A boost for the emerging field of RNA nanotechnology

Abstract

This Nano Focus article highlights recent advances in RNA nanotechnology as presented at the First International Conference of RNA Nanotechnology and Therapeutics, which took place in Cleveland, OH, USA (October 23-25, 2010) ( http://www.eng.uc.edu/nanomedicine/RNA2010/ ), chaired by Peixuan Guo and co-chaired by David Rueda and Scott Tenenbaum. The conference was the first of its kind to bring together more than 30 invited speakers in the frontier of RNA nanotechnology from France, Sweden, South Korea, China, and throughout the United States to discuss RNA nanotechnology and its applications. It provided a platform for researchers from academia, government, and the pharmaceutical industry to share existing knowledge, vision, technology, and challenges in the field and promoted collaborations among researchers interested in advancing this emerging scientific discipline. The meeting covered a range of topics, including biophysical and single-molecule approaches for characterization of RNA nanostructures; structure studies on RNA nanoparticles by chemical or biochemical approaches, computation, prediction, and modeling of RNA nanoparticle structures; methods for the assembly of RNA nanoparticles; chemistry for RNA synthesis, conjugation, and labeling; and application of RNA nanoparticles in therapeutics. A special invited talk on the well-established principles of DNA nanotechnology was arranged to provide models for RNA nanotechnology. An Administrator from National Institutes of Health (NIH) National Cancer Institute (NCI) Alliance for Nanotechnology in Cancer discussed the current nanocancer research directions and future funding opportunities at NCI. As indicated by the feedback received from the invited speakers and the meeting participants, this meeting was extremely successful, exciting, and informative, covering many groundbreaking findings, pioneering ideas, and novel discoveries.

Figure 1

Approaches in RNA nanotechnology. Figure modified with permission from ref (4). Copyright 2010 Nature Publishing Group.

Figure 2

Role of phi29 DNA packaging RNA (pRNA) in RNA nanotechnology. Six pRNA assemble into a hexameric ring to gear the DNA translocating machine. pRNA monomers can be re-engineered to fold into well-defined structures, such as dimers, hexamers, and arrays via hand-in-hand or foot-to-foot interactions between two interlocking loops. pRNA-based nanoparticles have been successfully constructed with functional modules and used as polyvalent vehicles to deliver a variety of therapeutic/detection molecules to cancer- and viral-infected cells. Image courtesy of YinYin Guo. Figure adapted with permission from ref (136). Copyright 2011 Elsevier.

Figure 3

(A) Representation of part of the computational RNA nanodesign pipeline associated with NanoTiler.⁻ What is depicted is one result, of many, which produces a closed-ring structure generated with the NanoTiler software that was derived in a totally automated fashion from a combinatorial search of motifs found in the RNAJunction database.(19) Sequences that were predicted for the shown structure were ultimately shown to be able to experimentally self-assemble into the depicted form. Panel A adapted with permission from ref (65). Copyright 2008 Elsevier. (B) Depiction of 10 stranded RNA cubes that were computer-designed with NanoTiler and later shown to be able to self-assemble.^, The layouts of the interacting strands are shown on the right. It should also be noted that the 10 stranded cubes, as shown here, were assembled in three forms: without a malachite green aptamer, with one malachite green aptamer, and with two malachite green aptamers. A properly formed aptamer fluoresces in the presence of a triarylmethane dye. The formation of the aptamers was used to confirm the cubes’ self-assembly and designed functionalization. Panel B adapted with permission from ref (38). Copyright 2011 Nature Publishing Group. (C) Depiction of the computer-designed, and later shown to be able to self-assemble, RNA hexagonal ring.^,, Each corner of the ring contains the motif derived from the RNAIi/RNAIIi kissing loop interaction that forms an angle of about 120°, which is conducive to hexagonal ring formation. Sides and dangling ends can be further functionalized. Panel C adapted from ref (69). Copyright 2011 American Chemical Society.

Figure 4

Watching the movement of single-molecular spiders at super-resolution (Nils Walter group). A multi-legged molecular nanoassembly walks along a prescribed substrate track on a DNA origami (A), where its fluorophore label (F1) is tracked at super-resolution relative to a label on the end of the track (F2) by total internal reflection fluorescence (TIRF) microscopy (B).^, The resulting trajectory compares well with a scale-drawn schematic of the origami (C). Figure modified with permission from ref (81). Copyright 2010 Elsevier.

Figure 5

Complex between the trans-activating responsive RNA element TAR, viral (Tat), and cell proteins (cyclin T1, CDK-9) is required for efficient transcription of the HIV-1 genome. Disruption of this complex leads to abortive transcription of the retroviral DNA. A high-affinity RNA hairpin aptamer (R06) specifically recognizes TAR. The transcription of a R06 construct in recipient cells reduces the production of β-galactosidase whose expression is driven by the HIV-1 promoter containing TAR, in response to retroviral infection. Therefore, targeting RNA hairpins offers an alternative to targeting proteins for the design of artificial modulators of gene expression.

Figure 6

(A) Internal nucleic acid reporter group, represented here by the fluorescent base analogue tC in pair with guanine looking down the long axis of a DNA duplex. (B) Same internal reporter group looking along the short axis of a DNA duplex.