Synergistic SHAPE/Single-Molecule Deconvolution of RNA Conformation under Physiological Conditions.

Structural RNA domains are widely involved in the regulation of biological functions, such as gene expression, gene modification, and gene repair. Activity of these dynamic regions depends sensitively on the global fold of the RNA, in particular, on the binding affinity of individual conformations to effector molecules in solution. Consequently, both the 1) structure and 2) conformational dynamics of noncoding RNAs prove to be essential in understanding the coupling that results in biological function. Toward this end, we recently reported observation of three conformational states in the metal-induced folding pathway of the tRNA-like structure domain of Brome Mosaic Virus, via single-molecule fluorescence resonance energy transfer studies. We report herein selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-directed structure predictions as a function of metal ion concentrations ([Mn+]) to confirm the three-state folding model, as well as test 2° structure models from the literature. Specifically, SHAPE reactivity data mapped onto literature models agrees well with the secondary structures observed at 0-10 mM [Mg2+], with only minor discrepancies in the E hairpin domain at low [Mg2+]. SHAPE probing and SHAPE-directed structure predictions further confirm the stepwise unfolding pathway previously observed in our single-molecule studies. Of special relevance, this means that reduction in the metal-ion concentration unfolds the 3' pseudoknot interaction before unfolding the long-range stem interaction. This work highlights the synergistic power of combining 1) single-molecule Förster resonance energy transfer and 2) SHAPE-directed structure-probing studies for detailed analysis of multiple RNA conformational states. In particular, single-molecule guided deconvolution of the SHAPE reactivities permits 2° structure predictions of isolated RNA conformations, thereby substantially improving on traditional limitations associated with current structure prediction algorithms.

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.

Single-Molecule FRET Reveals Three Conformations for the TLS Domain of Brome Mosaic Virus Genome.

Metabolite-dependent conformational switching in RNA riboswitches is now widely accepted as a critical regulatory mechanism for gene expression in bacterial systems. More recently, similar gene regulation mechanisms have been found to be important for viral systems as well. One of the most abundant and best-studied systems is the tRNA-like structure (TLS) domain, which has been found to occur in many plant viruses spread across numerous genera. In this work, folding dynamics for the TLS domain of Brome Mosaic Virus have been investigated using single-molecule fluorescence resonance energy transfer techniques. In particular, burst fluorescence methods are exploited to observe metal-ion ([M(n+)])-induced folding in freely diffusing RNA constructs resembling the minimal TLS element of brome mosaic virus RNA3. The results of these experiments reveal a complex equilibrium of at least three distinct populations. A stepwise, or consecutive, thermodynamic model for TLS folding is developed, which is in good agreement with the [M(n+)]-dependent evolution of conformational populations and existing structural information in the literature. Specifically, this folding pathway explains the metal-ion dependent formation of a functional TLS domain from unfolded RNAs via two consecutive steps: 1) hybridization of a long-range stem interaction, followed by 2) formation of a 3'-terminal pseudoknot. These two conformational transitions are well described by stepwise dissociation constants for [Mg(2+)] (K1 = 328 ± 30 µM and K2 = 1092 ± 183 µM) and [Na(+)] (K1 = 74 ± 6 mM and K2 = 243 ± 52 mM)-induced folding. The proposed thermodynamic model is further supported by inhibition studies of the long-range stem interaction using a complementary DNA oligomer, which effectively shifts the dynamic equilibrium toward the unfolded conformation. Implications of this multistep conformational folding mechanism are discussed with regard to regulation of virus replication.

Conformationally dynamic π-conjugation: probing structure-property relationships of fluorescent tris(N-salicylideneaniline)s.

We recently reported the design and synthesis of a series of conformationally dynamic chromophores that are built on the C(3)-symmetric tris(N-salicylideneaniline) platform. This system utilizes cooperative structural folding-unfolding motions for fluorescence switching, which is driven by the assembly and disassembly of hydrogen bonds between the rigid core and rotatable peripheral part of the molecule. Here, we report detailed time-resolved spectroscopic studies to investigate the structure-property relationships of a series of functionalized tris(N-salicylideneaniline)s. Time-resolved fluorescence decay spectroscopy was applied to determine the main relaxation mechanisms of these π-extended fluorophores, and to address the effects of hydrogen bonding, steric constraints, and extension of the π-conjugation on their relaxation dynamics. Our results agree well with the conformational switching model that was previously suggested from steady-state experiments. Notably, extension of the π-conjugation from peripheral aryl groups resulted in the stabilization of the excited states, as evidenced by longer lifetimes and lower nonradiative decay constants. As a consequence, an increase in the fluorescence quantum yields was observed, which could be explained by the suppression of the torsional motions about the C-N bonds from an overall increase in the quinoid character of the excited states. A combination of time-resolved and steady-state techniques also revealed intermolecular interactions through π-π stacking at higher concentrations, which provide additional de-excitation pathways that become more pronounced in solid samples.

Multivalent rubber-like RNA nanoparticles for targeted co-delivery of paclitaxel and MiRNA to silence the drug efflux transporter and liver cancer drug resistance.

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. IC50 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.

