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

Deciphering RNA Secondary Structure

  • RNA Nanotechnology

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    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).

    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.”

  • tRNA Identification

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    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.

    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.”

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