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Coronavirus infectious disease 2019 (COVID-19), caused by severe acute respiratory virus type 2 (SARS-CoV-2), has caused a global public health crisis. As an RNA virus, the high gene mutability of SARS-CoV-2 poses significant challenges to the development of broad-spectrum vaccines and antiviral therapeutics. There remains a lack of specific therapeutics directly targeting SARS-CoV-2. With the ability to efficiently inhibit the expression of target genes in a sequence-specific way, small interfering RNA (siRNA) therapy has exhibited significant potential in antiviral and other disease treatments. In this work, we presented a highly effective self-assembled siRNA nanoparticle targeting multiple highly conserved regions of SARS-CoV-2. The siRNA sequences targeting viral conserved regions were first screened and evaluated by their thermodynamic features, off-target effects, and secondary structure toxicities. RNA motifs including siRNA sequences were then designed and self-assembled into siRNA nanoparticles. These siRNA nanoparticles demonstrated remarkable uniformity and stability and efficiently entered cells directly through cellular endocytic pathways. Moreover, these nanoparticles effectively inhibited the replication of SARS-CoV-2, exhibiting a superior inhibitory effect compared to free siRNA. These results demonstrated that these self-assembled siRNA nanoparticles targeting highly conserved regions of SARS-CoV-2 represent highly effective antiviral candidates for the treatment of infections, and are promisingly effective against current and future viral variants. [ABSTRACT FROM AUTHOR]

Design strategies for DNA and RNA nanostructures have developed along parallel lines for the past 30 years, from small structural motifs derived from biology to large ‘origami’ structures with thousands to tens of thousands of bases. With the recent publication of numerous RNA origami structures and improved design methods-even permitting co-transcriptional folding of kilobase-sized structures – the RNA nanotechnolgy field is at an inflection point. Here, we review the key achievements which inspired and enabled RNA origami design and draw comparisons with the development and applications of DNA origami structures. We further present the available computational tools for the design and the simulation, which will be key to the growth of the RNA origami community. Finally, we portray the transition from RNA origami structure to function. Several functional RNA origami structures exist already, their expression in cells has been demonstrated and first applications in cell biology have already been realized. Overall, we foresee that the fast-paced RNA origami field will provide new molecular hardware for biophysics, synthetic biology and biomedicine, complementing the DNA origami toolbox.

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.

Coronavirus infectious disease 2019 (COVID-19), caused by severe acute respiratory virus type 2 (SARS-CoV-2), has caused a global public health crisis. As an RNA virus, the high gene mutability of SARS-CoV-2 poses significant challenges to the development of broad-spectrum vaccines and antiviral therapeutics. There remains a lack of specific therapeutics directly targeting SARS-CoV-2. With the ability to efficiently inhibit the expression of target genes in a sequence-specific way, small interfering RNA (siRNA) therapy has exhibited significant potential in antiviral and other disease treatments. In this work, we presented a highly effective self-assembled siRNA nanoparticle targeting multiple highly conserved regions of SARS-CoV-2. The siRNA sequences targeting viral conserved regions were first screened and evaluated by their thermodynamic features, off-target effects, and secondary structure toxicities. RNA motifs including siRNA sequences were then designed and self-assembled into siRNA nanoparticles. These siRNA nanoparticles demonstrated remarkable uniformity and stability and efficiently entered cells directly through cellular endocytic pathways. Moreover, these nanoparticles effectively inhibited the replication of SARS-CoV-2, exhibiting a superior inhibitory effect compared to free siRNA. These results demonstrated that these self-assembled siRNA nanoparticles targeting highly conserved regions of SARS-CoV-2 represent highly effective antiviral candidates for the treatment of infections, and are promisingly effective against current and future viral variants.

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.

RNAs are involved in the crucial processes of disease progression and have emerged as powerful therapeutic targets and diagnostic biomarkers. However, efficient delivery of therapeutic RNA to the targeted location and precise detection of RNA markers remains challenging. Recently, more and more attention has been paid to applying nucleic acid nanoassemblies in diagnosing and treating. Due to the flexibility and deformability of nucleic acids, the nanoassemblies could be fabricated with different shapes and structures. With hybridization, nucleic acid nanoassemblies, including DNA and RNA nanostructures, can be applied to enhance RNA therapeutics and diagnosis. This review briefly introduces the construction and properties of different nucleic acid nanoassemblies and their applications for RNA therapy and diagnosis and makes further prospects for their development.

