Peixuan Guo

The emergence of new viral diseases and viral drug resistance presents a critical challenge in human health. Recent structural and mechanistic studies have revealed remarkable parallels and structural similarities among viral dsDNA- and dsRNA-packaging motors, ATPases, ATP-binding cassette drug transporters (ABCs), and ATP-generating machines, particularly in their asymmetrical, multi-homosubunit hexameric arrangement and sequential revolving mechanisms. Both dsDNA and dsRNA viruses package their genomes into preformed hexameric ATPase motors. This review examines how understanding these evolutionarily convergent nanomotors can inform novel therapeutic strategies. We first review the structures and mechanisms of representative ds-genome packaging motors from bacteriophage phi29, human herpesvirus, dsRNA viruses, cystoviruses, and other systems. We systematically compare viral DNA packaging motors with ABC transporters, cataloguing over 30 major ATPase systems across eight functional categories. Their commonalities—multi-homosubunit architecture with sequential revolving mechanisms regulated by ATP—are summarized and classified by therapeutic potential. We then describe a novel drug development method targeting multi-homosubunit biomachines through the “Series-Circuit-Christmas-Bulb” mechanism, where Z > 1 (subunit stoichiometry) and K = 1 (drugged subunits needed for inhibition). Blocking one subunit disrupts the entire sequential chain, analogous to one single series-circuit Christmas lightbulb failure, disabling the entire strand. Mathematical validation using phi29 systems demonstrates 10 ⁷ -10 ⁸ fold inhibition efficiency. Practical examples include FDA-approved bedaquiline targeting bacterial F ₁ F ₀ ATPase for tuberculosis treatment and RNA nanoparticle-based delivery systems targeting P-glycoprotein for cancer drug resistance. The convergence of nano-structural insights, computational methods, and advanced delivery systems presents a unique framework for developing potent therapies against viral infections and drug resistance mechanisms.

Thermostable RNA motifs have been shown to be a unique material for building nanoparticles for many applications (Guo lab, Nature Nanotechnology, 2020 PMID: 21102465; 2021 PMID: 21909084). Here, we report the discovery of a G-rich ultra-stable human ncRNA G-quadruplex that binds ATP with a strong potential to become a drug-carrying nanoparticle. Motion is a key feature of living systems, which can be achieved with ATPase biomotors. Humans produce, metabolize, and consume ATP per day in an amount equivalent to their body weight. Due to the abundance of noncoding RNA (ncRNA) discovered in the human body, it is expected that many short or long noncoding RNAs will be involved in ATP binding and hydrolysis. This is based on the fact that, although all currently reported ATPases are proteins, only 1.5% of the human DNA genome codes for proteins, while the remaining 98.5% codes for ncRNAs that play crucial roles in regulating life functions. We discovered an endogenous human ncRNA G-quadruplex that binds ATP. This ATP-binding RNA was discovered via in vitro screening of endogenous ncRNAs extracted from human cells. This ATP-binding ncRNA, which was found to be G-quadruplex-forming, was characterized by ATP column binding and circular dichroism. The tightly folded, compact RNA nanoparticles will provide a new class of functional nanomaterials as both targets and carriers for drug conjugation and therapeutic delivery.

Chemotherapeutics are widely used in cancer treatments, but their toxicity, bioavailability and solubility present challenges. RNA nanotechnology has emerged as a promising modality for targeted delivery of chemotherapeutics. Structurally, RNA is thermostable, while conformationally it is dynamic and flexible. RNA's unique deformability and motility lead to rapid spontaneous tumor accumulation and glomerular excretion, thus fast body clearance, while its anionic charge and favorable small size prevent accumulation in vital organs, resulting in undetectable toxicity. We developed branched 4-way junction (4WJ) nanoparticles that were stable with a melting temperature >80 °C, even when conjugated with 24 drugs per 4WJ. Each 4WJ RNA component strand can conjugate six molecules of hydrophobic chemotherapeutic drugs, such as camptothecin, paclitaxel and SN-38. Thus, each 4WJ carries a total of 24 drug molecules spaced to prevent aggregation. RNA conjugation improved paclitaxel water solubility 32,000-fold. This protocol describes the construction of 4WJ RNA drug complexes for cancer therapy. Specific procedures include the modification of chemical drugs, conjugation of multiple prodrug molecules to each synthesized RNA component strand, assembly of RNA nanoparticles and their purification and characterization. Prodrugs are conjugated to RNA nanoparticles via efficient click chemistry, creating an ester linker that is cleaved by esterases in tumor tissues or cells, allowing the prodrugs to return back to their original structures and chemistry upon delivery and release, minimizing toxicity. Inclusion of tumor targeting ligands demonstrated specific delivery of high payload chemotherapeutics to tumors, controlled release of chemical drugs and strong tumor inhibition.