Self-assembly of four generations of RNA dendrimers for drug shielding with controllable layer-by-layer release.

Chemical dendrimers have been shown to be a promising drug delivery platform due to their advantageous properties such as monodispersity, multivalency and branched structure. Taking advantage of self-assembly and its intrinsic negative charge, we used RNA as the building block for dendrimer construction to eliminate complex synthesis procedures and cationic charge-related toxicity. Oligo ribonucleotides produced by solid phase chemical synthesis allow the large-scale manufacture of homologous RNA dendrimers. Employing concepts from RNA nanotechnology enabled the controllable production of dendrimers with generations from G1, G2, G3, to G4 with layer-by-layer release capability. The conjugation of functional groups into individual RNA strands and the incorporation of functionalized RNA strands into the dendrimers at different sites have been reported. Anticancer drugs loaded into RNA dendrimers showed comparable cancer cell inhibition effect to free drugs. Encapsulation of cell binding ligands and hydrophobic drugs within the dendrimer significantly reduced the efficiency of cell binding and protein binding respectively, demonstrating the shielding effect of RNA dendrimers. The results imply a potential application of RNA dendrimer for delivery, shielding and controlled release of hydrophobic drugs in vivo.

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.

Ultra-thermostable RNA nanoparticles for solubilizing and high-yield loading of paclitaxel for breast cancer therapy.

Paclitaxel is widely used in cancer treatments, but poor water-solubility and toxicity raise serious concerns. Here we report an RNA four-way junction nanoparticle with ultra-thermodynamic stability to solubilize and load paclitaxel for targeted cancer therapy. Each RNA nanoparticle covalently loads twenty-four paclitaxel molecules as a prodrug. The RNA-paclitaxel complex is structurally rigid and stable, demonstrated by the sub-nanometer resolution imaging of cryo-EM. Using RNA nanoparticles as carriers increases the water-solubility of paclitaxel by 32,000-fold. Intravenous injections of RNA-paclitaxel nanoparticles with specific cancer-targeting ligand dramatically inhibit breast cancer growth, with nearly undetectable toxicity and immune responses in mice. No fatalities are observed at a paclitaxel dose equal to the reported LD50. The use of ultra-thermostable RNA nanoparticles to deliver chemical prodrugs addresses issues with RNA unfolding and nanoparticle dissociation after high-density drug loading. This finding provides a stable nano-platform for chemo-drug delivery as well as an efficient method to solubilize hydrophobic drugs.

RNA-based micelles: A novel platform for paclitaxel loading and delivery.

RNA can serve as powerful building blocks for bottom-up fabrication of nanostructures for biotechnological and biomedical applications. In addition to current self-assembly strategies utilizing base pairing, motif piling and tertiary interactions, we reported for the first time the formation of RNA based micellar nanoconstruct with a cholesterol molecule conjugated onto one helical end of a branched pRNA three-way junction (3WJ) motif. The resulting amphiphilic RNA micelles consist of a hydrophilic RNA head and a covalently linked hydrophobic lipid tail that can spontaneously assemble in aqueous solution via hydrophobic interaction. Taking advantage of pRNA 3WJ branched structure, the assembled RNA micelles are capable of escorting multiple functional modules. As a proof of concept for delivery for therapeutics, Paclitaxel was loaded into the RNA micelles with significantly improved water solubility. The successful construction of the drug loaded RNA micelles was confirmed and characterized by agarose gel electrophoresis, atomic force microscopy (AFM), dynamic light scattering (DLS), and fluorescence Nile Red encapsulation assay. The estimate critical micelle formation concentration ranges from 39 nM to 78 nM. The Paclitaxel loaded RNA micelles can internalize into cancer cells and inhibit their proliferation. Further studies showed that the Paclitaxel loaded RNA micelles induced cancer cell apoptosis in a Caspase-3 dependent manner but RNA micelles alone exhibited low cytotoxicity. Finally, the Paclitaxel loaded RNA micelles targeted to tumor in vivo without accumulation in healthy tissues and organs. There is also no or very low induction of pro-inflammatory response. Therefore, multivalence, cancer cell permeability, combined with controllable assembly, low or non toxic nature, and tumor targeting are all promising features that make our pRNA micelles a suitable platform for potential drug delivery.

Reactivity-based fluoride detection: evolving design principles for spring-loaded turn-on fluorescent probes.

A covalently triggered fluorescence turn-on detection scheme has been implemented for a tris(N-salicylideneamine)-derived dynamic fluorophore. Selective cleavage of strategically placed Si-O bonds by fluoride ion induces spring-loaded conformational transitions that are tightly coupled to fluorescence enhancement.

Discovery of a new method for potent drug development using power function of stoichiometry of homomeric biocomplexes or biological nanomotors.

