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 107-108 fold inhibition efficiency. Practical examples include FDA-approved bedaquiline targeting bacterial F1F0 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.

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.

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.

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.

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.

Cancer patients benefit from significantly higher survival rates if tumors are detected at early stages and prior to metastasis. Positron emission tomography (PET), computed tomography (CT), Single-Photon Emission Computed Tomography (SPECT), and other imaging techniques allow for noninvasive diagnosis of various tumors in relatively short periods. Targeted delivery of radiation is also an important approach in cancer therapy. Due to the dynamic nature of RNA, RNA nanoparticles demonstrate spontaneous tumor targeting, resulting in rapid accumulation in tumors without the use of targeting ligands. Incorporating tumor-targeting ligands on RNA nanoparticles generates enhanced tumor accumulation and targeting. Here a unique technology to specifically label three-way junction (3WJ) RNA nanoparticles is reported to carry radioisotopes or other imaging markers for imaging. Two RNA nanoparticles were constructed to target prostate specific membrane antigen (PSMA) via a PSMA RNA aptamer or conjugated tert-Butyl-DCL (DCL). The spontaneous cancer homing resulted in the detection of tumors with high sensitivity in mouse models, which can be applied to any cancer subtype at an early stage. Tumor accumulation occurred due to the motile and deformable nature of RNA nanoparticles, allowing for passage of the high concentration of leaky vasculature in the tumor environment. Furthermore, RNA nanoparticles conjugated with a NOTA radioisotope chelator were incubated with 68Ga in a pH- and temperature-controlled environment to prevent 68Ga non-specific interactions with the negatively charged phosphodiester backbone of RNA. The low pH during 68Ga3+ conjugation neutralized the negative charge of the phosphate backbone on the RNA, ensuring only specific radioisotope chelation to NOTA. To prove the concept of the proposed system, 68Ga-labeled 3WJ was tested in a prostate cancer animal model by PET/CT. The 68Ga-SF5 3WJ accumulated in and identified prostate cancer tumors with high sensitivity, resolution, specificity, and reliability. The proof-of-concept study reported in this paper is an important step in the direction of developing novel radiotherapeutic agents for various cancers. The radioisotope- or fluorophore-labelled nanoparticles were excreted from the body quickly, thus reducing the chance of toxicity and side effects. This molecular imaging platform, based on RNA nanoparticles, shows great promise in early diagnosis, staging, and precise treatment of any tumor subtype.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that has no therapeutic targets, relies on chemotherapeutics for treatment, and is in dire need of novel therapeutic approaches for improved patient outcomes. Extracellular vesicles (EVs) serve as intercellular communicators and have been proposed as ideal drug delivery vehicles. Here, EVs were engineered with RNA nanotechnology to develop TNBC tumor inhibitors. Using super resolved-structured illumination microscopy, EVs were optimized for precise Survivin small interfering RNA (siRNA) conjugated to chemotherapeutics loading and CD44 aptamer ligand decoration, thereby enhancing specificity toward TNBC cells. Conventional treatments typically employ chemotherapy drugs gemcitabine (GEM) and paclitaxel (PTX) at dosages on the order of mg/kg respectively, per injection (intravenous) in mice. In contrast, engineered EVs encapsulating these drugs saw functional tumor growth inhibition at significantly reduced concentrations: 2.2 μg/kg for GEM or 5.6 μg/kg for PTX, in combination with 21.5 μg/kg survivin-siRNA in mice. The result is a substantial decrease in the chemotherapeutic dose required, by orders of magnitude, compared with standard regimens. In vivo and in vitro evaluations in a TNBC orthotopic xenograft mouse model demonstrated the efficacy of this decreased dosage strategy, indicating the potential for decreased chemotherapy-associated toxicity.

