In Vitro and In Vivo Evaluation of the Pathology and Safety Aspects of Three- and Four-Way Junction RNA Nanoparticles.

RNA therapeutics has advanced into the third milestone in pharmaceutical drug development, following chemical and protein therapeutics. RNA itself can serve as therapeutics, carriers, regulators, or substrates in drug development. Due to RNA's motile, dynamic, and deformable properties, RNA nanoparticles have demonstrated spontaneous targeting and accumulation in cancer vasculature and fast excretion through the kidney glomerulus to urine to prevent possible interactions with healthy organs. Furthermore, the negatively charged phosphate backbone of RNA results in general repulsion from negatively charged lipid cell membranes for further avoidance of vital organs. Thus, RNA nanoparticles can spontaneously enrich tumor vasculature and efficiently enter tumor cells via specific targeting, while those not entering the tumor tissue will clear from the body quickly. These favorable parameters have led to the expectation that RNA has low or little toxicity. RNA nanoparticles have been well characterized for their anticancer efficacy; however, little detail on RNA nanoparticle pathology and safety is known. Here, we report the in vitro and in vivo assessment of the pathology and safety aspects of different RNA nanoparticles including RNA three-way junction (3WJ) harboring 2'-F modified pyrimidine, folic acid, and Survivin siRNA, as well as the RNA four-way junction (4WJ) harboring 2'-F modified pyrimidine and 24 copies of SN38. Both animal models and patient serum were investigated. In vitro studies include hemolysis, platelet aggregation, complement activation, plasma coagulation, and interferon induction. In vivo studies include hematoxylin and eosin (H&E) staining, hematological and biochemical analysis as the serum profiling, and animal organ weight study. No significant toxicity, side effect, or immune responses were detected during the extensive safety evaluations of RNA nanoparticles. These results further complement previous cancer inhibition studies and demonstrate RNA nanoparticles as an effective and safe drug delivery vehicle for future clinical translations.

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.

Processing and decay of 6S-1 and 6S-2 RNAs in Bacillus subtilis.

Noncoding 6S RNAs regulate transcription by binding to the active site of bacterial RNA polymerase holoenzymes. Processing and decay of 6S-1 and 6S-2 RNA were investigated in Bacillus subtilis by northern blot and RNA-seq analyses using different RNase knockout strains, as well as by in vitro processing assays. For both 6S RNA paralogs, we identified a key-but mechanistically different-role of RNase J1. RNase J1 catalyzes 5'-end maturation of 6S-1 RNA, yet relatively inefficient and possibly via the enzyme's "sliding endonuclease" activity. 5'-end maturation has no detectable effect on 6S-1 RNA function, but rather regulates its decay: The generated 5'-monophosphate on matured 6S-1 RNA propels endonucleolytic cleavage in its apical loop region. The major 6S-2 RNA degradation pathway is initiated by endonucleolytic cleavage in the 5'-central bubble to trigger 5'-to-3'-exoribonucleolytic degradation of the downstream fragment by RNase J1. The four 3'-exonucleases of B. subtilis-RNase R, PNPase, YhaM, and particularly RNase PH-are involved in 3'-end trimming of both 6S RNAs, degradation of 6S-1 RNA fragments, and decay of abortive transcripts (so-called product RNAs, ∼14 nt in length) synthesized on 6S-1 RNA during outgrowth from stationary phase. In the case of the growth-retarded RNase Y deletion strain, we were unable to infer a specific role of RNase Y in 6S RNA decay. Yet, a participation of RNase Y in 6S RNA decay still remains possible, as evidence for such a function may have been obscured by overlapping substrate specificities of RNase Y, RNase J1, and RNase J2.

Potential of Nucleic Acid Receptor Ligands to Improve Vaccination Efficacy against the Filarial Nematode Litomosoides sigmodontis.

More than two-hundred-million people are infected with filariae worldwide. However, there is no vaccine available that confers long-lasting protection against filarial infections. Previous studies indicated that vaccination with irradiated infective L3 larvae reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as an adjuvant improves the efficacy of vaccination with irradiated L3 larvae of the rodent filaria Litomosoides sigmodontis with the aim of identifying novel vaccination strategies for filarial infections. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-ß RNA levels. To investigate the impact on parasite clearance, BALB/c mice received three subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a markedly greater reduction in adult-worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). In conclusion, activation of nucleic acid-sensing immune receptors boosts the protective immune response against L. sigmodontis and nucleic acid-receptor agonists as vaccine adjuvants represent a promising novel strategy to improve the efficacy of vaccines against filariae and potentially other helminths.

