Life science is often focused on the microscopic level. Single-molecule technology has been used to observe components at the micro- or nanoscale. Single-molecule imaging provides unprecedented information about the behavior of individual molecules in contrast to the information from ensemble methods that average the information of many molecules in various states. A typical feature of living systems is motion. The lack of synchronicity of motion biomachines in living systems makes it challenging to image the motion process with high resolution. Thus, single-molecule technology is especially useful for real-time study on motion mechanism of biomachines, such as viral DNA packaging motor, or other ATPases. The most common optical instrumentations in single-molecule studies are optical tweezers and single molecule total internal refection fluorescence microscopy (smTIRF). Optical tweezers are the force-based technique. The analysis of RNA using optical tweezer has led to the discovery of the rubbery or amoeba property of RNA nanoparticles for compelling vessel extravasation to enhance tumor targeting and fast renal excretion. The rubbery property of RNA lends mechanistic evidence for RNAs use as an ideal reagent in cancer treatment with undetectable toxicity. Single molecule photobleaching allows for the direct counting of biomolecules. This technique was invented for single molecule counting of RNA in the phi29 DNA packaging motor to resolve the debate between five and six copies of RNA in the motor. The technology has subsequently extended to counting components in biological machines composed of protein, DNA, and other macromolecules. In combination with statistical analysis, it reveals biomolecular mechanisms in detail and leads to the development of ultra-sensitive sensors in diagnosis and forensics. This review focuses on the applications of optical tweezers and fluorescence-based techniques as single-molecule technologies to resolve mechanistic questions related to RNA and DNA nanostructures.

Ribonucleic acid (RNA) nanotechnology platforms have the potential of harboring therapeutics for in vivo delivery in disease treatment. However, the nonspecific interaction between the harbored hydrophobic drugs and cells or other components before reaching the diseased site has been an obstacle in drug delivery. Here we report an encapsulation strategy to prevent such nonspecific hydrophobic interactions in vitro and in vivo based on a self-assembled three-dimensional (3D) RNA nanocage. By placing an RNA three-way junction (3WJ) in the cavity of the nanocage, the conjugated hydrophobic molecules were specifically positioned within the nanocage, preventing their exposure to the biological environment. The assembly of the nanocages was characterized by native polyacrylamide gel electrophoresis (PAGE), atomic force microscopy (AFM), and cryogenic electron microscopy (cryo-EM) imaging. The stealth effect of the nanocage for hydrophobic molecules in vitro was evaluated by gel electrophoresis, flow cytometry, and confocal microscopy. The in vivo sheathing effect of the nanocage for hydrophobic molecules was assessed by biodistribution profiling in mice. The RNA nanocages with hydrophobic biomolecules underwent faster clearance in liver and spleen in comparison to their counterparts. Therefore, this encapsulation strategy holds promise for in vivo delivery of hydrophobic drugs for disease treatment.

Nanotubes are miniature materials with significant potential applications in nanotechnological, medical, biological and material sciences. The quest for manufacturing methods of nano-mechanical modules is in progress. For example, the application of carbon nanotubes has been extensively investigated due to the precise width control, but the precise length control remains challenging. Here we report two approaches for the one-pot self-assembly of RNA nanotubes. For the first approach, six RNA strands were used to assemble the nanotube by forming a 11 nm long hollow channel with the inner diameter of 1.7 nm and the outside diameter of 6.3 nm. For the second approach, six RNA strands were designed to hybridize with their neighboring strands by complementary base pairing and formed a nanotube with a six-helix hollow channel similar to the nanotube assembled by the first approach. The fabricated RNA nanotubes were characterized by gel electrophoresis and atomic force microscopy (AFM), confirming the formation of nanotube-shaped RNA nanostructures. Cholesterol molecules were introduced into RNA nanotubes to facilitate their incorporation into lipid bilayer. Incubation of RNA nanotube complex with the free-standing lipid bilayer membrane under applied voltage led to discrete current signatures. Addition of peptides into the sensing chamber revealed discrete steps of current blockage. Polyarginine peptides with different lengths can be detected by current signatures, suggesting that the RNA-cholesterol complex holds the promise of achieving single molecule sensing of peptides.

Stimuli-responsive release of drugs from a nanocarrier in spatial-, temporal-, and dosage-controlled fashions is of great interest in the pharmaceutical industry. Paclitaxel is one of the most effective and popular chemotherapeutic drugs against a number of cancers such as metastatic or nonmetastatic breast cancer, non-small cell lung cancer, refractory ovarian cancer, AIDS-related Kaposi's sarcoma, and head and neck cancers. Here, by taking the advantage of RNA nanotechnology in biomedical and material science, we developed a three-dimensional pyramid-shaped RNA nanocage for a photocontrolled release of cargo, using paclitaxel as a model drug. The light-triggered release of paclitaxel or fluorophore Cy5 was achieved by incorporation of photocleavable spacers into the RNA nanoparticles. Upon irradiation with ultraviolet light, cargos were rapidly released (within 5 min). In vitro treatment of breast cancer cells with the RNA nanoparticles harboring photocleavable paclitaxel showed higher cytotoxicity as compared to RNA nanoparticles without the photocleavable spacer. The methodology provides proof of concept for the application of the light-triggered controlled release of drugs from RNA nanocages.