Methods for Single-Molecule Sensing and Detection Using Bacteriophage Phi29 DNA Packaging Motor.

Bacteriophage phi29 DNA packaging motor consists of a dodecameric portal channel protein complex termed connector that allows transportation of genomic dsDNA and a hexameric packaging RNA (pRNA) ring to gear the motor. The elegant design of the portal protein has facilitated its applications for real-time single-molecule detection of biopolymers and chemicals with high sensitivity and selectivity. The robust self-assembly property of the pRNA has enabled biophysical studies of the motor complex to determine the stoichiometry and structure/folding of the pRNA at single-molecule level. This chapter focuses on biophysical and analytical methods for studying the phi29 motor components at the single-molecule level, such as single channel conductance assays of membrane-embedded connectors; single molecule photobleaching (SMPB) assay for determining the stoichiometry of phi29 motor components; fluorescence resonance energy transfer (FRET) assay for determining the structure and folding of pRNA; atomic force microscopy (AFM) for imaging pRNA nanoparticles of various size, shape, and stoichiometry; and bright-field microscopy with magnetomechanical system for direct visualization of viral DNA packaging process. The phi29 system with explicit engineering capability has incredible potentials for diverse applications in nanotechnology and nanomedicine including, but not limited to, DNA sequencing, drug delivery to diseased cells, environmental surveillance, and early disease diagnosis.

RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity.

In response to injuries to the CNS, astrocytes enter a reactive state known as astrogliosis, which is believed to be deleterious in some contexts. Activated astrocytes overexpress intermediate filaments including glial fibrillary acidic protein (GFAP) and vimentin (Vim), resulting in entangled cells that inhibit neurite growth and functional recovery. Reactive astrocytes also secrete inflammatory molecules such as Lipocalin 2 (Lcn2), which perpetuate reactivity and adversely affect other cells of the CNS. Herein, we report proof-of-concept use of the packaging RNA (pRNA)-derived three-way junction (3WJ) motif as a platform for the delivery of siRNAs to downregulate such reactivity-associated genes. In vitro, siRNA-3WJs induced a significant knockdown of Gfap, Vim, and Lcn2 in a model of astroglial activation, with a concomitant reduction in protein expression. Knockdown of Lcn2 also led to reduced protein secretion from reactive astroglial cells, significantly impeding the perpetuation of inflammation in otherwise quiescent astrocytes. Intralesional injection of anti-Lcn2-3WJs in mice with contusion spinal cord injury led to knockdown of Lcn2 at mRNA and protein levels in vivo. Our results provide evidence for siRNA-3WJs as a promising platform for ameliorating astroglial reactivity, with significant potential for further functionalization and adaptation for therapeutic applications in the CNS.

Engineering of protein nanopores for sequencing, chemical or protein sensing and disease diagnosis.

Biological systems contain highly-ordered structures performing diverse functions. The elegant structures of biomachines have inspired the development of nanopores as single molecule sensors. Over the years, the utility of nanopores for detecting a wide variety of analytes have rapidly emerged for sensing, sequencing and diagnostic applications. Several protein channels with diverse shapes and sizes, such as motor channels from bacteriophage Phi29, SPP1, T3, and T4, as well as α-hemolysin, MspA, aerolysin, FluA, OmpF/G, CsgG, ClyA, have been continually investigated and developed as nanopores. Herein, we focus on advances in biological nanopores for single molecule sensing and DNA sequencing from a protein engineering standpoint for changing pore sizes, altering charge distributions, enhancing sensitivity, improving stability, and imparting new detection capabilities.

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

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

RNA versatility, flexibility, and thermostability for practice in RNA nanotechnology and biomedical applications.

In recent years, RNA has attracted widespread attention as a unique biomaterial with distinct biophysical properties for designing sophisticated architectures in the nanometer scale. RNA is much more versatile in structure and function with higher thermodynamic stability compared to its nucleic acid counterpart DNA. Larger RNA molecules can be viewed as a modular structure built from a combination of many 'Lego' building blocks connected via different linker sequences. By exploiting the diversity of RNA motifs and flexibility of structure, varieties of RNA architectures can be fabricated with precise control of shape, size, and stoichiometry. Many structural motifs have been discovered and characterized over the years and the crystal structures of many of these motifs are available for nanoparticle construction. For example, using the flexibility and versatility of RNA structure, RNA triangles, squares, pentagons, and hexagons can be constructed from phi29 pRNA three-way-junction (3WJ) building block. This review will focus on 2D RNA triangles, squares, and hexamers; 3D and 4D structures built from basic RNA building blocks; and their prospective applications in vivo as imaging or therapeutic agents via specific delivery and targeting. Methods for intracellular cloning and expression of RNA molecules and the in vivo assembly of RNA nanoparticles will also be reviewed. WIREs RNA 2018, 9:e1452. doi: 10.1002/wrna.1452 This article is categorized under: RNA Methods > RNA Nanotechnology RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

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

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

Advances in nanopore sensing promises to transform healthcare.

