Single-molecule study reveals novel rod-like structures formed by the thrombin aptamer repeat sequence
Thrombin aptamer (TBA) has attracted much attention due to its various applications. The structures and properties of long ssDNA chains with multiple TBA repeat sequences are interesting and distinct from their monomers. Due to the complexity of the sample system, it is quite difficult to reveal the structure of such a long-chain ssDNA using traditional method. In this work, we investigated the repeated ssDNA by single-molecule magnetic tweezers and AFM imaging. To do that we developed the polymerase change-rolling circle amplification (PC-RCA) synthetic method and prepared two-end modified repeated ssDNA. The rod-like G4 structures formed by intramolecular stacking of repeat sequence were for the first time identified. This novel structure is different from those higher-order quadruplex structures formed by G-tetrads or loop-mediated interactions. It is also quite interesting to find that the increase of TBA copy number can unitize the diversity of TBA conformation to fittest binding structure for thrombin. The methodology developed in this work can be used for studying other repeat sequences in the genome, such as telomeric DNA as well as interactions of ssDNA with the binding molecule.