Nanotechnology is the use of materials that have unique nanoscale properties. In recent years, nanotechnologies have shown promising results for human health, especially in cancer treatment. The self-assembly characteristic of RNA is a powerful bottom-up approach to the design and creation of nanostructures through interdisciplinary biological, chemical and physical techniques. The use of RNA nanotechnology in therapeutics is about to be realized. This review discusses different kinds of nano-based drug delivery systems and their characteristic features.

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. This review illustrated the RNA-decorated exosome to display specific ligand/marker for targeted delivery of therapeutics to cells, thus ensuring safety & reducing cytotoxicity. [Display omitted] [ABSTRACT FROM AUTHOR]

RNAs are involved in the crucial processes of disease progression and have emerged as powerful therapeutic targets and diagnostic biomarkers. However, efficient delivery of therapeutic RNA to the targeted location and precise detection of RNA markers remains challenging. Recently, more and more attention has been paid to applying nucleic acid nanoassemblies in diagnosing and treating. Due to the flexibility and deformability of nucleic acids, the nanoassemblies could be fabricated with different shapes and structures. With hybridization, nucleic acid nanoassemblies, including DNA and RNA nanostructures, can be applied to enhance RNA therapeutics and diagnosis. This review briefly introduces the construction and properties of different nucleic acid nanoassemblies and their applications for RNA therapy and diagnosis and makes further prospects for their development. Nucleic acid nanoassemblies with different shapes and structures are fabricated for enhanced RNA-based gene silencing, gene expression, gene editing, gene detection, and imaging strategies. [Display omitted] [ABSTRACT FROM AUTHOR]

Design strategies for DNA and RNA nanostructures have developed along parallel lines for the past 30 years, from small structural motifs derived from biology to large 'origami' structures with thousands to tens of thousands of bases. With the recent publication of numerous RNA origami structures and improved design methods-even permitting co-transcriptional folding of kilobase-sized structures – the RNA nanotechnolgy field is at an inflection point. Here, we review the key achievements which inspired and enabled RNA origami design and draw comparisons with the development and applications of DNA origami structures. We further present the available computational tools for the design and the simulation, which will be key to the growth of the RNA origami community. Finally, we portray the transition from RNA origami structure to function. Several functional RNA origami structures exist already, their expression in cells has been demonstrated and first applications in cell biology have already been realized. Overall, we foresee that the fast-paced RNA origami field will provide new molecular hardware for biophysics, synthetic biology and biomedicine, complementing the DNA origami toolbox. [ABSTRACT FROM AUTHOR]

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) serves to further the development of a wide variety of functional nucleic acids and other related nanotechnology platforms. To aid in the dissemination of the most recent advancements, a biennial discussion focused on biomotors, viral assembly, and RNA nanobiotechnology has been established where international experts in interdisciplinary fields such as structural biology, biophysical chemistry, nanotechnology, cell and cancer biology, and pharmacology share their latest accomplishments and future perspectives. The results summarized here highlight advancements in our understanding of viral biology and the structure–function relationship of frame-shifting elements in genomic viral RNA, improvements in the predictions of SHAPE analysis of 3D RNA structures, and the understanding of dynamic RNA structures through a variety of experimental and computational means. Additionally, recent advances in the drug delivery, vaccine design, nanopore technologies, biomotor and biomachine development, DNA packaging, RNA nanotechnology, and drug delivery are included in this critical review. We emphasize some of the novel accomplishments, major discussion topics, and present current challenges and perspectives of these emerging fields.

Naturally occurring ribozymes with defined three-dimensional (3D) structures serve as promising platforms for the design and construction of artificial RNA nanostructures. We constructed a hexameric ribozyme nanostructure by face-to-face dimerization of a pair of triangular ribozyme trimers, unit RNAs of which were derived from the Tetrahymena group I ribozyme. In this study, we have expanded the dimerization strategy to a square-shaped ribozyme tetramer by introducing four pillar units. The resulting box-shaped nanostructures, which contained eight ribozyme units, can be assembled from either four or two components of their unit RNAs. [ABSTRACT FROM AUTHOR]

