Dan Shu

Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics

RNA nanoparticles have applications in the treatment of cancers and viral infection; however, the instability of RNA nanoparticles has hindered their development for therapeutic applications. The lack of covalent linkage or crosslinking in nanoparticles causes dissociation in vivo. Here we show that the packaging RNA of bacteriophage phi29 DNA packaging motor can be assembled from 3–6 pieces of RNA oligomers without the use of metal salts. Each RNA oligomer contains a functional module that can be a receptor-binding ligand, aptamer, short interfering RNA or ribozyme. When mixed together, they self-assemble into thermodynamically stable tri-star nanoparticles with a three-way junction core. These nanoparticles are resistant to 8 M urea denaturation, are stable in serum and remain intact at extremely low concentrations. The modules remain functional in vitro and in vivo, suggesting that the three-way junction core can be used as a platform for building a variety of multifunctional nanoparticles. We studied 25 different three-way junction motifs in biological RNA and found only one other motif that shares characteristics similar to the three-way junction of phi29 pRNA. The three-way junction domain of the phi29 bacteriophage can be assembled from three pieces of RNA oligomers to form stable multifunctional nanoparticles that are useful for the treatment of different diseases.

Bottom-up Assembly of RNA Arrays and Superstructures as Potential Parts in Nanotechnology

DNA and protein have been extensively scrutinized for feasibility as parts in nanotechnology, but another natural building block, RNA, has been largely ignored. RNA can be manipulated to form versatile shapes, thus providing an element of adaptability to DNA nanotechnology, which is predominantly based upon a double-helical structure. The DNA-packaging motor of bacterial virus phi29 contains six DNA-packaging RNAs (pRNA), which together form a hexameric ring via loop/loop interaction. Here we report that this pRNA can be redesigned to form a variety of structures and shapes, including twins, tetramers, rods, triangles, and 3D arrays several microns in size via interaction of programmed helical regions and loops. Three dimensional RNA array formation required a defined nucleotide number for twisting of the interactive helix and a palindromic sequence. Such arrays are unusually stable and resistant to a wide range of temperatures, salt concentrations, and pH.

Counting of six pRNAs of phi29 DNA-packaging motor with customized single-molecule dual-view system

Direct imaging or counting of RNA molecules has been difficult owing to its relatively low electron density for EM and insufficient resolution in AFM. Bacteriophage phi29 DNA‐packaging motor is geared by a packaging RNA (pRNA) ring. Currently, whether the ring is a pentagon or hexagon is under fervent debate. We report here the assembly of a highly sensitive imaging system for direct counting of the copy number of pRNA within this 20‐nm motor. Single fluorophore imaging clearly identified the quantized photobleaching steps from pRNA labeled with a single fluorophore and concluded its stoichiometry within the motor. Almost all of the motors contained six copies of pRNA before and during DNA translocation, identified by dual‐color detection of the stalled intermediates of motors containing Cy3‐pRNA and Cy5‐DNA. The stalled motors were restarted to observe the motion of DNA packaging in real time. Heat‐denaturation analysis confirmed that the stoichiometry of pRNA is the common multiple of 2 and 3. EM imaging of procapsid/pRNA complexes clearly revealed six ferritin particles that were conjugated to each pRNA ring.

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

The DNA-packaging pRNA of bacterial virus phi29, which forms dimers and then hexamers, contains two independent tightly self-folded domains. Circularly permuted pRNAs were constructed without impacting pRNA folding. Connecting the pRNA 5′/3′ ends with variable sequences did not disturb its folding and function. These unique features, which help prevent two common problems – exonuclease degradation and misfolding in the cell, make pRNA an ideal vector to carry therapeutic RNAs. A pRNA-based vector was designed to carry hammerhead ribozymes that cleave the hepatitis B virus (HBV) polyA signal. The chimeric HBV-targeting ribozyme was connected to the pRNA 5′/3′ ends as circularly permuted pRNA. Two cis-cleaving ribozymes were used to flank and process the chimeric ribozyme. The hammerhead ribozyme including its two arms for HBV targeting was able to fold correctly while escorted by the pRNA. The chimeric ribozyme cleaved the polyA signal of HBV mRNA in vitro almost completely. Cell culture studies showed that the chimeric ribozyme was able to enhance the inhibition of HBV replication when compared with the ribozyme not escorted by pRNA, as demonstrated by Northern blot and e-antigen assays. pRNA could also carry another hammerhead ribozyme to cleave other RNA substrate. These findings suggest that pRNA can be used as a vector for imparting stability to ribozymes, antisense, and other therapeutic RNA molecules in vivo.

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.

One-way traffic of a viral motor channel for double-stranded DNA translocation.

Linear double-stranded DNA (dsDNA) viruses package their genome into a procapsid using an ATP-driven nanomotor. Here we report that bacteriophage phi29 DNA packaging motor exercises a one-way traffic property for dsDNA translocation from N-terminal entrance to C-terminal exit with a valve mechanism in DNA packaging, as demonstrated by voltage ramping, electrode polarity switching, and sedimentation force assessment. Without the use of gating control as found in other biological channels, the observed single direction dsDNA transportation provides a novel system with a natural valve to control dsDNA loading and gene delivery in bioreactors, liposomes, or high throughput DNA sequencing apparatus.

