Cracking the Code: Enhancing Molecular Tools for Progress in Nanobiotechnology.

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.

Posterior eye delivery of angiogenesis-inhibiting RNA nanoparticles via subconjunctival injection.

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.

Targeting oncogenic KRAS in non-small cell lung cancer with EGFR aptamer-conjugated multifunctional RNA nanoparticles.

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. Three-way 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-3WJ-siKRASG12C 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.

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 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.

Ligand-displaying-exosomes using RNA nanotechnology for targeted delivery of multi-specific drugs for liver cancer regression.

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.

The dynamic, motile and deformative properties of RNA nanoparticles facilitate the third milestone of drug development.

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.

Rational design for controlled release of Dicer-substrate siRNA harbored in phi29 pRNA-based nanoparticles.

Small interfering RNA (siRNA) for silencing genes and treating disease has been a dream since ranking as a top Breakthrough of the Year in 2002 by Science. With the recent FDA approval of four siRNA-based drugs, the potential of RNA therapeutics to become the third milestone in pharmaceutical drug development has become a reality. However, the field of RNA interference (RNAi) therapeutics still faces challenges such as specificity in targeting, intracellular processing, and endosome trapping after targeted delivery. Dicer-substrate siRNAs included onto RNA nanoparticles may be able to overcome these challenges. Here, we show that pRNA-based nanoparticles can be designed to efficiently harbor the Dicer-substrate siRNAs in vitro and in vivo to the cytosol of tumor cells and release the siRNA. The structure optimization and chemical modification for controlled release of Dicer-substrate siRNAs in tumor cells were also evaluated through molecular beacon analysis. Studies on the length requirement of the overhanging siRNA revealed that at least 23 nucleotides at the dweller's arm were needed for dicer processing. The above sequence parameters and structure optimization were confirmed in recent studies demonstrating the release of functional Survivin siRNA from the pRNA-based nanoparticles for cancer inhibition in non-small-cell lung, breast, and prostate cancer animal models.

Thermostability, Tunability, and Tenacity of RNA as Rubbery Anionic Polymeric Materials in Nanotechnology and Nanomedicine-Specific Cancer Targeting with Undetectable Toxicity.

RNA nanotechnology is the bottom-up self-assembly of nanometer-scale architectures, resembling LEGOs, composed mainly of RNA. The ideal building material should be (1) versatile and controllable in shape and stoichiometry, (2) spontaneously self-assemble, and (3) thermodynamically, chemically, and enzymatically stable with a long shelf life. RNA building blocks exhibit each of the above. RNA is a polynucleic acid, making it a polymer, and its negative-charge prevents nonspecific binding to negatively charged cell membranes. The thermostability makes it suitable for logic gates, resistive memory, sensor set-ups, and NEM devices. RNA can be designed and manipulated with a level of simplicity of DNA while displaying versatile structure and enzyme activity of proteins. RNA can fold into single-stranded loops or bulges to serve as mounting dovetails for intermolecular or domain interactions without external linking dowels. RNA nanoparticles display rubber- and amoeba-like properties and are stretchable and shrinkable through multiple repeats, leading to enhanced tumor targeting and fast renal excretion to reduce toxicities. It was predicted in 2014 that RNA would be the third milestone in pharmaceutical drug development. The recent approval of several RNA drugs and COVID-19 mRNA vaccines by FDA suggests that this milestone is being realized. Here, we review the unique properties of RNA nanotechnology, summarize its recent advancements, describe its distinct attributes inside or outside the body and discuss potential applications in nanotechnology, medicine, and material science.

Non-Small-Cell Lung Cancer Regression by siRNA Delivered Through Exosomes That Display EGFR RNA Aptamer.