Viruses and other microorganisms are constantly mutating and emerging in different regions. Here, we apply nanotechnology to examine the primary, secondary, and tertiary structures of novel noncoding RNA nanoparticles and find that primary sequences (1D) vary widely between viruses, while secondary (2D) and tertiary structures (3D) are highly conserved. The uniqueness of the phi29 system makes this reported study possible. RNA evolution studies are relevant to the concept of RNA as the origin of life. The recent discovery that RNA is a motile and recombinant entity supports the hypothesis that RNA is the substance of life. Previously, we reported noncoding RNA nanoparticles packaging RNA (pRNA) of the Bacillus virus phi29 (Nature Nanotechnology, 2011, PMID: 21909084; Nature Nanotechnology, 2010, PMID: 21102465; Science, 1987, PMID: 3107124). Phi29 can infect spore-forming Bacillus subtilis, which has long been hidden in the soil with slow or no evolution due to the protection of spores. They are living fossils. The pRNA of the phi29 DNA packaging motor drives the viral motor for DNA transport. In this study, we used existing datasets to search for more pRNAs. Their primary sequence diversity makes it challenging to identify them from the whole genomes of other species. Using the top-down (1D) approach and the bottom-up assembly (3D) approaches, we found that their 2D and 3D structures are highly conserved. Structural conservation enabled us to apply the two-dimensional structure-based approach to find these ncRNAs from databases and identify 12 new pRNAs. The presence of two additional components in the genome, a motor channel protein and a motor ATPase, further confirmed the authenticity of these pRNAs and supported the conclusion that these pRNAs are motor-driven components. Highly conserved and paired left and right loops for assembling the pRNA hexamer were identified in all 12 pRNAs. Artificial modification of the pairing and determination of the virion production activity of the mutated phi29 pRNA further confirmed the conclusion that the secondary and tertiary structures are highly conserved. Understanding the retention, conservation, and variation of viral non-coding RNA sequences and structure can help us trace the evolutionary history of the virus, find lineage information, and provide important information about the origin of the viruses. It can also provide knowledge for the design of disease prevention and treatment by providing the background for in vivo RNA nanotechnology.

SiRNA has been widely studied in cancer gene silencing over the last 25 years. However, few siRNA‐based therapeutics have been approved by the FDA. An RNA‐micelle platform provides a powerful therapeutic tool through simple one‐step, high‐yield production that is capable of efficient colorectal cancer lung metastasis treatment, a current lethal condition with a short survival rate post‐diagnosis. Here, it is reported that the use of RNA‐micelles co‐carrying siRNA and nucleoside analogues to completely inhibit lung metastasis of colorectal cancer (CRC). The major advantage of the RNA‐micelle is the successful co‐delivery of siRNA and chemotherapeutic agent in a single delivery vehicle while specifically targeting CRC cells via incorporation of an oncogenic surface receptor ligand. It was found that RNA‐micelles could combine to silence survivin protein expression via siRNA delivery, which in turn increased the efficacy of the delivered chemotherapeutic agent. Both siRNA and high‐payload nucleoside‐analogues were incorporated onto a single micelle, which remained stable during in vivo circulation, rather than individual RNA nanoparticles, thus generating this advantageous synergistic cancer regression. This platform provides a powerful therapeutic tool to address colorectal cancer lung metastasis, a currently serious, lethal disease that has a very poor prognosis following diagnosis.