Nucleoside analogs are potent inhibitors for cancer treatment, but the main obstacles to their application in humans are their toxicity, nonspecificity, and lack of targeted delivery tools. Here, we report the use of RNA four-way junctions (4WJs) to deliver two nucleoside analogs, floxuridine (FUDR) and gemcitabine (GEM), with high payloads through routine and simple solid-state RNA synthesis and nanoparticle assembly. The design of RNA nanotechnology for the co-delivery of nucleoside analogs and the chemotherapeutic drug paclitaxel (PTX) resulted in synergistic effects and high efficacy in the treatment of Triple-Negative Breast Cancer (TNBC). The 4WJ-drug complexes were confirmed to have efficient tumor spontaneous targeting and no toxicity because the motility of RNA nanoparticles has been previously shown to enable these RNA-drug complexes to spontaneously accumulate in tumor blood vessels. The negative charge of RNA enables those RNA complexes that are not targeted to tumor vasculature to circulate in the blood and enter the urine through the kidney glomerulus, without accumulating in organs, therefore being nontoxic. Drug incorporation into RNA 4WJ can be precisely controlled with a defined loading amount, location, and ratio. The incorporation of nucleoside analogs into 4WJ only requires one step using nucleoside analogue phosphoramidites during solid-phase RNA synthesis, without the need for additional conjugation and purification processes.

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., Competing Interests: Declaration of interests P.G.’s Sylvan Frank Endowed Chair in Pharmaceutics and Drug Delivery position is supported by the Chen’s Foundation. P.G. is the consultant, licensor, and grantee of Oxford Nanopore Technologies. P.G. also has a patent EP3440090B1 RNA ligand-displaying exosomes for specific delivery of therapeutics to cell by RNA nanotechnology., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Existing delivery methods for RNAi therapeutics encounter challenges, including stability, specificity, and off-target effects, which restrict their clinical effectiveness. In this study, a novel miR-133a zipper nanoparticle (NP) system that integrates miRNA zipper technology with rolling circle transcription (RCT) to achieve targeted delivery and specific regulation of miR-133a in adipocytes, is presented. This innovative approach can greatly enhance the delivery and release of miR-133a zippers, increasing the expression of thermogenic genes and mitochondrial biogenesis. he miR-133a zipper NP is utilized for the delivery of miRNA zipper-blocking miR-133a, an endogenous inhibitor of Prdm16 expression, to enhance the thermogenic activity of adipocytes by modulating their transcriptional program. Inhibition of miR-133a through the miR-133a zipper NP leads to more significant upregulation of thermogenic gene expression (Prdm16 and Ucp1) than with the free miR-133a zipper strand. Furthermore, miR-133a zipper NPs increase the number of mitochondria and induce heat production, reducing the size of 3D adipose spheroids. In short, this study emphasizes the role of RNA NPs in improving RNAi stability and specificity and paves the way for broader applications in gene therapy. Moreover, this research represents a significant advancement in RNAi-based treatments, pointing toward a promising direction for future therapeutic strategies., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

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.

RNA nanotechnology has shown great progress over the past decade. Diverse controllable and multifunctional RNA nanoparticles have been developed for various applications in many areas. For example, RNA nanoparticles can participate in the construction of drug delivery nanoplatforms. Recently, a three-way junction packaging RNA (3WJ-pRNA) has been exploited for its characteristics of self-assembly and ultrahigh stability in many aspects. 3WJ-pRNA is the 3WJ part of bacteriophage φ29 pRNA and joins different components of φ29 as a linker element. In this work, we used all-atom MD simulation to study the thermal stability of 3WJ-pRNA and the underlying mechanisms. While 3WJ-pRNA can remain in its original structure without Mg2+ ions at room temperature, only Mg-bound 3WJ-pRNA still maintains its initial three-way junction structure at a higher temperature (T = 400 K). The Mg-free 3WJ-pRNA undergoes dramatic deformation under high temperature condition. The contribution of Mg ions can be largely attributed to the protective effect of two Mg clamps on the hydrogen bond and base stacking interactions in helices. Taken together, our results reveal the extraordinary thermal stability of 3WJ-pRNA, which can be regulated by Mg2+ ions. Comprehensive depictions of thermal stability of pRNA and the regulation mechanism are helpful for the further development of controllable RNA nanoparticle drug delivery platforms. [ABSTRACT FROM AUTHOR]