Lung cancer is the second most common cancer in both men and women and is the leading cause of cancer death in the United States. The development of drug resistance to commonly used chemotherapeutics in non-small-cell lung cancer (NSCLC) poses significant health risks and there is a dire need to improve patient outcomes. In this study, we report the use of RNA nanotechnology to display ligand on exosome that was loaded with small interfering RNA (siRNA) for NSCLC regression in animal trials. Cholesterol was used to anchor the ligand targeting epidermal growth factor receptor on exosomes that were loaded with siRNA to silence the antiapoptotic factor survivin. The cytosolic delivery of siRNA overcame the problem of endosome trapping, leading to potent gene knockdown, chemotherapy sensitization, and tumor regression, thus achieving a favorable IC50 of 20 nmol/kg siRNA encapsulated by exosome particles in the in vivo gene knockdown assessment.

RNA nanotechnology to build a dodecahedral genome of single-stranded RNA virus.

The quest for artificial RNA viral complexes with authentic structure while being non-replicative is on its way for the development of viral vaccines. RNA viruses contain capsid proteins that interact with the genome during morphogenesis. The sequence and properties of the protein and genome determine the structure of the virus. For example, the Pariacoto virus ssRNA genome assembles into a dodecahedron. Virus-inspired nanotechnology has progressed remarkably due to the unique structural and functional properties of viruses, which can inspire the design of novel nanomaterials. RNA is a programmable biopolymer able to self-assemble sophisticated 3D structures with rich functionalities. RNA dodecahedrons mimicking the Pariacoto virus quasi-icosahedral genome structures were constructed from both native and 2'-F modified RNA oligos. The RNA dodecahedron easily self-assembled using the stable pRNA three-way junction of bacteriophage phi29 as building blocks. The RNA dodecahedron cage was further characterized by cryo-electron microscopy and atomic force microscopy, confirming the spontaneous and homogenous formation of the RNA cage. The reported RNA dodecahedron cage will likely provide further studies on the mechanisms of interaction of the capsid protein with the viral genome while providing a template for further construction of the viral RNA scaffold to add capsid proteins for the assembly of the viral nucleocapsid as a model. Understanding the self-assembly and RNA folding of this RNA cage may offer new insights into the 3D organization of viral RNA genomes. The reported RNA cage also has the potential to be explored as a novel virus-inspired nanocarrier.

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

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.

One-Pot Production of RNA Nanoparticles via Automated Processing and Self-Assembly.

From the original sequencing of the human genome, it was found that about 98.5% of the genome did not code for proteins. Subsequent studies have now revealed that a much larger portion of the genome is related to short or long noncoding RNAs that regulate cellular activities. In addition to the milestones of chemical and protein drugs, it has been proposed that RNA drugs or drugs targeting RNA will become the third milestone in drug development ( Shu , Y. ; Adv. Drug Deliv. Rev. 2014 , 66 , 74 . ). Currently, the yield and cost for RNA nanoparticle or RNA drug production requires improvement in order to advance the RNA field in both research and clinical translation by reducing the multiple tedious manufacturing steps. For example, with 98.5% incorporation efficiency of chemical synthesis of a 100 nucleotide RNA strand, RNA oligos will result with 78% contamination of aborted byproducts. Thus, RNA nanotechnology is one of the remedies, because large RNA can be assembled from small RNA fragments via bottom-up self-assembly. Here we report the one-pot production of RNA nanoparticles via automated processing and self-assembly. The continuous production of RNA by rolling circle transcription (RCT) using a circular dsDNA template is coupled with self-cleaving ribozymes encoded in the concatemeric RNA transcripts. Production was monitored in real-time. Automatic production of RNA fragments enabled their assembly either in situ or via one-pot co-transcription to obtain RNA nanoparticles of desired motifs and functionalities from bottom-up assembly of multiple RNA fragments. In combination with the RNA nanoparticle construction process, a purification method using a large-scale electrophoresis column was also developed.

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression.

Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

Assessment and comparison of thermal stability of phosphorothioate-DNA, DNA, RNA, 2'-F RNA, and LNA in the context of Phi29 pRNA 3WJ.