Active targeting-mediated nanodelivery takes advantages of ligand-receptor specificity to avoid non-specific distribution, holding great promise for the treatment of a spectrum of diseases. RNA nanoparticles have demonstrated rapid spontaneous tumor targeting and very little organ accumulation due to rapid renal clearance of non-tumor accumulated RNA nanoparticles. However available ligands for specific cells are limited, yet many chemical entities possess receptor targeting capability and remains unexplored. To provide specific tumor accumulation, a multivalent targeting strategy on RNA nanoparticles to control their in vivo fate is implemented. Methotrexate (MTX), a clinically approved chemotherapy was used as a tumor-targeting ligand through conjugation to our RNA nanoparticle with controlled conjugation of various copy numbers. As copies of conjugated MTX increased on the nanoparticle, the specific binding to overexpressed folate receptor was enhanced as demonstrated by flow cytometry analysis and confocal microscopy imaging. Increasing the amounts of conjugated MTX did not significantly change the nanoparticle size, Zeta potential, or cytokine induction. Increased amounts of conjugated MTX resulted in improved cell inhibition due to MTX release following cell internalization. However, increasing conjugated MTX to the RNA nanoparticles reduced the melting temperature of RNA nanoparticles and increased in vitro serum protein binding to the nanoparticles. Thus, in vivo biodistribution profiles of RNA nanoparticles revealed different behaviors based on MTX conjugation in cancer targeting and clearance. Increased copies of MTX changed the ability of nanoparticles to target tumors, accumulate in healthy organs, and rapidly clear through the urine. Nanoparticle design must be closely considered for optimized cancer targeting and therapy, providing the rationale for a proper design of RNA nanodelivery in cancer treatment.

Previous studies of RNA nanoparticles have demonstrated the potential of these nanoparticles in ocular delivery via the subconjunctival route. Sustained ocular delivery is beneficial for chronic eye disease treatment, and utilizing a reservoir implant in the periocular space (e.g., episcleral implant) can prolong ocular delivery of these nanoparticles. The objectives of the present study were to (a) demonstrate the fabrication of the reservoir implants, (b) evaluate the performance of the implants with model permeants and RNA nanoparticles in vitro, and (c) investigate the applicability of hindered transport theory for the release kinetics from the implants. In vitro release testing was performed with the implants to determine the release kinetics and implant membrane permeability. In addition to RNA nanoparticles, model permeants fluorescein-isothiocyanate (FITC) labeled dextrans (10, 40, and 150 kDa) were examined. The results indicated that the rates of permeant release from the implants were a function of the (a) size and structure of the permeant/nanoparticle and (b) type and pore size of the implant membrane. The model analyses provided insights into implant membrane transport and ocular pharmacokinetics of the nanoparticles for transscleral delivery. The results suggested the potential of prolonged delivery of the RNA nanoparticles with the episcleral implant approach.

Neovascularization contributes to various posterior eye segment diseases such as age-related macular degeneration and diabetic retinopathy. RNA nanoparticles were demonstrated previously to enter the corneal and retinal cells after subconjunctival injection for ocular delivery. In the present study, antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) were conjugated to RNA nanoparticles. The objectives were to investigate the clearance and distribution of these angiogenesis-inhibiting RNA nanoparticles after subconjunctival injection in vivo and their antiangiogenic effects for inhibiting ocular neovascularization in vitro. The results in the whole-body fluorescence imaging study showed that the clearance of RNA nanoparticles was size-dependent with no significant differences between RNA nanoparticles with and without the aptamers except for pRNA-3WJ. The distribution study of RNA nanoparticles by confocal microscopy of the dissected eye tissues in vivo indicated cell internalization of the larger RNA nanoparticles in the retina and retinal pigment epithelium after subconjunctival injection, and the larger nanoparticles with aptamers showed higher levels of cell internalization than those without. In the cell proliferation assay in vitro, RNA nanoparticles with multiple aptamers had higher antiangiogenic effects. With both longer retention time and high antiangiogenic effect, SQR-VEGF-Ang2 could be a promising RNA nanoparticle for posterior eye delivery.