Immunotherapy has become a revolutionary subject in cancer therapy during the past few years. Immune checkpoint-targeting antibodies (Abs) could boost anticancer immune responses. However, certain protein-based immunotherapies revealed side effects and unfavorable biodistribution, so effective non-protein options with lower side effects are highly sought after. RNA’s ability to form various three-dimensional configurations allows for the creation of a variety of ligands to bind different cell receptors. The rubber-like properties of RNA nanoparticles (NPs) allow for swift lodging to cancer vasculature with little accumulation in vital organs, resulting in a favorable pharmacokinetic/pharmacodynamic (PK/PD) profile and safe pharmacological parameters. Multi-specific drugs are expected to be the fourth wave of biopharmaceutical innovation. Herein, we report the development of multi-specific Ab-like RNA NPs carrying multiple ligands for immunotherapy. The stoichiometries and stereo conformations of the checkpoint-activating RNA NPs were optimized for T cell activation. When compared to mono- and bi-specific RNA NPs, the tri-specific Ab-like RNA NPs bound to the trimeric T cell receptor with the highest efficiency, showed the optimal T cell activation, and promoted the strongest anti-tumor function of immune cells. Animal trials demonstrated that the tri-specific RNA NPs inhibited cancer growth. This Ab-like RNA NP platform represents an alternative to protein Abs for tumor immunotherapy.

RNA nanotechnology harnesses the unique chemical and structural properties of RNA to build nanoassemblies and supramolecular structures with dynamic and functional capabilities. This review focuses on design and assembly approaches to building RNA structures, the RNA chemical modifications used to enhance stability and functionality, and modern-day applications in therapeutics, biosensing, and bioimaging. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) mediated by small interfering RNA (siRNA) duplexes is a powerful therapeutic modality, but the translation of siRNAs from the bench into clinical application has been hampered by inefficient delivery in vivo. An innovative delivery strategy involves fusing siRNAs to a three‐way junction (3WJ) motif derived from the phi29 bacteriophage prohead RNA (pRNA). Chimeric siRNA‐3WJ molecules are presumed to enter the RNAi pathway through Dicer cleavage. Here, we fused siRNAs to the phi29 3WJ and two phylogenetically related 3WJs. We confirmed that the siRNA‐3WJs are substrates for Dicer in vitro. However, our results reveal that siRNA‐3WJs transfected into Dicer‐deficient cell lines trigger potent gene silencing. Interestingly, siRNA‐3WJs transfected into an Argonaute 2‐deficient cell line also retain some gene silencing activity. siRNA‐3WJs are most efficient when the antisense strand of the siRNA duplex is positioned 5′ of the 3WJ (5′‐siRNA‐3WJ) relative to 3′ of the 3WJ (3′‐siRNA‐3WJ). This work sheds light on the functional properties of siRNA‐3WJs and offers a design rule for maximizing their potency in the human RNAi pathway. [ABSTRACT FROM AUTHOR]

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 2ʹ-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. [ABSTRACT FROM AUTHOR]

Bacteriophage RNA polymerases, in particular T7 RNA polymerase (RNAP), are well-characterized and popular enzymes for many RNA applications in biotechnology both in vitro and in cellular settings. These monomeric polymerases are relatively inexpensive and have high transcription rates and processivity to quickly produce large quantities of RNA. T7 RNAP also has high promoter-specificity on double-stranded DNA (dsDNA) such that it only initiates transcription downstream of its 17-base promoter site on dsDNA templates. However, there are many promoter-independent T7 RNAP transcription reactions involving transcription initiation in regions of single-stranded DNA (ssDNA) that have been reported and characterized. These promoter-independent transcription reactions are important to consider when using T7 RNAP transcriptional systems for DNA nanotechnology and DNA computing applications, in which ssDNA domains often stabilize, organize, and functionalize DNA nanostructures and facilitate strand displacement reactions. Here we review the existing literature on promoter-independent transcription by bacteriophage RNA polymerases with a specific focus on T7 RNAP, and provide examples of how promoter-independent reactions can disrupt the functionality of DNA strand displacement circuit components and alter the stability and functionality of DNA-based materials. We then highlight design strategies for DNA nanotechnology applications that can mitigate the effects of promoter-independent T7 RNAP transcription. The design strategies we present should have an immediate impact by increasing the rate of success of using T7 RNAP for applications in DNA nanotechnology and DNA computing.