The question of whether RNA is more stable or unstable compared to DNA or other nucleic acids has long been a subject of extensive scrutiny and public attention. Recently, thermodynamically stable and degradation-resistant RNA motifs have been utilized in RNA nanotechnology to build desired architectures and integrate multiple functional groups. Here we report the effects of phosphorothioate deoxyribonucleotides (PS-DNA), deoxyribonucleotides (DNA), ribonucleotides (RNA), 2'-F nucleotides (2'-F), and locked nucleic acids (LNA) on the thermal and in vivo stability of the three-way junction (3WJ) of bacteriophage phi29 motor packaging RNA. It was found that the thermal stability gradually increased following the order of PS-DNA/PS-DNA < DNA/DNA < DNA/RNA < RNA/RNA < RNA/2'-F RNA < 2'-F RNA/2'-F RNA < 2'-F RNA/LNA < LNA/LNA. This proposition is supported by studies on strand displacement and the melting of homogeneous and heterogeneous 3WJs. By simply mixing different chemically modified oligonucleotides, the thermal stability of phi29 pRNA 3WJ can be tuned to cover a wide range of melting temperatures from 21.2°C to over 95°C. The 3WJLNA was resistant to boiling temperature denaturation, urea denaturation, and 50% serum degradation. Intravenous injection of fluorescent LNA/2'-F hybrid 3WJs into mice revealed its exceptional in vivo stability and presence in urine. It is thus concluded that incorporation of LNA nucleotides, alone or in combination with 2'-F, into RNA nanoparticles derived from phi29 pRNA 3WJ can extend the half-life of the RNA nanoparticles in vivo and improve their pharmacokinetics profile.

Favorable biodistribution, specific targeting and conditional endosomal escape of RNA nanoparticles in cancer therapy.

The past decades have witnessed the successful transition of several nanotechnology platforms into the clinical trials. However, specific delivery of therapeutics to tumors is hindered by several barriers including cancer recognition and tissue penetration, particle heterogeneity and aggregation, and unfavorable pharmacokinetic profiles such as fast clearance and organ accumulation. With the advent of RNA nanotechnology, a series of RNA nanoparticles have been successfully constructed to overcome many of the aforementioned challenges for in vivo cancer targeting with favorable biodistribution profiles. Compared to other nanodelivery platforms, the physiochemical properties of RNA nanoparticles can be tuned with relative ease for investigating the in vivo behavior of nanoparticles upon systemic injection. The size, shape, and surface chemistry, especially hydrophobic modifications, exert significant impacts on the in vivo fate of RNA nanoparticles. Rationally designed RNA nanoparticles with defined stoichiometry and high homogeneity have been demonstrated to specifically target tumor cells while avoiding accumulation in healthy vital organs after systemic injection. RNA nanoparticles were proven to deliver therapeutics such as siRNA and anti-miRNA to block tumor growth in several animal models. Although the release of anti-miRNA from the RNA nanoparticles has achieved high efficiency of tumor regression in multiple animal models, the efficiency of endosomal escape for siRNA delivery needs further improvement. This review focuses on the advances and perspectives of this promising RNA nanotechnology platform for cancer targeting and therapy.

Advancement of the Emerging Field of RNA Nanotechnology.

The field of RNA nanotechnology has advanced rapidly during the past decade. A variety of programmable RNA nanoparticles with defined shape, size, and stoichiometry have been developed for diverse applications in nanobiotechnology. The rising popularity of RNA nanoparticles is due to a number of factors: (1) removing the concern of RNA degradation in vitro and in vivo by introducing chemical modification into nucleotides without significant alteration of the RNA property in folding and self-assembly; (2) confirming the concept that RNA displays very high thermodynamic stability and is suitable for in vivo trafficking and other applications; (3) obtaining the knowledge to tune the immunogenic properties of synthetic RNA constructs for in vivo applications; (4) increased understanding of the 4D structure and intermolecular interaction of RNA molecules; (5) developing methods to control shape, size, and stoichiometry of RNA nanoparticles; (6) increasing knowledge of regulation and processing functions of RNA in cells; (7) decreasing cost of RNA production by biological and chemical synthesis; and (8) proving the concept that RNA is a safe and specific therapeutic modality for cancer and other diseases with little or no accumulation in vital organs. Other applications of RNA nanotechnology, such as adapting them to construct 2D, 3D, and 4D structures for use in tissue engineering, biosensing, resistive biomemory, and potential computer logic gate modules, have stimulated the interest of the scientific community. This review aims to outline the current state of the art of RNA nanoparticles as programmable smart complexes and offers perspectives on the promising avenues of research in this fast-growing field.