International Conference on Nanopore Technology (Shenzhen), 30 March-1 April 2017, Shenzhen, China The International Conference on Nanopore Technology (Shenzhen) was held from 30 March to 1 April 2017 in Shenzhen, China. The goal of the meeting was threefold: leverage the unique properties of nanopore technology to promote transformative advances in medicine, encourage cross-disciplinary collaborations in the research community within China and abroad; and discuss critical challenges that need to be addressed to rapidly advance the field. The meeting was chaired by Peixuan Guo, Endowed chair professor and Director of The Center for RNA Nanobiotechnology & Nanomedicine at The Ohio State University, USA and co-chaired by Xian-En Zhang, distinguished professor of the Institute of Biophysics, Chinese Academy of Sciences, China. The conference was attended by more than 300 academic researchers, hospital administrators, government leaders and scientists from many disciplines across the country from both academic institutions and industry.

Using Planar Phi29 pRNA Three-Way Junction to Control Size and Shape of RNA Nanoparticles for Biodistribution Profiling in Mice.

RNA is rapidly emerging as a versatile building block for nanoparticle assembly due to its simplicity in base pairing, while exhibiting diversity in function such as enzymatic activity similar to some proteins. Recent advances in RNA nanotechnology have generated significant interests in applying RNA nanoparticles for various applications in nanotechnology and nanomedicine. In particular, assessing the effect of size and shape on cell entry and intracellular trafficking as well as in vivo biodistribution of nanoparticles is challenging due to the lack of nanoparticles rich in structure while varying in size and shape. RNA nanotechnology exemplified by the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor has provided a different prospect in nanoparticle designs. Of note, there is a robust three-way junction (3WJ) motif in pRNA which can serve as an adaptable scaffold to construct thermodynamically stable 2D planar and 3D globular RNA architectures with tunable shapes and sizes, and harboring various targeting, therapeutic, and imaging modules. This chapter focuses on the methods for constructing pRNA-3WJ based nanoparticles with controllable sizes and shapes, and assessment of their biodistribution profiles in cancer mouse models after systemic injection and ocular mouse models following subconjunctival injection.

Channel of viral DNA packaging motor for real time kinetic analysis of peptide oxidation states.

Nanopore technology has become a powerful tool in single molecule sensing, and protein nanopores appear to be more advantageous than synthetic counterparts with regards to channel amenability, structure homogeneity, and production reproducibility. However, the diameter of most of the well-studied protein nanopores is too small to allow the passage of protein or peptides that are typically in multiple nanometers scale. The portal channel from bacteriophage SPP1 has a large channel size that allows the translocation of peptides with higher ordered structures. Utilizing single channel conductance assay and optical single molecule imaging, we observed translocation of peptides and quantitatively analyzed the dynamics of peptide oligomeric states in real-time at single molecule level. The oxidative and the reduced states of peptides were clearly differentiated based on their characteristic electronic signatures. A similar Gibbs free energy (ΔG0) was obtained when different concentrations of substrates were applied, suggesting that the use of SPP1 nanopore for real-time quantification of peptide oligomeric states is feasible. With the intrinsic nature of size and conjugation amenability, the SPP1 nanopore has the potential for development into a tool for the quantification of peptide and protein structures in real time.

Advancement of the Emerging Field of RNA Nanotechnology.

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

Three-step channel conformational changes common to DNA packaging motors of bacterial viruses T3, T4, SPP1, and Phi29.

The DNA packaging motor of dsDNA bacterial viruses contains a head-tail connector with a channel for the genome to enter during assembly and to exit during host infection. The DNA packaging motor of bacterial virus phi29 was recently reported to use the "One-way revolving" mechanism for DNA packaging. This raises a question of how dsDNA is ejected during infection if the channel acts as a one-way inward valve. Here we report a three step conformational change of the portal channel that is common among DNA translocation motors of bacterial viruses T3, T4, SPP1, and phi29. The channels of these motors exercise three discrete steps of gating, as revealed by electrophysiological assays. The data suggest that the three step channel conformational changes occur during DNA entry process, resulting in a structural transition in preparation for DNA movement in the reverse direction during ejection.

Oriented single directional insertion of nanochannel of bacteriophage SPP1 DNA packaging motor into lipid bilayer via polar hydrophobicity.