Injectable anticoagulants are widely used in medical procedures to prevent unwanted blood clotting. However, many lack safe, effective reversal agents. Here, we present new data on a previously described RNA origami-based, direct thrombin inhibitor (HEX01). We describe a new, fast-acting, specific, single-molecule reversal agent (antidote) and present in vivo data for the first time, including efficacy, reversibility, preliminary safety, and initial biodistribution studies. HEX01 contains multiple thrombin-binding aptamers appended on an RNA origami. It exhibits excellent anticoagulation activity in vitro and in vivo. The new single-molecule, DNA antidote (HEX02) reverses anticoagulation activity of HEX01 in human plasma within 30 s in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulation mainly in the liver over 24 h and with 10-fold lower concentrations in the kidneys. Additionally, we show that the HEX01/HEX02 system is non-cytotoxic to epithelial cell lines and non-hemolytic in vitro. Furthermore, we found no serum cytokine response to HEX01/HEX02 in a murine model. HEX01 and HEX02 represent a safe and effective coagulation control system with a fast-acting, specific reversal agent showing promise for potential drug development., Competing Interests: Declaration of interests T.H.L. and A.K. disclose the existence of a startup company, Helixomer Inc., that they founded to commercialize the HEX01/HEX02 anticoagulation system. T.H.L., A.K., and NC State University acknowledge that a patent and a provisional patent on these nucleic acid sequences and technology designs have been filed (U.S. Application 17/285,371 and U.S. Provisional Application, 63/230,153)., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

DNA droplets, artificial liquid-like condensates of well-engineered DNA sequences, allow the critical aspects of phase-separated biological condensates to be harnessed programmably, such as molecular sensing and phase-state regulation. In contrast, their RNA-based counterparts remain less explored despite more diverse molecular structures and functions ranging from DNA-like to protein-like features. Here, we design and demonstrate computational RNA droplets capable of two-input AND logic operations. We use a multibranched RNA nanostructure as a building block comprising multiple single-stranded RNAs. Its branches engaged in RNA-specific kissing-loop (KL) interaction enables the self-assembly into a network-like microstructure. Upon two inputs of target miRNAs, the nanostructure is programmed to break up into lower-valency structures that are interconnected in a chain-like manner. We optimize KL sequences adapted from viral sequences by numerically and experimentally studying the base-wise adjustability of the interaction strength. Only upon receiving cognate microRNAs, RNA droplets selectively show a drastic phase-state change from liquid to dispersed states due to dismantling of the network-like microstructure. This demonstration strongly suggests that the multistranded motif design offers a flexible means to bottom-up programming of condensate phase behavior. Unlike submicroscopic RNA-based logic operators, the macroscopic phase change provides a naked-eye-distinguishable readout of molecular sensing. Our computational RNA droplets can be applied to in situ programmable assembly of computational biomolecular devices and artificial cells from transcriptionally derived RNA within biological/artificial cells.

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.

Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on the translocation of canonical B-form or A-form nucleic acids, while direct RNA analysis is emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of noncanonical RNA:DNA hybrids in electrophoretic transport to the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that RD duplexes present a noncanonical helix, with distinct transport properties from B-form DD molecules. We find that RD and DD molecules, with the same contour length, move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, atomistic molecular dynamics simulations, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and molecular dynamics simulations, we find the effective force per unit length applied by the electric field to a fragment of RD or DD duplex in nanopores with various geometries or shapes to be approximately the same. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and the molecular understanding of electrophoretic transport.

