Peixuan Guo

The emerging field of RNA nanotechnology

Like DNA, RNA can be designed and manipulated to produce a variety of different nanostructures. Moreover, RNA has a flexible structure and possesses catalytic functions that are similar to proteins. Although RNA nanotechnology resembles DNA nanotechnology in many ways, the base-pairing rules for constructing nanoparticles are different. The large variety of loops and motifs found in RNA allows it to fold into numerous complicated structures, and this diversity provides a platform for identifying viable building blocks for various applications. The thermal stability of RNA also allows the production of multivalent nanostructures with defined stoichiometry. Here we review techniques for constructing RNA nanoparticles from different building blocks, we describe the distinct attributes of RNA inside the body, and discuss potential applications of RNA nanostructures in medicine. We also offer some perspectives on the yield and cost of RNA production.

Inter-RNA Interaction of Phage φ29 pRNA to Form a Hexameric Complex for Viral DNA Transportation

Ds-DNA viruses package their DNA into a preformed protein shell (procapsid) during maturation. Bacteriophage phi29 requires an RNA (pRNA) to package its genomic DNA into the procapsid. We report here that the pRNA upper and lower loops are involved in RNA/RNA interactions. Mutation in only one loop results in inactive pRNAs. However, mixing of two, three and six inactive mutant pRNAs restores DNA packaging activity as long as an interlocking hexameric ring can be predicted to form by base pairing of the mutated loops in separate RNA molecules. The stoichiometry of pRNA for the packaging of one viral DNA genome is six. Homogeneous pRNA purified from a single band in denaturing gels showed six bands when rerun in native gels. These results suggest that six pRNAs form a hexameric ring by the intermolecular interaction of two RNA loops to serve as part of the DNA transportation machinery.

Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics

RNA nanoparticles have applications in the treatment of cancers and viral infection; however, the instability of RNA nanoparticles has hindered their development for therapeutic applications. The lack of covalent linkage or crosslinking in nanoparticles causes dissociation in vivo. Here we show that the packaging RNA of bacteriophage phi29 DNA packaging motor can be assembled from 3–6 pieces of RNA oligomers without the use of metal salts. Each RNA oligomer contains a functional module that can be a receptor-binding ligand, aptamer, short interfering RNA or ribozyme. When mixed together, they self-assemble into thermodynamically stable tri-star nanoparticles with a three-way junction core. These nanoparticles are resistant to 8 M urea denaturation, are stable in serum and remain intact at extremely low concentrations. The modules remain functional in vitro and in vivo, suggesting that the three-way junction core can be used as a platform for building a variety of multifunctional nanoparticles. We studied 25 different three-way junction motifs in biological RNA and found only one other motif that shares characteristics similar to the three-way junction of phi29 pRNA. The three-way junction domain of the phi29 bacteriophage can be assembled from three pieces of RNA oligomers to form stable multifunctional nanoparticles that are useful for the treatment of different diseases.

Engineering RNA for Targeted siRNA Delivery and Medical Application

Abstract RNA engineering for nanotechnology and medical applications is an exciting emerging research field. RNA has intrinsically defined features on the nanometre scale and is a particularly interesting candidate for such applications due to its amazing diversity, flexibility and versatility in structure and function. Specifically, the current use of siRNA to silence target genes involved in disease has generated much excitement in the scientific community. The intrinsic ability to sequence-specifically downregulate gene expression in a temporally- and spatially controlled fashion has led to heightened interest and rapid development of siRNA-based therapeutics. Although methods for gene silencing have been achieved with high efficacy and specificity in vitro, the effective delivery of nucleic acids to specific cells in vivo has been a hurdle for RNA therapeutics. This article covers different RNA-based approaches for diagnosis, prevention and treatment of human disease, with a focus on the latest developments of non-viral carriers of siRNA for delivery in vivo. The applications and challenges of siRNA therapy, as well as potential solutions to these problems, the approaches for using phi29 pRNA-based vectors as polyvalent vehicles for specific delivery of siRNA, ribozymes, drugs or other therapeutic agents to specific cells for therapy will also be addressed.

Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores.

Biological pores have been used to study the transport of DNA and other molecules but most pores have channels that allow only the movement of small molecules and single-stranded DNA and RNA. The bacteriophage phi29 DNA-packaging motor, which allows double-stranded DNA to enter and exit during a viral infection, contains a connector protein that has a 3.6 – 6.0 nm wide channel. Here we show that a modified version of the connector protein, when reconstituted into liposomes and inserted into planar lipid bilayers, can act as conductive channels to allow the translocation of double-stranded DNA. Single-channel conductance assays and quantitative PCR confirmed the translocation through the pore. The measured conductance of a single connector channel was 4.8 nS in 1 M KCl. This engineered and membrane-adapted phage connector is expected to have interesting applications in nanotechnology and nanomedicine, such as MEMS sensing, microreactors, gene delivery, drug loading, and DNA sequencing.

Bottom-up Assembly of RNA Arrays and Superstructures as Potential Parts in Nanotechnology

DNA and protein have been extensively scrutinized for feasibility as parts in nanotechnology, but another natural building block, RNA, has been largely ignored. RNA can be manipulated to form versatile shapes, thus providing an element of adaptability to DNA nanotechnology, which is predominantly based upon a double-helical structure. The DNA-packaging motor of bacterial virus phi29 contains six DNA-packaging RNAs (pRNA), which together form a hexameric ring via loop/loop interaction. Here we report that this pRNA can be redesigned to form a variety of structures and shapes, including twins, tetramers, rods, triangles, and 3D arrays several microns in size via interaction of programmed helical regions and loops. Three dimensional RNA array formation required a defined nucleotide number for twisting of the interactive helix and a palindromic sequence. Such arrays are unusually stable and resistant to a wide range of temperatures, salt concentrations, and pH.

