Computational and Experimental RNA Nanobiology

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Recently we developed a new type of RNA nanostructure that forms a truncated tetrahedron in collaboration with Luc Jaeger, UC Santa Barbara. The structure was built from our hexameric ring where 4 sides of the tetrahedral structure each contain the hexmeric ring, but each ring contains 3 H-shaped crossover connectors to the other rings. This type of construct allows for the incorporation of up to 12 functional entities such as Dicer substrates, beacons and/or aptamers. We found that cells take up these constructs better than some of the other RNA nanoconstructs. The hypothesis that nanoparticle shape and size matter regarding functionality seems to be true. Due, at least in part, to the better uptake and we believe better processivity, we found that knockdown of targeted genes to induce cell death, using incorporated Dicer substrate PLK1 is more efficacious than some of our other particles. Several different methods were used to stunningly verify the assembly of this particle including an atomic force microscope (AFM) and Cryo-EM in collaborations with the Optical Microscopy and Image Analysis Lab and with the Electron Microscopy Lab respectively, of CCR. ---To achieve control over deliverable functionality and stability of RNA-based nanoparticles, the properties of DNA and RNA were merged in the development of computationally designed nanoparticles that were constructed from RNA/DNA hybrids. These molecules allow higher stability in blood serum, attachment of fluorescent markers for tracking, and the ability to split the components of functional elements inactivating them, but allowing later activation under the control of complementary toeholds by which the kinetics of re-association can be tuned. Diceable substrate siRNA could be split into two components, each consisting of an RNA/DNA hybrid. Complementary RNA single-stranded toeholds rather than DNA can be used in the construction of the hybrids. The two hybrids, when transfected into cells recombine into two products due to the toeholds and the computationally determined thermodynamic difference between the hybrids and the products. From the perspective of thermodynamics, the use of RNA toeholds is advantageous as it reduces the length of the single stranded ends required to unzip the hybrids and generate the functional RNA element. In addition, less DNA is needed since the DNA portion of the hybrid is shortened. This has the added advantage of reducing innate cellular immune responses as a result of shorter double stranded DNA helical products. From a design perspective, the RNA toehold can be part of the functional DS RNA, or other potential RNA moiety, reducing the size and simplifying the design of the resulting hybrid duplexes. RNA-based hybrids containing 3 Dicer substrate siRNAs for synergistic simultaneous targeting of apoptosis-related genes in HT29 tumors have been tested in vivo after significant testing in cell cultures, in a comprehensive mouse study funded, in part, by the Invention Development Program. Results look encouraging showing retardation of tumor growth both intratumorally and more so by tail vein injection. Further studies are being performed using alternative delivery agents. ----Since we can control immune response with RNA-based nanoparticles, we have been collaborating with Joost Oppenheim, CCR, to take advantage of these properties to activate the immune system for anti-cancer treatment. Working with Joost Oppenheim's group we found significant in vivo results showing a cure in 3 out 10 immune competent mice. Most of the other mice showed signficant regression ot their tumors. Further experiments have been undertaken and are continuing to further characterize the pathways of action and the functionality related to different RNA ring constructs.---Besides using small molecules to target various RNA motifs for potential drugs as described under another project heading, we have been exploring, by the use of 3D modeling, molecular dynamics and molecular docking, the use of small molecule ligands that dock to specific RNA motifs that in turn alters the dynamics, shape and functionality of the nano constructs built from these motifs. We have shown that such affects are possible, i,e. changing of the biophysical characteristics of the targets. Further exploration of this type of nano design is being investigated.---The delivery of RNA-based nanoconstructs in cell culture and in vivo is essential for the development of therapeutic methodologies using these agents. Non-modified naked RNAs have short half-lives in blood serum due to nucleases and have difficulty crossing cell membranes due to their negative charge. Thus, we are developing lipid and polymer formulations. We have worked with Jonathan Lovell, U of Buffalo, on the development of sulfonated photoactivatable polymers for the delivery of our RNA-based nanoparticles. The concept being that the use of focused near infrared light on tumors that have been administered these photoactivatable polymers will specifically release drugs into the tumor with little of any release in normal tissues that have not be effected by the light. We have shown minimal leakage without laser treatment and significant functionality when laser treated.---We have also tested bolaamphiphile vesicles GLH-19 and GLH-20 formulations for delivery of siRNA to tumors and to the brain in collaboration with Eli Heldman, Ben Gurion University. We showed good delivery to both locations, including the brain which is difficult to target due to issues related to crossing the blood-brain barrier. The stability of the various formulations tested were also analyzed by molecular dynamics, which explained quite well the results we were seeing experimentally.---A relatively new lipid based on the use of oxime-ethers is being explored in collaboration with Michael Nantz, University of Louisville and Xiulang Lu, University of Connecticut. Results look quite promising for their use as a delivery agent in target lung cancer. A paper was recently submitted describing these results.--- A collaboration with Xandra Breakefield, Harvard Medical school is also being pursued to develop techniques to load exosomes with RNA for to be used as potential delivery agents. The technique utilizes highly positively charged proteins to enable this loading. Preliminary results look promising.