Breast cancer has been identified as the primary cause of female mortality on a global scale. Recent statistics indicate that a significant number of women, approximately 8.2 million, have been diagnosed with BC in the past five years. Scientists around the world have been motivated to investigate alternative management strategies because of the high prevalence and poor neoadjuvant chemotherapeutic efficacy of BC [1, 2]. Over the last decade, tumor-derived exosomes (TDEs) have been reported in an exponential increase of scientific studies relating to BC pathophysiology and treatment [3]. Exosomes (Exos) are extracellular vesicles that have shown promising results in delivering a payload to cancer cells with great efficacy. Presented in this report are innovative exosomes, adorned with transforming growth factor-a (TGFA), which act as efficient carriers for the targeted delivery of Docetaxel (DTX) and miR155 to breast tumors. This novel approach has demonstrated promising outcomes in inhibiting breast cancer. The Exos targeting capability is facilitated by TGFA, which binds to the overexpressed epidermal growth factor receptors (EGFRs) on the breast cancer cell surface. To achieve specific targeting, a 3-way junction (3WJ) RNA nanoparticle was designed to harbor DTX and miR-155. The 3WJ RNA-DTX complex was then loaded into the Exos, and RNA nanotechnology was employed to decorate its surface with TGFA. The multi-specific Exos have been found to possess the ability to selectively bind and efficiently deliver payload into liver cancer cells. This is due to their unique multi-specific effect of miR-155, DTX, TGFA, and Exos, which results in the highest degree of cancer cell inhibition. This research proved that Exos, a natural cargo vehicle, can be effectively used to target difficult-to treat cancers with anticancer therapeutics. The multi-specific strategy employed reduces the required dose of chemical drugs carried by Exos, indicating high efficiency and low toxicity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Despite RNA's diverse secondary and tertiary structures and its complex conformational changes, nature utilizes a limited set of structural "motifs"--helices, junctions, and tertiary contact modules--to build diverse functional RNAs. Thus, in-depth descriptions of a relatively small universe of RNA motifs may lead to predictive models of RNA tertiary conformational landscapes. Motifs may have different properties depending on sequence and secondary structure, giving rise to subclasses that expand the universe of RNA building blocks. Yet we know very little about motif subclasses, given the challenges in mapping conformational properties in high throughput. Previously, we used "RNA on a massively parallel array" (RNA-MaP), a quantitative, high-throughput technique, to study thousands of helices and two-way junctions. Here, we adapt RNA-MaP to study the thermodynamic and conformational properties of tetraloop/tetraloop receptor (TL/TLR) tertiary contact motifs, analyzing 1,493 TLR sequences from different classes. Clustering analyses revealed variability in TL specificity, stability, and conformational behavior. Nevertheless, natural GAAA/11ntR TL/TLRs, while varying in tertiary stability by ~2.5 kcal/mol, exhibited conserved TL specificity and conformational properties. Thus, RNAs may tune stability without altering the overall structure of these TL/TLRs. Furthermore, their stability correlated with natural frequency, suggesting thermodynamics as the dominant selection pressure. In contrast, other TL/TLRs displayed heterogenous conformational behavior and appear to not be under strong thermodynamic selection. Our results build toward a generalizable model of RNA-folding thermodynamics based on the properties of isolated motifs, and our characterized TL/TLR library can be used to engineer RNAs with predictable thermodynamic and conformational behavior. [ABSTRACT FROM AUTHOR]

Combinations of different Nucleic Acid Nanoparticles (NANPs) and delivery agents allows to direct desirable and avoid undesirable immunological effects and therapeutic actions. [Display omitted] Nucleic acid nanoparticles (NANPs) represent a highly versatile molecular platform for the targeted delivery of various therapeutics. However, despite their promise, further clinical translation of this innovative technology can be hindered by immunological off-target effects. All human cells are equipped with an arsenal of receptors that recognize molecular patterns specific to foreign nucleic acids and understanding the rules that guide this recognition offer the key rationale for the development of therapeutic NANPs with tunable immune stimulation. Numerous recent studies have provided increasing evidence that in addition to NANPs' physicochemical properties and therapeutic effects, their interactions with cells of the immune system can be regulated through multiple independently programmable architectural parameters. The results further suggest that defined immunomodulation by NANPs can either support their immunoquiescent delivery or be used for conditional stimulation of beneficial immunological responses. [ABSTRACT FROM AUTHOR]

The field of RNA therapeutics has been emerging as the third milestone in pharmaceutical drug development. RNA nanoparticles have displayed motile and deformable properties to allow for high tumor accumulation with undetectable healthy organ accumulation. Therefore, RNA nanoparticles have the potential to serve as potent drug delivery vehicles with strong anti-cancer responses. Herein, we report the physicochemical basis for the rational design of a branched RNA four-way junction (4WJ) nanoparticle that results in advantageous high-thermostability and -drug payload for cancer therapy, including metastatic tumors in the lung. The 4WJ nanostructure displayed versatility through functionalization with an anti-cancer chemical drug, SN38, for the treatment of two different cancer models including colorectal cancer xenograft and orthotopic lung metastases of colon cancer. The resulting 4WJ RNA drug complex spontaneously targeted cancers effectively for cancer inhibition with and without ligands. The 4WJ displayed fast renal excretion, rapid body clearance, and little organ accumulation with undetectable toxicity and immunogenicity. The safety parameters were documented by organ histology, blood biochemistry, and pathological analysis. The highly efficient cancer inhibition, undetectable drug toxicity, and favorable Chemical, Manufacturing, and Control (CMC) production of RNA nanoparticles document a candidate with high potential for translation in cancer therapy. [ABSTRACT FROM AUTHOR]