Both siRNA and miRNA can serve as powerful gene-silencing reagents but their specific delivery to cancer cells in vivo without collateral damage to healthy cells remains challenging. We report here the application of RNA nanotechnology for specific and efficient delivery of anti-miRNA seed-targeting sequence to block the growth of prostate cancer in mouse models. Utilizing the thermodynamically ultra-stable three-way junction of the pRNA of phi29 DNA packaging motor, RNA nanoparticles were constructed by bottom-up self-assembly containing the anti-prostate-specific membrane antigen (PSMA) RNA aptamer as a targeting ligand and anti-miR17 or anti-miR21 as therapeutic modules. The 16 nm RNase-resistant and thermodynamically stable RNA nanoparticles remained intact after systemic injection in mice and strongly bound to tumors with little or no accumulation in healthy organs 8 hours postinjection, and subsequently repressed tumor growth at low doses with high efficiency.

Nature continually refines its processes for optimal efficiency, especially within biological systems. This article explores the collaborative efforts of researchers worldwide, aiming to mimic nature's efficiency by developing smarter and more effective nanoscale technologies and biomaterials. Recent advancements highlight progress and prospects in leveraging engineered nucleic acids and proteins for specific tasks, drawing inspiration from natural functions. The focus is developing improved methods for characterizing, understanding, and reprogramming these materials to perform user-defined functions, including personalized therapeutics, targeted drug delivery approaches, engineered scaffolds, and reconfigurable nanodevices. Contributions from academia, government agencies, biotech, and medical settings offer diverse perspectives, promising a comprehensive approach to broad nanobiotechnology objectives. Encompassing topics from mRNA vaccine design to programmable protein-based nanocomputing agents, this work provides insightful perspectives on the trajectory of nanobiotechnology toward a future of enhanced biomimicry and technological innovation.

RNA nanotechnology is the bottom-up self-assembly of nanoscale RNA structures, the main framework of which is mainly composed of RNA. Scaffolds, ligands, therapeutics, and modulators can all be composed of RNA. Classical RNA research has focused on interactions and 2D/3D structures within RNA. RNA nanotechnology can elucidate and exploit RNA-RNA interactions and quaternary (4D) structures. RNA technology is like “Lego bricks”. Ideal materials for building blocks should have the following attributes: 1. Diversity. 2. Capable of constructing structures with various shapes, sizes, and modifiable stoichiometry. 3. Mix together to self-assemble. 4. Thermodynamically, chemically, and enzymatically stable, with long shelf life. The potential of RNA NPs (nanoparticles) in various drug delivery applications including anticancer drug delivery is enormous. Therefore, RNA NPs can be developed in cancer research for chemotherapy and immunotherapy. The next decade is expected to witness many clinical trials in this field. RNA instability is no longer an issue and can be overcome through various chemical modifications. If we look at the big picture, the therapeutic potential of RNA, well-defined RNA NPs, and recent successes in stabilizing them not only support that RNA nanotechnology at the beginning of a new era but also suggest that future therapeutic prospects may predominate. Only 1.5% of the human genome encodes proteins. A significant portion of the remaining 98.5% of so-called “junk DNA” codes for important small or long non-coding RNAs. All substances that are toxic to cells are known to stop cancer growth. Currently, most cancer chemotherapies are highly toxic to normal cells and viral organoids. RNA nanoparticles have been shown to be a motile, dynamic, and deformable material that spontaneously runs enriched with cancer vasculature and is rapidly excreted into the urine via the glomerulus with little accumulation in vital organs. Therefore, RNA nanoparticles can efficiently and specifically target tumor tissues while being rapidly cleared from the body. Thus, RNA technology can convert toxic drugs in chemotherapy into non-toxic drugs. The promise of RNA as the third milestone in drug development has come true. Importantly, RNA itself can be used as a drug, such as siRNA, miRNA, aptamer, ribozyme, anti-miRNA, etc. Another area of interest is the use of small chemical drugs as ligands targeting non-coding RNA or mRNA. This field has been attractive and emerging but progress has been slow. This segment is expected to witness significant growth in the coming years.