Structurally distinct PARP7 inhibitors provide new insights into the function of PARP7 in regulating nucleic acid-sensing and IFN-ß signaling.

The mono-ADP-ribosyltransferase PARP7 has emerged as a key negative regulator of cytosolic NA-sensors of the innate immune system. We apply a rational design strategy for converting a pan-PARP inhibitor into a potent selective PARP7 inhibitor (KMR-206). Consistent with studies using the structurally distinct PARP7 inhibitor RBN-2397, co-treatment of mouse embryonic fibroblasts with KMR-206 and NA-sensor ligands synergistically induced the expression of the type I interferon, IFN-ß. In mouse colon carcinoma (CT-26) cells, KMR-206 alone induced IFN-ß. Both KMR-206 and RBN-2397 increased PARP7 protein levels in CT-26 cells, demonstrating that PARP7's catalytic activity regulates its own protein levels. Curiously, treatment with saturating doses of KMR-206 and RBN-2397 achieved different levels of PARP7 protein, which correlated with the magnitude of type I interferon gene expression. These latter results have important implications for the mechanism of action of PARP7 inhibitors and highlights the usefulness of having structurally distinct chemical probes for the same target.

E. coli 6S RNA complexed to RNA polymerase maintains product RNA synthesis at low cellular ATP levels by initiation with noncanonical initiator nucleotides.

The E. coli 6S RNA is an RNA polymerase (RNAP) inhibitor that competes with σ70-dependent DNA promoters for binding to RNAP holoenzyme (RNAP:σ70). The 6S RNA when bound is then used as a template to synthesize a short product RNA (pRNA; usually 13-nt-long). This pRNA changes the 6S RNA structure, triggering the 6S RNA:pRNA complex to release and allowing DNA-dependent housekeeping gene expression to resume. In high nutrient conditions, 6S RNA turnover is extremely rapid but becomes very slow in low nutrient environments. This leads to a large accumulation of inhibited RNAP:σ70 in stationary phase. As pRNA initiates synthesis with ATP, we and others have proposed that the 6S RNA release rate strongly depends on ATP levels as a proxy for sensing the cellular metabolic state. By purifying endogenous 6S RNA:pRNA complexes using RNA Mango and using reverse transcriptase to generate pRNA-cDNA chimeras, we demonstrate that 6S RNA:pRNA formation can be simultaneous with 6S RNA 5' maturation. More importantly, we find a dramatic accumulation of capped pRNAs during stationary phase. This indicates that ATP levels in stationary phase are low enough for noncanonical initiator nucleotides (NCINs) such as NAD+ and NADH to initiate pRNA synthesis. In vitro, mutation of the conserved 6S RNA template sequence immediately upstream of the pRNA transcriptional start site can increase or decrease the pRNA capping efficiency, suggesting that evolution has tuned the biological 6S RNA sequence for an optimal capping rate. NCIN-initiated pRNA synthesis may therefore be essential for cell viability in low nutrient conditions.

Next generation sequencing reveals miR-431-3p/miR-1303 as immune-regulating microRNAs for active tuberculosis.

OBJECTIVES: RNA therapeutics is an emerging field that widens the range of treatable targets and would improve disease outcome through bypassing the antibiotic bactericidal targets to kill Mycobacterium tuberculosis (M.tb). METHODS: We screened for microRNA with immune-regulatory functions against M.tb by next generation sequencing of peripheral blood mononuclear cells, followed by validation in an independent cohort. RESULTS: Twenty three differentially expressed microRNAs were identified between 12 active pulmonary TB patients and 4 healthy subjects, and 35 microRNAs before and after 6-month anti-TB therapy. Enriched predicted target pathways included proteoglycan, HIF-1 signaling, longevity-regulating, central carbon metabolism, and autophagy. We validated miR-431-3p down-regulation and miR-1303 up-regulation accompanied with corresponding changes in their predicted target genes in an independent validation cohort of 46 active TB patients, 30 latent TB infection subjects, and 24 non-infected healthy subjects. In vitro experiments of transfections with miR-431-3p mimic/miR-1303 short interfering RNA in THP-1 cells under ESAT-6 stimuli showed that miR-431-3p and miR-1303 were capable to augment and suppress autophagy/apoptosis/phagocytosis of macrophage via targeting MDR1/MMP16/RIPOR2 and ATG5, respectively. CONCLUSIONS: This study provides a proof of concept for microRNA-based host-directed immunotherapy for active TB disease. The combined miR-431-3p over-expression and miR-1303 knock-down revealed new vulnerabilities of treatment-refractory TB disease.