The fast-developing field of RNA nanotechnology requires the adoption and development of novel and faster computational approaches to modeling and characterization of RNA-based nano-objects. We report the first application of Elastic Network Modeling (ENM), a structure-based dynamics model, to RNA nanotechnology. With the use of an Anisotropic Network Model (ANM), a type of ENM, we characterize the dynamic behavior of non-compact, multi-stranded RNA-based nanocubes that can be used as nano-scale scaffolds carrying different functionalities. Modeling the nanocubes with our tool NanoTiler and exploring the dynamic characteristics of the models with ANM suggested relatively minor but important structural modifications that enhanced the assembly properties and thermodynamic stabilities. In silico and in vitro, we compared nanocubes having different numbers of base pairs per side, showing with both methods that the 10 bp-long helix design leads to more efficient assembly, as predicted computationally. We also explored the impact of different numbers of single-stranded nucleotide stretches at each of the cube corners and showed that cube flexibility simulations help explain the differences in the experimental assembly yields, as well as the measured nanomolecule sizes and melting temperatures. This original work paves the way for detailed computational analysis of the dynamic behavior of artificially designed multi-stranded RNA nanoparticles.

RNA nanotechnology has shown great progress over the past decade. Diverse controllable and multifunctional RNA nanoparticles have been developed for various applications in many areas. For example, RNA nanoparticles can participate in the construction of drug delivery nanoplatforms. Recently, a three-way junction packaging RNA (3WJ-pRNA) has been exploited for its characteristics of self-assembly and ultrahigh stability in many aspects. 3WJ-pRNA is the 3WJ part of bacteriophage φ29 pRNA and joins different components of φ29 as a linker element. In this work, we used all-atom MD simulation to study the thermal stability of 3WJ-pRNA and the underlying mechanisms. While 3WJ-pRNA can remain in its original structure without Mg2+ ions at room temperature, only Mg-bound 3WJ-pRNA still maintains its initial three-way junction structure at a higher temperature (T = 400 K). The Mg-free 3WJ-pRNA undergoes dramatic deformation under high temperature condition. The contribution of Mg ions can be largely attributed to the protective effect of two Mg clamps on the hydrogen bond and base stacking interactions in helices. Taken together, our results reveal the extraordinary thermal stability of 3WJ-pRNA, which can be regulated by Mg2+ ions. Comprehensive depictions of thermal stability of pRNA and the regulation mechanism are helpful for the further development of controllable RNA nanoparticle drug delivery platforms.

Ribozymes are catalytic RNAs that are attractive platforms for the construction of nanoscale objects with biological functions. We designed a dimeric form of the Tetrahymena group I ribozyme as a unit structure in which two ribozymes were connected in a tail-to-tail manner with a linker element. We introduced a kink-turn motif as a bent linker element of the ribozyme dimer to design a closed trimer with a triangular shape. The oligomeric states of the resulting ribozyme dimers (kUrds) were analyzed biochemically and observed directly by atomic force microscopy (AFM). Formation of kUrd oligomers also triggered trans-splicing reactions, which could be monitored with a reporter system to yield a fluorescent RNA aptamer as the trans-splicing product. [ABSTRACT FROM AUTHOR]

This Minireview discusses the design and applications of binary (also known as split) light‐up aptameric sensors (BLAS). BLAS consist of two RNA or DNA strands and a fluorogenic organic dye added as a buffer component. When associated, the two strands form a dye‐binding site, followed by an increase in fluorescence of the aptamer‐bound dye. The design is cost‐efficient because it uses short oligonucleotides and does not require conjugation of organic dyes with nucleic acids. In some applications, BLAS design is preferable over monolithic sensors because of simpler assay optimization and improved selectivity. RNA‐based BLAS can be expressed in cells and used for the intracellular monitoring of biological molecules. BLAS have been used as reporters of nucleic acid association events in RNA nanotechnology and nucleic‐acid‐based molecular computation. Other applications of BLAS include the detection of nucleic acids, proteins, and cancer cells, and potentially they can be tailored to report a broad range of biological analytes. [ABSTRACT FROM AUTHOR]

