15 + 8) was two-to-two combination and MB(15 + 6) was one-to-two combination. When the ratio of SS and cDNA (15 + 8) was 1:1, the quenching efficiency reached maximum. But with the molar ratio of SS and cDNA(8 + 8) increasing, the quenching efficiency increased continuously. Under the optimal conditions that we studied, the detection limit of thrombin by MB(8 + 8) and MB(15 + 8) was 0.19 and 1.2 nM, respectively. In addition, the assay proved to be selective, and the average recovery of thrombin detected by MB(8 + 8) and MB(15 + 8) in diluted serum was 95.4 and 94.5%, respectively.

Automated summary

A blocked aptamer-functionalized molecular beacon (MB) was designed as fluorescence sensors to detect thrombins by binding-induced turn-on structural transformation. The length of the complementary cDNA greatly influenced the structure of MBs, with MB(8 + 8) and MB(15 + 8) forming quenching MBs. The quenching efficiency of MBs was found to increase with the molar ratio of SS and cDNA, with MB(8 + 8) and MB(15 + 8) showing detection limits of 0.19 and 1.2 nM for thrombin, respectively. Selectivity and recovery rates in diluted serum samples were also demonstrated.