Mg2+ Ions Regulating 3WJ-PRNA to Construct Controllable RNA Nanoparticle Drug Delivery Platforms.

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.

Advancements in 3WJ-based RNA nanotechnology and its application for cancer diagnosis and therapy.

BACKGROUND: This attractive and intriguing Ribonucleic acid (RNA) nanotechnology has been conceptualized over the last two decades and with our increasing understanding of RNA structure and function and improvements of RNA nanotechnology it is now possible to use this in clinical settings. METHODS: Here we review the unique properties and the recent advances in RNA nanotechnology and then look at its scientific and preclinical applications for tumor diagnosis and targeted delivery and RNA-based therapy using RNA nanoparticles with diverse structures and functions. Finally, we discuss the future perspectives and challenges to RNA nanotechnology. RESULTS: RNA can be designed and manipulated in a similar way to DNA while having different rules for base-pairing and displaying functions similar to proteins. Rationally designed RNA nanoparticles based on the three-way junction (3WJ) motif as the core scaffold have been extensively explored in the field of nanomedicine and targeted cancer diagnosis and therapy. CONCLUSIONS: RNA nanostructures based on 3WJs demonstrate promising future applications due to their thermal stability, molecular-level plasticity, multifunctional chemotherapeutic drug delivery and other intrinsic characteristics, which will greatly improve the treatment of cancer and promote further major breakthroughs in this field.

Structural and Functional Insight into the Mechanism of Bacillus subtilis 6S-1 RNA Release from RNA Polymerase.

Here we investigated the refolding of Bacillus subtilis 6S-1 RNA and its release from σA-RNA polymerase (σA-RNAP) in vitro using truncated and mutated 6S-1 RNA variants. Truncated 6S-1 RNAs, only consisting of the central bubble (CB) flanked by two short helical arms, can still traverse the mechanistic 6S RNA cycle in vitro despite ~10-fold reduced σA-RNAP affinity. This indicates that the RNA's extended helical arms including the '-35'-like region are not required for basic 6S-1 RNA functionality. The role of the 'central bubble collapse helix' (CBCH) in pRNA-induced refolding and release of 6S-1 RNA from σA-RNAP was studied by stabilizing mutations. This also revealed base identities in the 5'-part of the CB (5'-CB), upstream of the pRNA transcription start site (nt 40), that impact ground state binding of 6S-1 RNA to σA-RNAP. Stabilization of the CBCH by the C44/45 double mutation shifted the pRNA length pattern to shorter pRNAs and, combined with a weakened P2 helix, resulted in more effective release from RNAP. We conclude that formation of the CBCH supports pRNA-induced 6S-1 RNA refolding and release. Our mutational analysis also unveiled that formation of a second short hairpin in the 3'-CB is detrimental to 6S-1 RNA release. Furthermore, an LNA mimic of a pRNA as short as 6 nt, when annealed to 6S-1 RNA, retarded the RNA's gel mobility and interfered with σA-RNAP binding. This effect incrementally increased with pLNA 7- and 8-mers, suggesting that restricted conformational flexibility introduced into the 5'-CB by base pairing with pRNAs prevents 6S-1 RNA from adopting an elongated shape. Accordingly, atomic force microscopy of free 6S-1 RNA versus 6S-1:pLNA 8- and 14-mer complexes revealed that 6S-1:pRNA hybrid structures, on average, adopt a more compact structure than 6S-1 RNA alone. Overall, our findings also illustrate that the wild-type 6S-1 RNA sequence and structure ensures an optimal balance of the different functional aspects involved in the mechanistic cycle of 6S-1 RNA.

Rapid preparation of 6S RNA-free B. subtilis σA-RNA polymerase and σA.

The regulatory 6S-1 and 6S-2 RNAs of B. subtilis bind to the housekeeping RNA polymerase holoenzyme (σA-RNAP) with submicromolar affinity. We observed copurification of endogenous 6S RNAs from a published B. subtilis strain expressing a His-tagged RNAP. Such 6S RNA contaminations in σA-RNAP preparations reduce the fraction of enzymes that are accessible for binding to DNA promoters. In addition, this leads to background RNA synthesis by σA-RNAP utilizing copurified 6S RNA as template for the synthesis of short abortive transcripts termed product RNAs (pRNAs). To avoid this problem we constructed a B. subtilis strain expressing His-tagged RNAP but carrying deletions of the two 6S RNA genes. The His-tagged, 6S RNA-free σA-RNAP holoenzyme can be prepared with sufficient purity and activity by a single affinity step. We also report expression and separate purification of B. subtilis σA that can be added to the His-tagged RNAP to maximize the amount of holoenzyme and, by inference, in vitro transcription activity.

