October 15, 2013 (Vol. 33, No. 18)

RNAs play an integral part in all kingdoms of life and mediate critical processes from gene regulation to genomic maintenance and protein synthesis.

RNAs also have an amazing diversity in structure and function. The importance of RNA was highlighted at the “International Conference of RNA Nanotechnology and Therapeutics.” At the conference, several cutting-edge RNA technologies and emerging trends were discussed.

Scientists described how to decipher RNA secondary structure via novel mathematic approaches or via in-gel analysis following electrophoresis. Others are modifying technologies such as Illumina sequencing to probe thousands of RNAs simultaneously, or employing tandem mass spectrometry for tRNA analysis. Finally, in the arena of therapeutics, self-assembling RNA nanoparticles are being designed for diagnostics and therapeutics.

RNA processing is a critical facet driving infection by HIV. In the course of infection, HIV RNA is multiply spliced, exported from the nucleus, and translated into viral accessory proteins such as Rev (regulator of expression of viral proteins). To further assist HIV replication, Rev shuttles back into the nucleus and binds the viral Rev response element (RRE), a ~350 nucleotide RNA segment necessary for export of full-length and singly spliced mRNAs.

HIV can be divided into two subgroups. While HIV-1 is responsible for the AIDS pandemic, its cousin, HIV-2, also can cause AIDS.

“Much is known about HIV-1 and Rev1, yet comparatively little is known about Rev2 and its interactions with the homologous RRE2,” said Stuart F.J. Le Grice, Ph.D., a researcher who holds senior positions at both the HIV Drug Resistance Program and the Center for Cancer Research.

“Similar to its HIV-1 counterpart, RRE2 transports HIV-2 viral RNA from the nucleus to the cytoplasm,” Dr. Le Grice continued.

“Our goal was to determine the structure of RRE2 RNA to better understand the nucleoprotein complexes involved in this process. To do this we utilized complementary chemical and biochemical probing techniques.”

Dr. Le Grice’s group employed an RNA probing method that had been pioneered by Kevin Weeks, Ph.D., Kenan Distinguished Professor, department of chemistry, University of North Carolina at Chapel Hill. The method, called SHAPE (selective 2′-hyroxyl acylation analyzed by primer extension), takes data from capillary electrophoresis experiments and automates the interpretation.

“SHAPE works exceeding well as long as there is a single conformation of the RNA. When we evaluated RRE2, we found three bands in our polyacrylamide gels,” said Dr. Le Grice. “One of our very talented post-docs, Sabrina Lusvarghi, Ph.D., came up with the idea to add a novel mathematical approach for determining the secondary structure of each RNA conformer.”

Coupling site-directed hydroxyl radical footprinting along with SHAPE profiling did the trick. The breakthrough, according to Dr. Le Grice, occurred when the method was used to identify transitional states of the RNA. The group found that RRE2 contains five peripheral stem loops linked by adjacent four-way and three-way junctions.

“We don’t know whether RNA structure changes in the context of a virus-infected cell, but do know there is a structural transition in vitro that provides a basis for the first 3D glimpses of this critical viral RNA element,” said Dr. Le Grice. “Approaches such as this will contribute toward developing RNA-based therapeutics against HIV.”

Novel RNA Modeling

Delineating the secondary structure of large, multidomain RNA can be complicated when more than one structural conformer is present or when molecular multimerization occurs. “Most RNAs in vivo likely adopt alternate functional conformations involving intermolecular or intramolecular interactions,” said Julia C. Kenyon Ph.D., a post-doctoral fellow in the laboratory of Andrew M. L. Lever, Ph.D., who heads the division of infectious diseases at the University of Cambridge’s department of medicine.

Traditionally, a variety of biochemical techniques are used to analyze RNA secondary structure, including nuclease mapping and chemical modifications. With these techniques, reactive sites are examined by primer extension or 3′ labeling with radioactive isotopes. With the SHAPE method, however, investigators take advantage of recently found compounds that acylate the ribose 2′-hydroxyl such as occurs in single-stranded regions.

Dr. Kenyon developed a novel method called in-gel SHAPE in which a mixed structural population of RNA molecules is first separated by native polyacrylamide gel electrophoresis and then conformers are subsequently individually isolated and analyzed. “By probing the individual RNAs in a population within the gel matrix, we can determine the structure of each conformer without the need for mutagenesis or variation of refolding conditions,” said Dr. Kenyon. “It’s a straightforward approach, and sometimes when we present this at meetings people comment, ‘Why hasn’t this been done before?’”