Liver cancer such as hepatocellular carcinoma (HCC) poorly responds to chemotherapeutics as there are no effective means to deliver the drugs to liver cancer. Here we report GalNAc decorated exosomes as cargo for targeted delivery of Paclitaxel (PTX) and miR122 to liver tumors as an effective means to inhibit the HCC. Exosomes (Exos) are nanosized extracellular vesicles that deliver a payload to cancer cells effectively. GalNAc provides Exos targeting ability by binding to the asialoglycoprotein-receptor (ASGP-R) overexpressed on the liver cancer cell surface. A 4-way junction (4WJ) RNA nanoparticle was constructed to harbor 24 copies of hydrophobic PTX and 1 copy of miR122. The 4WJ RNA-PTX complex was loaded into the Exos, and its surface was decorated with GalNAc using RNA nanotechnology to obtain specific targeting. The multi-specific Exos selectively bind and efficiently delivered the payload into the liver cancer cells and exhibited the highest cancer cell inhibition due to the multi-specific effect of miR122, PTX, GalNAc, and Exos. The same was reflected in mice xenograft studies, the liver cancer was efficiently inhibited after systemic injection of the multi-specific Exos. The required effective dose of chemical drugs carried by Exos was significantly reduced, indicating high efficiency and low toxicity. The multi-specific strategy demonstrates that Exos can serve as a natural cargo vehicle for the targeted delivery of anticancer therapeutics to treat difficult-to-treat cancers.

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 lipid-bilayer 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.

KRAS mutations are one of the most common oncogenic driver mutations in human cancers, including non-small cell lung cancer (NSCLC), and have established roles in cancer pathogenesis and therapeutic resistance. The development of effective inhibitors of mutant KRAS represents a significant challenge. Threeway junction (3WJ)-based multi-functional RNA nanoparticles have the potential to serve as an effective in vivo siRNA delivery platform with the ability to enhance tumor targeting specificity and visualize biodistribution through an imaging moiety. Herein, we assembled novel EGFRapt-3WJ-siKRASG12C mutation targeted nanoparticles to target EGFR-expressing human NSCLC harboring a KRASG12C mutation to silence KRASG12C expression in a tumor cell-specific fashion. We found that EGFRapt-3WJsiKRASG12C nanoparticles potently depleted cellular KRASG12C expression, resulting in attenuation of downstream MAPK pathway signaling, cell proliferation, migration/invasion ability, and sensitized NSCLC cells to chemoradiotherapy. In vivo, these nanoparticles induced tumor growth inhibition in KRASG12C NSCLC tumor xenografts. Together, this study suggests that the 3WJ pRNA-based platform has the potential to suppress mutant KRAS activity for the treatment of KRAS-driven human cancers, and warrants further development for clinical translation.

Besides mRNA, rRNA, and tRNA, cells contain many other noncoding RNA that display critical roles in the regulation of cellular functions. Human genome sequencing revealed that the majority of non-protein-coding DNA actually codes for non-coding RNAs. The dynamic nature of RNA results in its motile and deformative behavior. These conformational transitions such as the change of base-pairing, breathing within complemented strands, and pseudoknot formation at the 2D level as well as the induced-fit and conformational capture at the 3D level are important for their biological functions including regulation, translation, and catalysis. The dynamic, motile and catalytic activity has led to a belief that RNA is the origin of life. We have recently reported that the deformative property of RNA nanoparticles enhances their penetration through the leaky blood vessel of cancers which leads to highly efficient tumor accumulation. This special deformative property also enables RNA nanoparticles to pass the glomerulus, overcoming the filtration size limit, resulting in fast renal excretion and rapid body clearance, thus low or no toxicity. The biodistribution of RNA nanoparticles can be further improved by the incorporation of ligands for cancer targeting. In addition to the favorable biodistribution profiles, RNA nanoparticles possess other properties including self-assembly, negative charge, programmability, and multivalency; making it a great material for pharmaceutical applications. The intrinsic negative charge of RNA nanoparticles decreases the toxicity of drugs by preventing nonspecific binding to the negative charged cell membrane and enhancing the solubility of hydrophobic drugs. The polyvalent property of RNA nanoparticles allows the multi-functionalization which can apply to overcome drug resistance. This review focuses on the summary of these unique properties of RNA nanoparticles, which describes the mechanism of RNA dynamic, motile and deformative properties, and elucidates and prepares to welcome the RNA therapeutics as the third milestone in pharmaceutical drug development.