LncRNA MEG3 regulates breast cancer proliferation and apoptosis through miR-141-3p/RBMS3 axis.

Maternally expressed 3 (MEG3) and RNA binding motif single stranded interacting protein 3 (RBMS3) are abnormally expressed in breast cancer susceptibility genes (BRCA), but the mechanism of the two in breast cancer (BC) is unclear. By performing in vivo and in vitro experiments, we found that MEG3 and RBMS3 were low-expressed, negatively correlated with high-expressed miR-141-3p, were positively correlated with each other in BC. MEG3 targeted miR-141-3p, and miR-141-3p targeted RBMS3. MEG3, which was mainly distributed in BC cytoplasm, could down-regulate miR-141-3p and up-regulate RBMS3, and reverse effect of miR-141-3p on related gene expressions and on promoting cancer development. Overexpressed MEG3 inhibited growth of xenografts, promoted cell apoptosis via regulating apoptosis related factors, and up-regulated RBMS3 expression but down-regulated miR-141-3p. The findings of this study showed that MEG3 inhibited proliferation and promoted apoptosis of BC cells through the miR-141-3p/RBMS3 axis, and MEG3 inhibited growth of xenografts through miR-141-3p.

Optical tweezer and TIRF microscopy for single molecule manipulation of RNA/DNA nanostructures including their rubbery property and single molecule counting.

Life science is often focused on the microscopic level. Single-molecule technology has been used to observe components at the micro- or nanoscale. Single-molecule imaging provides unprecedented information about the behavior of individual molecules in contrast to the information from ensemble methods that average the information of many molecules in various states. A typical feature of living systems is motion. The lack of synchronicity of motion biomachines in living systems makes it challenging to image the motion process with high resolution. Thus, single-molecule technology is especially useful for real-time study on motion mechanism of biomachines, such as viral DNA packaging motor, or other ATPases. The most common optical instrumentations in single-molecule studies are optical tweezers and single molecule total internal refection fluorescence microscopy (smTIRF). Optical tweezers are the force-based technique. The analysis of RNA using optical tweezer has led to the discovery of the rubbery or amoeba property of RNA nanoparticles for compelling vessel extravasation to enhance tumor targeting and fast renal excretion. The rubbery property of RNA lends mechanistic evidence for RNAs use as an ideal reagent in cancer treatment with undetectable toxicity. Single molecule photobleaching allows for the direct counting of biomolecules. This technique was invented for single molecule counting of RNA in the phi29 DNA packaging motor to resolve the debate between five and six copies of RNA in the motor. The technology has subsequently extended to counting components in biological machines composed of protein, DNA, and other macromolecules. In combination with statistical analysis, it reveals biomolecular mechanisms in detail and leads to the development of ultra-sensitive sensors in diagnosis and forensics. This review focuses on the applications of optical tweezers and fluorescence-based techniques as single-molecule technologies to resolve mechanistic questions related to RNA and DNA nanostructures.

Similarities and differences between 6S RNAs from Bradyrhizobium japonicum and Sinorhizobium meliloti.

6S RNA, a conserved and abundant small non-coding RNA found in most bacteria, regulates gene expression by inhibiting RNA polymerase (RNAP) holoenzyme. 6S RNAs from α-proteobacteria have been studied poorly so far. Here, we present a first in-depth analysis of 6S RNAs from two α-proteobacteria species, Bradyrhizobium japonicum and Sinorhizobium meliloti. Although both belong to the order Rhizobiales and are typical nitrogen-fixing symbionts of legumes, their 6S RNA expression profiles were found to differ: B. japonicum 6S RNA accumulated in the stationary phase, thus being reminiscent of Escherichia coli 6S RNA, whereas S. meliloti 6S RNA level peaked at the transition to the stationary phase, similarly to Rhodobacter sphaeroides 6S RNA. We demonstrated in vitro that both RNAs have hallmarks of 6S RNAs: they bind to the σ70-type RNAP holoenzyme and serve as templates for de novo transcription of so-called product RNAs (pRNAs) ranging in length from ∼13 to 24 nucleotides, with further evidence of the synthesis of even longer pRNAs. Likewise, stably bound pRNAs were found to rearrange the 6S RNA structure to induce its dissociation from RNAP. Compared with B. japonicum 6S RNA, considerable conformational heterogeneity was observed for S. meliloti 6S RNA and its complexes with pRNAs, even though the two 6S RNAs share ∼75% sequence identity. Overall, our findings suggest that the two rhizobial 6S RNAs have diverged with respect to their regulatory impact on gene expression throughout the bacterial life cycle.