During the last stage of replication of double-stranded RNA or DNA viruses, their genome is packaged into a preassembled protein capsid. The bacterial virus phi29 dsDNA-packaging motor uses a noncoding packaging RNA (pRNA) molecule to gear its genomic DNA translocation. In this study, we constructed chimeric pRNAs by fusing the pRNA of bacterial virus M2 and that of phi29. The chimeric pRNAs can form dimers or trimers. The dimeric or trimeric pRNAs were active in the packaging of the phi29 dsDNA genome into the purified procapsid, which was subsequently converted into the infectious viruses, as proven by counting plaque-forming units (PFUs). These data show that the stoichiometry of the chimeric pRNAs on the motor is six subunits, a multiple of 2 and 3. Furthermore, AFM studies on pRNA fused to an RNA-triangle revealed hexamer formation. But how do the six identical RNAs anchor on the 12-subunit connector with the double stoichiometry? Structural analysis in combination with enzymatic and chemical probing data revealed that each native pRNA contributes two domains to bind to the 12-subunit DNA-packaging channel at three positively charged residues RKR, proving the formation of the hexameric ring. Resolving the hexamer versus pentamer debate clarifies the mechanism of dsDNA translocation in living organisms.

Packaging of double-stranded DNA (dsDNA) into viral capsids is crucial in dsDNA viruses, including herpesviruses, adenoviruses, poxviruses, and bacteriophages. An ATPase motor compacts genomes. The phi29 DNA packaging motor, a model system, employs a hexameric pRNA (packaging RNA) ring and ATPase, sharing a revolving mechanism observed in herpesvirus genome packaging, bacterial DNA transport, Holliday junction resolution, and plasmid conjugation. Channel gating terminates translocation and readies a reversed pore for dsDNA exit; its mechanism is unclear. We report a packaging efficiency difference between dsDNA and RNA/DNA hybrids. Single-channel electrophysiology and sucrose gradient ultracentrifugation reveal that packaging fails if both ends are dsRNA, but succeeds if either 5′ or 3′ end is DNA. As long as one strand is DNA, RNA/DNA hybrids are packaged, with a higher copy number than dsDNA. Single-pore conductance assays show that this efficiency results from the absence of channel gating. The channel remains open during RNA/DNA translocation and does not close after hybrid packaging, implying dsDNA’s role in gating and conformational changes. This gating arises from dsDNA’s interaction with three flexible loops of the motor channel. These findings offer a structural and chemical foundation for designing containers to package RNA/DNA hybrids for gene/RNAi delivery, therapy, synthetic biology, nanotechnology, and single-particle sensing.

RNA nanoparticles, derived from the packaging RNA three-way junction motif (pRNA-3WJ) of the bacteriophage phi29 DNA packaging motor, have been demonstrated to be thermodynamically and chemically stable, with promise as a nanodelivery system. Background/Objectives: A previous study showed that RNA nanoparticles with antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) inhibited cell proliferation via WST-1 assay. To further investigate the antiangiogenic potential of these RNA nanoparticles, a modified three-dimensional (3D) spheroid sprouting assay model of human umbilical vein endothelial cells was utilized in the present study. Methods: Three groups of RNA nanoparticles were evaluated, namely, pRNA-3WJ series, RNA square series (polygon-type RNA nanoparticles), and 8WJ series (multiple-way junction RNA nanoparticles), which were conjugated with a single anti-VEGF, the combination of one anti-VEGF and one anti-Ang2, or multiple anti-VEGF aptamers. The core scaffold RNA nanoparticles (without aptamers) were used as the references, and bevacizumab was used as the positive control. Results: The results demonstrated the inhibition effects of the RNA nanoparticles on endothelial cell tube formation at 67 nM in a 3D spheroid sprouting model. The results in the 3D spheroid sprouting assay are consistent with those of the WST-1 proliferation assays. Conclusions: Among the RNA nanoparticles evaluated, 3WJ-3VEGF and SQR-VEGF-Ang2 had inhibition effects equivalent to bevacizumab and were promising for anti-angiogenesis treatment.