A Viral RNA That Binds ATP and Contains a Motif Similar to an ATP-binding Aptamer from SELEX

The intriguing process of free energy conversion, ubiquitous in all living organisms, is manifested in ATP binding and hydrolysis. ATPase activity has long been recognized to be a capability limited to proteins. However, the presence of an astonishing variety of unknown RNA species in cells and the finding that RNA has catalytic activity have bred the notion that RNA might not be excluded from the group of ATPases. All DNA-packaging motors of double-stranded DNA phages involve two nonstructural components with certain characteristics typical of ATPases. In bacterial virus phi29, one of these two components is an RNA (pRNA). Here we report that this pRNA is able to bind ATP. A comparison between the chemically selected ATP-binding RNA aptamer and the central region of pRNA reveals similarity in sequence and structure. The replacement of the central region of pRNA with the sequence from ATP-binding RNA aptamer produced chimeric aptRNA that is able to both bind ATP and assemble infectious viruses in the presence of ATP. RNA mutation studies revealed that changing only one base essential for ATP binding caused both ATP binding and viral assembly to cease, suggesting that the ATP binding motif is the vital part of the pRNA that forms a hexamer to drive the phi29 DNA-packaging motor. This is the first demonstration of a natural RNA molecule that binds ATP and the first case to report the presence of a SELEX-derived RNA aptamer in living organisms.

Regression of Gastric Cancer by Systemic Injection of RNA Nanoparticles Carrying both Ligand and siRNA.

Gastric cancer is the second leading cause of cancer-related death worldwide. RNA nanotechnology has recently emerged as an important field due to recent finding of its high thermodynamic stability, favorable and distinctive in vivo attributes. Here we reported the use of the thermostable three-way junction (3WJ) of bacteriophage phi29 motor pRNA to escort folic acid, a fluorescent image marker and BRCAA1 siRNA for targeting, imaging, delivery, gene silencing and regression of gastric cancer in animal models. In vitro assay revealed that the RNA nanoparticles specifically bind to gastric cancer cells, and knock-down the BRCAA1 gene. Apoptosis of gastric cancer cells was observed. Animal trials confirmed that these RNA nanoparticles could be used to image gastric cancer in vivo, while showing little accumulation in crucial organs and tissues. The volume of gastric tumors noticeably decreased during the course of treatment. No damage to important organs by RNA nanoparticles was detectible. All the results indicated that this novel RNA nanotechnology can overcome conventional cancer therapeutic limitations and opens new opportunities for specific delivery of therapeutics to stomach cancer without damaging normal cells and tissues, reduce the toxicity and side effect, improve the therapeutic effect, and exhibit great potential in clinical tumor therapy.

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

Both siRNA and miRNA can serve as powerful gene-silencing reagents but their specific delivery to cancer cells in vivo without collateral damage to healthy cells remains challenging. We report here the application of RNA nanotechnology for specific and efficient delivery of anti-miRNA seed-targeting sequence to block the growth of prostate cancer in mouse models. Utilizing the thermodynamically ultra-stable three-way junction of the pRNA of phi29 DNA packaging motor, RNA nanoparticles were constructed by bottom-up self-assembly containing the anti-prostate-specific membrane antigen (PSMA) RNA aptamer as a targeting ligand and anti-miR17 or anti-miR21 as therapeutic modules. The 16 nm RNase-resistant and thermodynamically stable RNA nanoparticles remained intact after systemic injection in mice and strongly bound to tumors with little or no accumulation in healthy organs 8 hours postinjection, and subsequently repressed tumor growth at low doses with high efficiency.

Programmable folding of fusion RNA in vivo and in vitro driven by pRNA 3WJ motif of phi29 DNA packaging motor

Misfolding and associated loss of function are common problems in constructing fusion RNA complexes due to changes in energy landscape and the nearest-neighbor principle. Here we report the incorporation and application of the pRNA-3WJ motif of the phi29 DNA packaging motor into fusion RNA with controllable and predictable folding. The motif included three discontinuous ∼18 nucleotide (nt) fragments, displayed a distinct low folding energy (Shu D et al., Nature Nanotechnology, 2011, 6:658–667), and folded spontaneously into a leading core that enabled the correct folding of other functionalities fused to the RNA complex. Three individual fragments dispersed at any location within the sequence allowed the other RNA functional modules to fold into their original structures with authentic functions, as tested by Hepatitis B virus ribozyme, siRNA, and aptamers for malachite green (MG), spinach, and streptavidin (STV). Only nine complementary nucleotides were present for any two of the three ∼18-nt fragments, but the three 9 bp branches were so powerful that they disrupted other double strands with more than 15 bp within the fusion RNA. This system enabled the production of fusion complexes harboring multiple RNA functionalities with correct folding for potential applications in biotechnology, nanomedicine and nanotechnology. We also applied this system to investigate the principles governing the folding of RNA in vivo and in vitro. Temporal production of RNA sequences during in vivo transcription caused RNA to fold into different conformations that could not be predicted with routine principles derived from in vitro studies.