Atomic force microscope-based single-molecule force spectroscopy of RNA unfolding

Single-molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) has emerged as an important tool for probing biomolecular interaction and exploring the forces, dynamics, and energy landscapes that underlie function and specificity of molecular interaction. These studies require attaching biomolecules on solid supports and AFM tips to measure unbinding forces between individual binding partners. Herein we describe efficient and robust protocols for probing RNA interaction by AFM and show their value on two well-known RNA regulators, the Rev-responsive element (RRE) from the HIV-1 genome and an adenine-sensing riboswitch. The results show the great potential of AFM–SMFS in the investigation of RNA molecular interactions, which will contribute to the development of bionanodevices sensing single RNA molecules.