A single-molecule study reveals novel rod-like structures formed by a thrombin aptamer repeat sequence

Thrombin aptamers (TBAs) have attracted much attention due to their various applications. The structures and properties of long ssDNA chains with multiple TBA repeat sequences are interesting and distinct from those of 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 methods. In this work, we investigated the repeated ssDNA by using 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 the 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 the TBA copy number can unitize the diversity of TBA conformation to the best-fit 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.