INTRODUCTION: Multidrug resistance and the appearance of incurable diseases inspire the quest for potent therapeutics. AREAS COVERED: We review a new methodology in designing potent drugs by targeting multi-subunit homomeric biological motors, machines or complexes with Z > 1 and K = 1, where Z is the stoichiometry of the target, and K is the number of drugged subunits required to block the function of the complex. The condition is similar to a series electrical circuit of Christmas decorations: failure of one light bulb causes the entire lighting system to lose power. In most multi-subunit, homomeric biological systems, a sequential coordination or cooperative action mechanism is utilized, thus K equals 1. Drug inhibition depends on the ratio of drugged to non-drugged complexes. When K = 1, and Z > 1, the inhibition effect follows a power law with respect to Z, leading to enhanced drug potency. The hypothesis that the potency of drug inhibition depends on the stoichiometry of the targeted biological complexes was recently quantified by Yang-Hui's Triangle (or binomial distribution), and proved using a highly sensitive in vitro phi29 viral DNA packaging system. Examples of targeting homomeric bio-complexes with high stoichiometry for potent drug discovery are discussed. EXPERT OPINION: Biomotors with multiple subunits are widespread in viruses, bacteria and cells, making this approach generally applicable in the development of inhibition drugs with high efficiency.

Using RNA nanoparticles with thermostable motifs and fluorogenic modules for real-time detection of RNA folding and turnover in prokaryotic and eukaryotic cells.

RNA nanotechnology is an emerging field at the interface of biochemistry and nanomaterials that shows immense promise for applications in nanomedicines, therapeutics and nanotechnology. Noncoding RNAs, such as siRNA, miRNA, ribozymes, and riboswitches, play important roles in the regulation of cellular processes. They carry out highly specific functions on a compact and efficient footprint. The properties of specificity and small size make them excellent modules in the construction of multifaceted RNA nanoparticles for targeted delivery and therapy. Biological activity of RNA molecules, however, relies on their proper folding. Therefore their thermodynamic and biochemical stability in the cellular environment is critical. Consequently, it is essential to assess global fold and intracellular lifetime of multifaceted RNA nanoparticles to optimize their therapeutic effectiveness. Here, we describe a method to express and assemble stable RNA nanoparticles in cells, and to assess the folding and turnover rate of RNA nanoparticles in vitro as well as in vivo in real time using a thermostable core motif derived from pRNA of bacteriophage Phi29 DNA packaging motor and fluorogenic RNA modules.

Functional assays for specific targeting and delivery of RNA nanoparticles to brain tumor.

Cumulative progress in nanoparticle development has opened a new era of targeted delivery of therapeutics to cancer cells and tissue. However, developing proper detection methods has lagged behind resulting in the lack of precise evaluation and monitoring of the systemically administered nanoparticles. RNA nanoparticles derived from the bacteriophage phi29 DNA packaging motor pRNA have emerged as a new generation of drugs for cancer therapy. Multifunctional RNA nanoparticles can be fabricated by bottom-up self-assembly of engineered RNA fragments harboring targeting (RNA aptamer or chemical ligand), therapeutic (siRNA, miRNA, ribozymes, and small molecule drugs), and imaging (fluorophore, radiolabels) modules. We have recently demonstrated that RNA nanoparticles can reach and target intracranial brain tumors in mice upon systemic injection with little or no accumulation in adjacent healthy brain tissues or in major healthy internal organs. Herein, we describe various functional imaging methods (fluorescence confocal microscopy, flow cytometry, fluorescence whole body imaging, and magnetic resonance imaging) to evaluate and monitor RNA nanoparticle targeting to intracranial brain tumors in mice. Such imaging techniques will allow in-depth evaluation of specifically delivered RNA therapeutics to brain tumors.

Two classes of nucleic acid translocation motors: rotation and revolution without rotation.

Biomotors are extensively involved in biological processes including cell mitosis, bacterial binary fission, DNA replication, DNA repair, homologous recombination, Holliday junction resolution, RNA transcription, and viral genome packaging. Traditionally, they were classified into two categories including linear and rotation motors. In 2013, a third class of motor by revolution mechanism without rotation was discovered. In this issue of "Structure and mechanisms of nanomotors in the cells", four comprehensive reviews are published to address the latest advancements of the structure and motion mechanism of a variety of biomotors in archaea, animal viruses, bacteria, and bacteriophages.

Photothermal imaging and measurement of protein shell stoichiometry of single HIV-1 Gag virus-like nanoparticles.

Virus life stages often constitute a complex chain of events, difficult to track in vivo and in real-time. Challenges are associated with spatial and time limitations of current probes: most viruses are smaller than the diffraction limit of optical microscopes while the entire time scale of virus dynamics spans over 8 orders of magnitude. Thus, virus processes such as entry, disassembly, and egress have generally remained poorly understood. Here we discuss photothermal heterodyne imaging (PHI) as a possible alternative to fluorescence microscopy in the study of single virus-like nanoparticle (VNP) dynamics, with relevance in particular to virus uncoating. Being based on optical absorption rather than emission, PHI could potentially surpass some of the current limitations associated with fluorescent labels. As proof-of-principle, single VNPs self-assembled from 60 nm DNA-functionalized gold nanoparticles (DNA-Au NPs) encapsulated in a Gag protein shell of the human immunodeficiency virus (HIV-1) were imaged, and their photothermal response was compared with DNA-Au NPs. For the first time, the protein stoichiometry of a single virus-like particle was estimated by a method other than electron microscopy.