Targeted delivery of RNAi to cancer cells using RNA-ligand displaying exosome

Exosome is an excellent vesicle for in vivo delivery of therapeutics,including RNAi and chemical drugs.The extremely high efficiency in cancer regression can partly be attributed to its fusion mechanism in delivering therapeutics to cytosol without endosome trapping.However,being composed of a lipidbilayer membrane without specific recognition capacity for aimed-cells,the entry into nonspecific cells can lead to potential side-effects and toxicity.Applying engineering approaches for targeting-capacity to deliver therapeutics to specific cells is desirable.Techniques with chemical modification in vitro and genetic engineering in cells have been reported to decorate exosomes with targeting ligands.RNA nanoparticles have been used to harbor tumor-specific ligands displayed on exosome surface.The negative charge reduces nonspecific binding to vital cells with negatively charged lipid-membrane due to the electrostatic repulsion,thus lowering the side-effect and toxicity.In this review,we focus on the uniqueness of RNA nanoparticles for exosome surface display of chemical ligands,small peptides or RNA aptamers,for specific cancer targeting to deliver anticancer therapeutics,highlighting recent advances in targeted delivery of siRNA and miRNA that overcomes the previous RNAi delivery roadblocks.Proper understanding of exosome engineering with RNA nanotechnology promises efficient therapies for a wide range of cancer subtypes.

Photo-controlled release of paclitaxel and model drugs from RNA pyramids

Stimuli-resp on sive release of drugs from a nano carrier in spatial-,temporal-,and dosage-controlled fashi ons 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 non metastatic breast can cer,non-small cell lung can cer,refractory ovaria n cancer,AIDS-related Kaposi's sarcoma,and head and neck can cers.Here,by taki ng the adva ntage of RNA nanotechno logy in biomedical and material scie nee,we developed a three-dime nsional 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 can cer cells with the RNA nano particles harbori ng photocleavable paclitaxel showed higher cytotoxicity as compared to RNA nanoparticles without the photocleavable spacer.The methodology provides proof of con cept for the applicati on of the light-triggered con trolled release of drugs from RNA nano cages.

Construction of RNA nanotubes

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.

Translation of the long-term fundamental studies on viral DNA packaging motors into nanotechnology and nanomedicine

Many years of fundamental studies on viral genome packaging motors have led to fruitful applications.The double-stranded DNA(dsDNA)viruses package their genomes into preformed protein shells via nanomotors including several elegant and meticulous coaxial modules.The motor is geared by the hexameric RNA ring.An open washer displayed as hexametric string of phi29 motor ATPase has been reported.The open washer linked into a filament as a queue with left-handed chirality along the dsDNA chain.It was found that a free 5′-and 3′-dsDNA end is not required for one gp16 dimer and four monomers to assemble into the hexametric washer on dsDNA.The above studies have inspired several applications in nanotechnology and nanomedicine.These applications include:(i)studies on the precision motor channels have led to their application in the single pore sensing;(ii)investigations into the hand-in-hand integration of the hexametric pRNA ring have resulted in the emergence of the new field of RNA nanotechnology;and(iii)the studies on the motor stoichiometry of homologous multi-subunits that subsequently have inspired the discovery of new methods in highly potent drug development.This review focuses on the structure and function of the viral DNA packaging motors and describes how fundamental studies inspired various applications.Given these advantages,more nanotechnological and biomedical applications using bacteriophage motor components are expected.

3D RNA nanocage for encapsulation and shielding of hydrophobic biomolecules to improve the in vivo biodistribution

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.

High resolution structure of hexameric herpesvirus DNA-packaging motor elucidates revolving mechanism and ends 20-year fervent debate

In this issue of the Protein&Cell Journal,a research team(from Institute of Biophysics,Chinese Academy of Sciences)led by professors Xiangxi Wang and Zihe Rao report the high-resolution structural and physical properties of the ATPdriven DNA packaging motor of the double-stranded(ds)herpesvirus(Yang et al.2020).The structures of the portal vertex,the channel hub of the DNA packaging motor were also investigated,revealing essential protein-protein interactions in the assembly and maturation of herpesvirus procapsid(Chen et al.2020;Wang et al.2020).Their impressive data clearly demonstrated that the herpesvirus DNA packaging motor forms a hexameric structure and utilizes the revolving mechanism instead of rotation(Fig.1).This is the first paper of its kind,with images in angstromscale resolution,to convincingly elucidate the structure data to end the 20-year debate on whether the structure or the viral DNA packaging motor is pentamer or hexamer,and whether the motion mechanism is rotation or revolution.