RNA Nanoparticles as Rubber for Compelling Vessel Extravasation to Enhance Tumor Targeting and for Fast Renal Excretion to Reduce Toxicity.

Rubber is a fascinating material in both industry and daily life. The development of elastomeric material in nanotechnology is imperative due to its economic and technological potential. By virtue of their distinctive physicochemical properties, nucleic acids have been extensively explored in material science. The Phi29 DNA packaging motor contains a 3WJ with three angles of 97°, 125°, and 138°. Here, the rubber-like property of RNA architectures was investigated using optical tweezers and in vivo imaging technologies. The 3WJ 97° interior angle was contracted or stretched to 60°, 90°, and 108° at will to build elegant RNA triangles, squares, pentagons, cubes, tetrahedrons, dendrimers, and prisms. RNA nanoarchitecture was stretchable and shrinkable by optical tweezer with multiple extension and relaxation repeats like a rubber. Comparing to gold and iron nanoparticles with the same size, RNA nanoparticles display stronger cancer-targeting outcomes, while less accumulation in healthy organs. Generally, the upper limit of renal excretion is 5.5 nm; however, the 5, 10, and 20 nm RNA nanoparticles passed the renal filtration and resumed their original structure identified in urine. These findings solve two previous mysteries: (1) Why RNA nanoparticles have an unusually high tumor targeting efficiency since their rubber or amoeba-like deformation property enables them to squeeze out of the leaky vasculature to improve the EPR effect; and (2) why RNA nanoparticles remain non-toxic since they can be rapidly cleared from the body via renal excretion into urine with little accumulation in the body. Considering its controllable shape and size plus its rubber-like property, RNA holds great promises for industrial and biomedical applications especially in cancer therapeutics delivery.

Combination of In Situ Lcn2 pRNA-RNAi Nanotherapeutics and iNSC Transplantation Ameliorates Experimental SCI in Mice.

Spinal cord injury (SCI) is a debilitating neurological condition characterized by different cellular and molecular mechanisms that interplay in exacerbating the progression of the pathology. No fully restorative therapies are yet available, and it is thus becoming recognized that combinatorial approaches aimed at addressing different aspects of SCI will likely results in greater functional outcomes. Here we employed packaging RNA-mediated RNA interference (pRNA-RNAi) nanotherapeutics to downregulate in situ the expression of lipocalin 2 (Lcn2), a known mediator of neuroinflammation and autocrine mediator of reactive astrogliosis, and to create a more amenable niche for the subsequent transplantation of induced neural stem cells (iNSCs). To our knowledge, this is the first approach that takes advantage of the modular and multifunctional pRNA three-way junction platform in the SCI niche, while also exploiting the therapeutic potential of immune-compatible and feasible iNSC transplants. We show the combination of such treatments in a mouse model of contusion thoracic SCI leads to significant improvement of locomotor function, albeit not better than single pRNA-RNAi treatment, and results in synergistic histopathological effects, such as reduction of glial scar volume, diminished pro-inflammatory response, and promotion of neuronal survival. Our results provide evidence for a novel combinatorial approach for treating SCI.

Mapping and Quantification of Non-Coding RNA Originating from the rDNA in Human Glioma Cells.

Ribosomal DNA is one of the most conserved parts of the genome, especially in its rRNA coding regions, but some puzzling pieces of its noncoding repetitive sequences harbor secrets of cell growth and development machinery. Disruptions in the neat mechanisms of rDNA orchestrating the cell functioning result in malignant conversion. In cancer cells, the organization of rRNA coding genes and their transcription somehow differ from that of normal cells, but little is known about the particular mechanism for this switch. In this study, we demonstrate that the region ~2 kb upstream of the rDNA promoter is transcriptionally active in one type of the most malignant human brain tumors, and we compare its expression rate to that of healthy human tissues and cell cultures. Sense and antisense non-coding RNA transcripts were detected and mapped, but their secondary structure and functions remain to be elucidated. We propose that the transcripts may relate to a new class of so-called promoter-associated RNAs (pRNAs), or have some other regulatory functions. We also hope that the expression of these non-coding RNAs can be used as a marker in glioma diagnostics and prognosis.