Aberrant expression of certain genes and microRNAs (miRNAs) has been shown to drive cancer development and progression, thus the modification of aberrant gene and miRNA expression presents an opportunity for therapeutic targeting. Ectopic modulation of a single dysregulated miRNA has the potential to revert therapeutically unfavorable gene expression in cancer cells by targeting multiple genes simultaneously. Although the use of noncoding RNA-based cancer therapy is a promising approach, the lack of a feasible delivery platform for small noncoding RNAs has hindered the development of this therapeutic modality. Recently, however, there has been an evolution in RNA nanotechnology, in which small noncoding RNA is loaded onto nanoparticles derived from the pRNA-3WJ viral RNA motif of the bacteriophage phi29. Preclinical studies have shown the capacity of this technology to specifically target tumor cells by conjugating these nanoparticles with ligands specific for cancer cells and resulting in the endocytic delivery of siRNA and miRNA inhibitors directly into the cell. Here we provide a systematic review of the various strategies, which have been utilized for miRNA delivery with a specific focus on the preclinical evaluation of promising RNA nanoparticles for glioblastoma (GBM) targeted therapy. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Analogous to the border customs, liver mainly functions as a filter to detoxify chemicals and metabolite administered orally or intravenously. Besides, the liver cancer cells overexpress the drug exporters which cause high drug effluxion from liver cancer cells, leading to chemoresistance and a diminished chemotherapeutic effect on liver cancer. Recently, we found that RNA nanoparticles display rubber-like property that can rapidly deliver therapeutics to tumor site efficiently and the rest of the RNA nanoparticle were cleared by renal excretion within half hour after systemic injection. Therefore, we designed a new multivalent RNA nanoparticle harboring three copies of hepatocyte targeting-ligands, one copy of miR122, and 24 copies of Paclitaxel to overcome the drug effluxion and chemoresistance thus, synergistically treating HCC. The hepatocyte targeting ligands introduce tumor specificity to the RNA nanoparticles as they selectively bind and internalize into liver cancer cells. The rubber-like RNA nanoparticles allow for enhanced targeting ability to the HCC tumors. The RNA nanoparticles carrying miR122 and PTX were delivered to the liver cancer cells efficiently due to their rubber-like property to enhance their EPR as well as the receptor-mediated endocytosis by hepatocyte targeting-ligands. The miR122 efficiently silenced the drug exporters and the oncogenic proteins. The synergistic effect between miR122 and PTX was confirmed by HSA (Highest Single Agent) synergy model. IC 50 was determined to be 460 nM. In vivo studies on mice xenografts revealed that the RNA nanoparticle predominantly accumulated in HCC tumor sites and efficiently inhibited the tumor growth after multiple IV injection. This demonstrates the potential of the rubber-like multivalent RNA nanoparticles to conquest the liver cancer, a currently incurable lethal disease. Unlabelled Image • Rubber-like 6WJ RNA nanoparticle carries 24 PTX, one miR122 and one chemical ligand. • RNA nanoparticle displaying hepatocyte targeting ligand specifically target to tumor. • Co-delivery of PTX and miR-122 exhibit synergetic therapeutic effect to liver cancer. • The targeted RNA nanoparticles inhibited liver cancer growth in liver cancer xenograft. [ABSTRACT FROM AUTHOR]

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences, which results in synthesis failure or delays by DNA synthesis vendors. This represents a major obstacle for the development of synthetic biology since repetitive elements are increasingly being used in the design of genetic circuits and design of biomolecular nanostructures. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden-Gate-compatible overhangs. Then, synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate Assembly. We demonstrate the method by constructing eight challenging genes with repetitive elements, e.g., multiple repeats of RNA aptamers and RNA origami scaffolds with multiple identical aptamers. The genes range in size from 133 to 456 base pairs and are assembled with fidelities of up to 87.5%. The method was developed to facilitate our own specific research but may be of general use for constructing challenging and repetitive genes and, thus, a valuable addition to the molecular cloning toolbox.

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. [ABSTRACT FROM AUTHOR]