To validate her in-gel technique, Dr. Kenyon used the well-characterized, structurally known HIV-1 trans-activation response element (TAR). “Our method provided an authentic and accurate structural analysis that was also highly reproducible. We then further examined the utility of in-gel SHAPE analysis for separating monomeric and dimeric species of HIV-1 packaging signal RNA,” said Dr. Kenyon. “We found extensive differences between monomer and dimer that supported a recently proposed structural switch model of the RNA genomic dimerization and packaging.”

Although current analyses are limited to RNAs of 1,000 or fewer nucleotides, the group is examining different gel matrixes to solve that challenge. “We plan on utilizing this approach in the future to study RNA/RNA interactions as well as RNA/protein interactions. It’s a great way to increase throughput over traditional methods,” said Dr. Kenyon.

RNA Mapping

Chemical probing is a valuable tool for inferring RNA functional secondary structures and tertiary RNA interactions. The gold standards of chemical probing are comparative sequence analysis, X-ray crystallography, and nuclear magnetic resonance (NMR). These techniques, however, require significant investigator insight and effort. Even worse, they are not always tractable for RNAs.

“We have created a whole new platform utilizing Illumina sequencing to probe thousands of RNA molecules at once,” reported Rhiju Das, Ph.D., assistant professor of biochemistry at Stanford University. “It takes about a day and only requires a tabletop Illumina machine followed by analysis with our software package, which is called MAPseeker.”

Initially, Julius B. Lucks, Ph.D., and his group at Cornell University’s department of chemical and biomolecular engineering developed an experimental protocol and bioinformatics pipeline to enable highly parallel chemical mapping. “We decided to extend this to RNAs of many distinct sequences,” said Dr. Das. “We also decided to advance this protocol into a MAP-seq (multiplexed accessibility probing) method in order to increase the rate at which large numbers of RNAs could be probed and to reduce bias.”

The strategy allows multiple chemical reactions, such as carbodiimide base modification and acylation of the 2′-hydroxyl to be carried out in parallel. Conditions that give near-quantitative, single-stranded DNA ligation are used, and an Illumina adapter sequence is added to the reverse-transcribed DNA. “Unique sequence identifiers, placed at the 3′ ends of RNAs, are then sequestered into hairpins,” said Dr. Das. “This helps prevent interference with the fold of the RNA segments.” Finally, analysis is carried out with MAPseeker, an automated tool that can, according to Dr. Das, “quantify all the reactivities of the nucleotides in each RNA of every condition.”

MAP-seq continues to evolve rapidly to enhance multiplexing, to probe oligonucleotides pools, and to tackle RNAs with lengths reaching into the kilobases. “Right now one limitation of the technology is size. The limit is about 100 nucleotides. That will likely be increased in the coming years,” said Dr. Das.

Dr. Das also has constructed a fun, Web-based site where anyone can type in an RNA sequence and then get MAP-seq structural data through his lab’s experimental pipeline. “It’s really becoming quite popular,” said Dr Das. “You can check it out at http://eterna.stanford.edu/web/.”

RNA Nanotechnology

RNA molecules can be designed and manipulated as easily as DNA. They also display versatility in structure and diversity in function (including enzymatic activity) similar to that of proteins. These properties make RNA suitable candidates to act as building blocks for applications in nanotechnology and nanomedicine.

“RNA can fold into well-defined tertiary structures with specialized functions,” said Peixuan Guo, Ph.D., who holds the William Farish Endowed Chair of Nanobiotechnology at the University of Kentucky’s Markey Cancer Center, and who also serves as a professor at the university’s College of Pharmacy. “We use this information to rationally design building blocks that self-assemble into RNA nanoparticles.”

Dr. Guo, a pioneer of RNA nanotechnology, published a paper in 1998 that described how a virus known as bacteriophage phi29 uses six RNAs strung together in the shape of a hexagon to create a kind of molecular motor. He has since used phi29 packaging RNA (pRNA) for siRNA or drug delivery to specific cells and single-molecule imaging. He has also incorporated the phi29 motor channel into a lipid membrane for single-molecule sensing and developed a new system with the potential for high-throughput dsDNA sequencing.

Although a number of techniques are available to design RNA nanoparticles, Dr. Guo’s group is focusing on three. “We focus on using RNA designs involving interlocking loops for hand-in-hand interactions, palindrome sequences for foot-to-foot interactions, and an RNA three-way junction for branch extensions,” said Dr. Guo. These techniques are used in Dr Guo’s lab to make toolkits to construct RNA architectures with diverse shapes and angles.