Translation of the long-term fundamental studies on viral DNA packaging motors into nanotechnology and nanomedicine.

Many years of fundamental studies on viral genome packaging motors have led to fruitful applications. The double-stranded DNA (dsDNA) viruses package their genomes into preformed protein shells via nanomotors including several elegant and meticulous coaxial modules. The motor is geared by the hexameric RNA ring. An open washer displayed as hexametric string of phi29 motor ATPase has been reported. The open washer linked into a filament as a queue with left-handed chirality along the dsDNA chain. It was found that a free 5'- and 3'-dsDNA end is not required for one gp16 dimer and four monomers to assemble into the hexametric washer on dsDNA. The above studies have inspired several applications in nanotechnology and nanomedicine. These applications include: (i) studies on the precision motor channels have led to their application in the single pore sensing; (ii) investigations into the hand-in-hand integration of the hexametric pRNA ring have resulted in the emergence of the new field of RNA nanotechnology; and (iii) the studies on the motor stoichiometry of homologous multi-subunits that subsequently have inspired the discovery of new methods in highly potent drug development. This review focuses on the structure and function of the viral DNA packaging motors and describes how fundamental studies inspired various applications. Given these advantages, more nanotechnological and biomedical applications using bacteriophage motor components are expected.

Co-delivery of antigen and dual adjuvants by aluminum hydroxide nanoparticles for enhanced immune responses.

Adjuvants that contain pathogen-associated molecular patterns (PAMPs) can enhance vaccination efficacy by binding to pattern recognition receptors (PRRs), thereby stimulating immune responses. Particularly effective may be the combination of multiple PAMPs that activate different PRRs, which occurs with natural pathogens. Here we hypothesized the enhanced effects would occur in two adjuvants that stimulate different PRRs: CpG oligodeoxynucleotide (CpG-ODN), which is Toll-like receptor 9 agonist; and 5'-triphosphate, short, double-stranded RNA (3pRNA), which activates retinoic acid-inducible gene I (RIG-I). The model antigen ovalbumin (OVA) was loaded and adjuvants were surface-adsorbed to aluminum hydroxide nanoparticles (hereafter NP-3pRNA-CpG) by electrostatic interaction with an average size of 120 nm and a negative surface charge for targeting lymph nodes. These nanoparticles were efficiently internalized by antigen-presenting cells (APCs) in the lymph nodes, and the resulting APC activation and antigen cross-presentation generated strong humoral immunity and cytotoxic T lymphocyte responses and IFN-γ secretion. NP-3pRNA-CpG significantly suppressed B16-OVA tumor growth and prolonged survival of tumor-bearing mice in therapeutic and prophylactic models, illustrating the enhanced effects of CpG-ODN and 3pRNA. Our study highlights the potential of combining multiple adjuvants for effective vaccine design.

Single Functionalized pRNA/Gold Nanoparticle for Ultrasensitive MicroRNA Detection Using Electrochemical Surface-Enhanced Raman Spectroscopy.

Controlling the selective one-to-one conjugation of RNA with nanoparticles is vital for future applications of RNA nanotechnology. Here, the monofunctionalization of a gold nanoparticle (AuNP) with a single copy of RNA is developed for ultrasensitive microRNA-155 quantification using electrochemical surface-enhanced Raman spectroscopy (EC-SERS). A single AuNP is conjugated with one copy of the packaging RNA (pRNA) three-way junction (RNA 3WJ). pRNA 3WJ containing one strand of the 3WJ is connected to a Sephadex G100 aptamer and a biotin group at each arm (SEPapt/3WJ/Bio) which is then immobilized to the Sephadex G100 resin. The resulting complex is connected to streptavidin-coated AuNP (STV/AuNP). Next, the STV/AuNP-Bio/3WJa is purified and reassembled with another 3WJ to form a single-labeled 3WJ/AuNP. Later, the monoconjugate is immobilized onto the AuNP-electrodeposited indium tin oxide coated substrate for detecting microRNA-155 based on EC-SERS. Application of an optimum potential of +0.2 V results in extraordinary amplification (≈7 times) of methylene blue (reporter) SERS signal compared to the normal SERS signal. As a result, a highly sensitive detection of 60 × 10-18 m microRNA-155 in 1 h in serum based on monoconjugated AuNP/RNA is achieved. Thus, the monofunctionalization of RNA onto nanoparticle can provide a new methodology for biosensor construction and diverse RNA nanotechnology development.