Counting of six pRNAs of phi29 DNA-packaging motor with customized single-molecule dual-view system

Direct imaging or counting of RNA molecules has been difficult owing to its relatively low electron density for EM and insufficient resolution in AFM. Bacteriophage phi29 DNA‐packaging motor is geared by a packaging RNA (pRNA) ring. Currently, whether the ring is a pentagon or hexagon is under fervent debate. We report here the assembly of a highly sensitive imaging system for direct counting of the copy number of pRNA within this 20‐nm motor. Single fluorophore imaging clearly identified the quantized photobleaching steps from pRNA labeled with a single fluorophore and concluded its stoichiometry within the motor. Almost all of the motors contained six copies of pRNA before and during DNA translocation, identified by dual‐color detection of the stalled intermediates of motors containing Cy3‐pRNA and Cy5‐DNA. The stalled motors were restarted to observe the motion of DNA packaging in real time. Heat‐denaturation analysis confirmed that the stoichiometry of pRNA is the common multiple of 2 and 3. EM imaging of procapsid/pRNA complexes clearly revealed six ferritin particles that were conjugated to each pRNA ring.

Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma is a poorly differentiated upper respiratory tract cancer that highly expresses human folate receptors (hFR). Binding of folate to hFR triggers endocytosis. The folate was conjugated into adenosine 5′-monophosphate (AMP) by 1,6-hexanediamine linkages. After reverse HPLC to reach 93% purity, the folate–AMP, which can only be used for transcription initiation but not for chain extension, was incorporated into the 5′-end of bacteriophage phi29 motor pRNA. A 16:1 ratio of folate–AMP to ATP in transcription resulted in more than 60% of the pRNA containing folate. A pRNA with a 5′-overhang is needed to enhance the accessibility of the 5′ folate for specific receptor binding. Utilizing the engineered left/right interlocking loops, polyvalent dimeric pRNA nanoparticles were constructed using RNA nanotechnology to carry folate, a detection marker, and siRNA targeting at an antiapoptosis factor. The chimeric pRNAs were processed into ds-siRNA by Dicer. Incubation of nasopharyngeal epidermal carcinoma (KB) cells with the dimer resulted in its entry into cancer cells, and the subsequent silencing of the target gene. Such a protein-free RNA nanoparticle with undetectable antigenicity has a potential for repeated long-term administration for nasopharyngeal carcinoma as the effectiveness and specificity were confirmed by ex vivo delivery in the animal trial.

Conditions and pathway to assemble pRNA hexamers that gear phage phi29 DNA translocating machine

Six RNA (pRNA) molecules form a hexamer, via hand-in-hand interaction, to gear bacterial virus phi29 DNA translocation machinery. Here we report the pathway and the conditions for the hexamer formation. Stable pRNA dimers and trimers were assembled in solution, isolated from native gels, and separated by sedimentation, providing a model system for the study of RNA dimers and trimers in a protein-free environment. Cryo-atomic force microscopy revealed that monomers displayed a ✓ outline, dimers exhibited an elongated shape, and trimers formed a triangle. Dimerization of pRNA was promoted by a variety of cations including spermidine, whereas procapsid binding and DNA packaging required specific divalent cations, including Mg2+, Ca2+, and Mn2+. Both the tandem and fused pRNA dimers with complementary loops designed to form even-numbered rings were active in DNA packaging, whereas those without complementary loops were inactive. We conclude that dimers are the building blocks of the hexamer, and the pathway of building a hexamer is: dimer → tetramer → hexamer. The Hill coefficient of 2.5 suggests that there are three binding sites with cooperative binding on the surface of the procapsid. The two interacting loops played a key role in recruiting the incoming dimer, whereas the procapsid served as the foundation for hexamer assembly.

Sequence requirement for hand-in-hand interaction in formation of RNA dimers and hexamers to gear phi29 DNA translocation motor.

Translocation of DNA or RNA is a ubiquitous phenomenon. One intricate translocation process is viral DNA packaging. During maturation, the lengthy genome of dsDNA viruses is translocated with remarkable velocity into a limited space within the procapsid. We have revealed that phi29 DNA packaging is accomplished by a mechanism similar to driving a bolt with a hex nut, which consists of six DNA-packaging pRNAs. Four bases in each of the two pRNA loops are involved in RNA/RNA interactions to form a hexagonal complex that gears the DNA translocating machine. Without considering the tertiary interaction, in some cases only two G/C pairs between the interacting loops could provide certain pRNAs with activity. When all four bases were paired, at least one G/C pair was required for DNA packaging. The maximum number of base pairings between the two loops to allow pRNA to retain wild-type activity was five, whereas the minimum number was five for one loop and three for the other. The findings were supported by phylogenetic analysis of seven pRNAs from different phages. A 75-base RNA segment, bases 23-97, was able to form dimer, to interlock into the hexamer, to compete with full-length pRNA for procapsid binding, and therefore to inhibit phi29 assembly in vitro. Our result suggests that segment 23-97 is a self-folded, independent domain involved in procapsid binding and RNA/RNA interaction in dimer and hexamer formation, whereas bases 1-22 and 98-120 are involved in DNA translocation but dispensable for RNA/RNA interaction. Therefore, this 75-base RNA could be a model for structural studies in RNA dimerization.