“Because of its many useful structural features, [phi29 pRNA] is often used as a backbone for the assembly of RNA nanoparticles,” said Dr. Guo. “We have developed our toolkits using pRNA as a delivery platform. After they incorporate the desired functionalities (such as siRNAs, miRNA, ribozymes, or ligands), the RNA nanoparticles are thermodynamically and chemically stable.”

Dr. Guo sees many applications for the technology. His group is initially focusing on cancers such as colon, lung, etc. According to Dr Guo, the size of pRNA-based nanoparticles is ideal for passively delivering them into tumors.

“Research over the last decade has demonstrated that the size of nanoparticles is a critical determinant of their in vivo behavior,” said Dr. Guo. “If nanoparticles are smaller than 10 nanometers, the result is nonspecific diffusion into tissue, while if they are larger than 100 nanometers, nanoparticles are less likely to gain access to cells. Our RNA nanoparticles are 20–50 nanometers. Their size allows enhanced permeability and retention effects that minimize off-target effects or toxicity.”

According to Dr. Guo, active delivery of RNA nanoparticles can be achieved by adding specific targeting moieties to the complex. “These functionalized RNA nanoparticles, with the combination of detection molecules, targeting groups, and therapeutic payload, are useful for the diagnosis of and therapy for cancer and viral disease.”


Some of the RNA nanoparticles constructed in Dr. Peixuan Guo’s lab at the University of Kentucky are shown utilizing the pRNA of the bacteriophage phi29 DNA-packaging motor. The two illustrations on the top left are side and top views of the hexametric structure of the phi29 DNA-packaging motor (Guo et al., Virology 2013, in press). The two illustrations on the bottom left are side and top views of the hexameric pRNA derived from X-ray crystallography (Zhang et al., RNA, 2013, 9: 1226). The right panels are AFM images of diverse pRNA nanoparticles constructed using three different toolkits, including hand-in-hand interactions, foot-to-foot interactions, and branch extension (scale bars: 10 nm) (Shu et al., Nature Nanotech 2013, 6: 658; Shu et al., Nature Protocols 2013, 8: 1635).

tRNA Identification

Transfer ribonucleic acids (tRNAs) can be exceedingly difficult to analyze. Patrick Limbach, Ph.D., professor of chemistry and Ohio Eminent Scholar at the University of Cincinnati, is utilizing a highly optimized tandem mass spectrometry approach to characterize tRNAs.

“Our long-term goal is to understand how tRNAs change in disease states,” said Dr. Limbach. “We really don’t know a lot about how tRNA populations change during stress and illness. They can be studied using next-generation sequencing, microRNA analysis, or RNAseq. However, this just gives information about the nucleic acid and not the many modifications that decorate tRNAs.”

Dr. Limbach noted that Collin Wetzel, a senior graduate student in his group, utilized tandem mass spectrometry to identify individual tRNAs from a pool of total tRNA in E. coli cell lysates. “In contrast to genomic and biochemical approaches, mass spectrometry-based methods can clearly identify complex modifications found in tRNAs because such modifications alter the mass-to-charge ratio of these ions, said Dr. Limbach. “Further, this type of strategy enables highly accurate identification of tRNA species in a manner that is conducive to high-throughput targeted analysis.”

The novel method allows identification of individual isoacceptor tRNAs via the detection of unique oligonucleotide sequences following a single enzymatic digestion. “We found that 44 out of the 47 isoaccepting tRNAs predicted to be in E. coli could be detected by targeting 22 precursor ions in less than 15 minutes,” said Dr. Limbach. “The tandem mass spectrometry allows monitoring specific transitions from precursor to product ions. Ultimately, targeted tandem mass spectrometry can monitor the specific transitions for any known pool of known tRNA sequences.”

Dr. Limbach also indicated that his group will begin characterizing tRNAs in eukaryotic cells: “We’d like eventually to bring this to the clinic by reducing the assay times to two to five minutes. We are continuing to refine and improve the technology.”


Collin Wetzel, a senior graduate student in the laboratory of Professor Patrick Limbach at the University of Cincinnati, prepares tRNA samples for analysis. Wetzel and Limbach recently described a new approach for tRNA identification using targeted tandem mass spectrometry. Their approach requires minimal sample preparation and provides a new avenue for future higher throughput tRNA analyses by mass spectrometry.

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