Mechanism of three-component collision to produce ultrastable pRNA three-way junction of Phi29 DNA-packaging motor by kinetic assessment.

RNA nanotechnology is rapidly emerging. Due to advantageous pharmacokinetics and favorable in vivo biodistribution, RNA nanoparticles have shown promise in targeted delivery of therapeutics. RNA nanotechnology applies bottom-up assembly, thus elucidation of the mechanism of interaction between multiple components is of fundamental importance. The tendency of diminishing concern about RNA instability has accelerated by the finding of the novel thermostable three-way junction (3WJ) motif of the phi29 DNA-packaging motor. The kinetics of these three components, each averaging 18 nucleotides (nt), was investigated to elucidate the mechanism for producing the stable 3WJ. The three fragments coassembled into the 3WJ with extraordinary speed and affinity via a two-step reaction mechanism, 3WJb + 3WJc ↔ 3WJbc + 3WJa ↔ 3WJabc The first step of reaction between 3WJb and 3WJc is highly dynamic since these two fragments only contain 8 nt for complementation. In the second step, the 3WJa, which contains 17 nt complementary to the 3WJbc complex, locks the unstable 3WJbc complex into a highly stable 3WJ. The resulting pRNA-3WJ is more stable than any of the dimer species as shown in the much more rapid association rates and slowest dissociation rate constant. The second step occurs at a very high association rate that is difficult to quantify, resulting in a rapid formation of a stable 3WJ. Elucidation of the mechanism of three-component collision in producing the ultrastable 3WJ proves a promising platform for bottom-up assembly of RNA nanoparticles as a new class of anion polymers for material science, electronic elements, or therapeutic reagents.

Specific Delivery of MiRNA for High Efficient Inhibition of Prostate Cancer by RNA Nanotechnology.

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.

Entropy-driven one-step formation of Phi29 pRNA 3WJ from three RNA fragments.

The emerging field of RNA nanotechnology necessitates creation of functional RNA nanoparticles but has been limited by particle instability. It has been shown that the three-way junction of bacteriophage phi29 motor pRNA has unusual stability and can self-assemble from three fragments with high efficiency. It is generally believed that RNA and DNA folding is energy landscape-dependent, and the folding of RNA is driven by enthalpy. Here we examine the thermodynamic characteristics of the 3WJ components as 2'-fluoro RNA, DNA, and RNA. It was seen that the three fragments existed either in 3WJ complex or as monomers, with the intermediate of dimers almost undetectable. It seems that the three fragments can lead to the formation of the 3WJ complex efficiently within a rapid time. A low dissociation constant (apparent KD) of 11.4 nM was determined for RNA, inclusion of 2'-F pyrimidines strengthened the KD to 4.5 nM, and substitution of DNA weakened it to 47.7 nM. The ΔG°37, were -36, -28, and -15 kcal/mol for 3WJ2'-F, 3WJRNA, and 3WJDNA, respectively. It is found that the formation of the three-component complex was governed by entropy, instead of enthalpy, as usually found in RNA complexes. Here entropy-driven is referring to a dominating entropic contribution to the increased stability of the 3WJ(2'-F and 3WJ(RNA) compared to the 3WJ(DNA,) instead of referring to the absolute role or total energy governing 3WJ folding. [corrected].