Insertion of biological nanopore into artificial membrane is of fundamental importance in nanotechnology. Many applications require control and knowledge of channel orientation. In this work, the insertion orientation of the bacteriophage SPP1 and phi29 DNA packaging motors into lipid membranes was investigated. Single molecule electrophysiological assays and Ni-NTA-nanogold binding assays revealed that both SPP1 and phi29 motor channels exhibited a one-way traffic property for TAT peptide translocation from N- to C-termini of the protein channels. SPP1 motor channels preferentially inserts into liposomes with their C-terminal wider region facing inward. Changing the hydrophobicity of the N- or C-termini of phi29 connector alters the insertion orientation, suggesting that the hydrophobicity and hydrophilicity of the termini of the protein channel governs the orientation of the insertion into lipid membrane. It is proposed that the specificity in motor channel orientation is a result of the hydrophilic/hydrophobic interaction at the air/water interface when the protein channels are incorporating into liposome membranes.

Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor.

Nanopore technology has become a highly sensitive and powerful tool for single molecule sensing of chemicals and biopolymers. Protein pores have the advantages of size amenability, channel homogeneity, and fabrication reproducibility. But most well-studied protein pores for sensing are too small for passage of peptide analytes that are typically a few nanometers in dimension. The funnel-shaped channel of bacteriophage phi29 DNA packaging motor has previously been inserted into a lipid membrane to serve as a larger pore with a narrowest N-terminal constriction of 3.6 nm and a wider C-terminal end of 6 nm. Here, the utility of phi29 motor channel for fingerprinting of various peptides using single molecule electrophysiological assays is reported. The translocation of peptides is proved unequivocally by single molecule fluorescence imaging. Current blockage percentage and distinctive current signatures are used to distinguish peptides with high confidence. Each peptide generated one or two distinct current blockage peaks, serving as typical fingerprint for each peptide. The oligomeric states of peptides can also be studied in real time at single molecule level. The results demonstrate the potential for further development of phi29 motor channel for detection of disease-associated peptide biomarkers.

Controllable self-assembly of RNA dendrimers.

We report programmable self-assembly of branched, 3D globular, monodisperse and nanoscale sized dendrimers using RNA as building blocks. The central core and repeating units of the RNA dendrimer are derivatives of the ultrastable three-way junction (3WJ) motif from the bacteriophage phi29 motor pRNA. RNA dendrimers were constructed by step-wise self-assembly of modular 3WJ building blocks initiating with a single 3WJ core (Generation-0) with overhanging sticky end and proceeding in a radial manner in layers up to Generation-4. The final constructs were generated under control without any structural defects in high yield and purity, as demonstrated by gel electrophoresis and AFM imaging. Upon incorporation of folate on the peripheral branches of the RNA dendrimers, the resulting constructs showed high binding and internalization into cancer cells. RNA dendrimers are envisioned to have a major impact in targeting, disease therapy, molecular diagnostics and bioelectronics in the near future. FROM THE CLINICAL EDITOR: Dendrimers are gaining importance as a carrier platform for diagnosis and therapeutics. The authors here reported building of their dendrimer molecules using RNA as building blocks. The addition of folate also allowed recognition and subsequent binding to tumor cells. This new construct may prove to be useful in many clinical settings.

Systemic Delivery of Anti-miRNA for Suppression of Triple Negative Breast Cancer Utilizing RNA Nanotechnology.

MicroRNAs play important roles in regulating the gene expression and life cycle of cancer cells. In particular, miR-21, an oncogenic miRNA is a major player involved in tumor initiation, progression, invasion and metastasis in several cancers, including triple negative breast cancer (TNBC). However, delivery of therapeutic miRNA or anti-miRNA specifically into cancer cells in vivo without collateral damage to healthy cells remains challenging. We report here the application of RNA nanotechnology for specific and efficient delivery of anti-miR-21 to block the growth of TNBC in orthotopic mouse models. The 15 nm therapeutic RNA nanoparticles contains the 58-nucleotide (nt) phi29 pRNA-3WJ as a core, a 8-nt sequence complementary to the seed region of miR-21, and a 39-nt epidermal growth factor receptor (EGFR) targeting aptamer for internalizing RNA nanoparticles into cancer cells via receptor mediated endocytosis. The RNase resistant and thermodynamically stable RNA nanoparticles remained intact after systemic injection into mice and strongly bound to tumors with little or no accumulation in healthy organs 8 h postinjection, and subsequently repressed tumor growth at low doses. The observed specific cancer targeting and tumor regression is a result of several key attributes of RNA nanoparticles: anionic charge which disallows nonspecific passage across negatively charged cell membrane; "active" targeting using RNA aptamers which increases the homing of RNA nanoparticles to cancer cells; nanoscale size and shape which avoids rapid renal clearance and engulfment by lung macrophages and liver Kupffer cells; favorable biodistribution profiles with little accumulation in healthy organs, which minimizes nonspecific side effects; and favorable pharmacokinetic profiles with extended in vivo half-life. The results demonstrate the clinical potentials of RNA nanotechnology based platform to deliver miRNA based therapeutics for cancer treatment.

RNA nanoparticle as a vector for targeted siRNA delivery into glioblastoma mouse model.

Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues.

Single pore translocation of folded, double-stranded, and tetra-stranded DNA through channel of bacteriophage phi29 DNA packaging motor.

The elegant architecture of the channel of bacteriophage phi29 DNA packaging motor has inspired the development of biomimetics for biophysical and nanobiomedical applications. The reengineered channel inserted into a lipid membrane exhibits robust electrophysiological properties ideal for precise sensing and fingerprinting of dsDNA at the single-molecule level. Herein, we used single channel conduction assays to quantitatively evaluate the translocation dynamics of dsDNA as a function of the length and conformation of dsDNA. We extracted the speed of dsDNA translocation from the dwell time distribution and estimated the various forces involved in the translocation process. A ∼35-fold slower speed of translocation per base-pair was observed for long dsDNA, a significant contrast to the speed of dsDNA crossing synthetic pores. It was found that the channel could translocate both dsDNA with ∼32% of channel current blockage and with ∼64% for tetra-stranded DNA (two parallel dsDNA). The calculation of both cross-sectional areas of the dsDNA and tetra-stranded DNA suggested that the blockage was purely proportional to the physical space of the channel lumen and the size of the DNA substrate. Folded dsDNA configuration was clearly reflected in their characteristic current signatures. The finding of translocation of tetra-stranded DNA with 64% blockage is in consent with the recently elucidated mechanism of viral DNA packaging via a revolution mode that requires a channel larger than the dsDNA diameter of 2 nm to provide room for viral DNA revolving without rotation. The understanding of the dynamics of dsDNA translocation in the phi29 system will enable us to design more sophisticated single pore DNA translocation devices for future applications in nanotechnology and personal medicine.

Overview of methods in RNA nanotechnology: synthesis, purification, and characterization of RNA nanoparticles.

RNA nanotechnology encompasses the use of RNA as a construction material to build homogeneous nanostructures by bottom-up self-assembly with defined size, structure, and stoichiometry; this pioneering concept demonstrated in 1998 (Guo et al., Molecular Cell 2:149-155, 1998; featured in Cell) has emerged as a new field that also involves materials engineering and synthetic structural biology (Guo, Nature Nanotechnology 5:833-842, 2010). The field of RNA nanotechnology has skyrocketed over the last few years, as evidenced by the burst of publications in prominent journals on RNA nanostructures and their applications in nanomedicine and nanotechnology. Rapid advances in RNA chemistry, RNA biophysics, and RNA biology have created new opportunities for translating basic science into clinical practice. RNA nanotechnology holds considerable promise in this regard. Increased evidence also suggests that substantial part of the 98.5 % of human genome (Lander et al. Nature 409:860-921, 2001) that used to be called "junk DNA" actually codes for noncoding RNA. As we understand more on how RNA structures are related to function, we can fabricate synthetic RNA nanoparticles for the diagnosis and treatment of diseases. This chapter provides a brief overview of the field regarding the design, construction, purification, and characterization of RNA nanoparticles for diverse applications in nanotechnology and nanomedicince.

Large scale purification of RNA nanoparticles by preparative ultracentrifugation.

Purification of large quantities of supramolecular RNA complexes is of paramount importance due to the large quantities of RNA needed and the purity requirements for in vitro and in vivo assays. Purification is generally carried out by liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), or agarose gel electrophoresis (AGE). Here, we describe an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation.

Methods and assays for specific targeting and delivery of RNA nanoparticles to cancer metastases.

In recent years, RNA nanotechnology has become increasingly attractive due to its potential for applications in nanomedicine. RNA nanotechnology refers to the design and synthesis of nanoparticles composed mainly of RNA via bottom-up self-assembly. RNA nanoparticle is a suitable candidate for targeted delivery of therapeutics to cancer cells due to its multivalency, which allows the combination of therapeutic, targeting, and detection moieties all into one nanoparticle. To date, a system capable of exclusively targeting metastatic cancers that have spread to distant organs or lymph nodes is in demand. In this chapter, we report methods for establishing the clinically relevant colorectal cancer mouse metastasis models and describe methods and assays for constructing multifunctional, thermodynamically and chemically stable RNA nanoparticles that specifically target colorectal cancer metastases in the liver. Systemic injection of RNA nanoparticles showed metastatic cells targeting with little or no accumulation in normal liver parenchyma several hours after injection, demonstrating the therapeutic potential of these RNA nanoparticles as a delivery system for the treatment of cancer metastases.