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. DS siRNAs (Diceable substrate siRNA) could be split into two components, each consisting of an RNA/DNA hybrid. Now complementary RNA single-stranded toeholds rather than DNA are 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. Previously designed RNA ring scaffolds carrying six hybrids, now contain RNA toeholds rather than DNA. Toeholds of 2, 4, 6, and 8 nucleotides were investigated for their efficiency of re-association. FRET revealed that longer toeholds led to improved re-association rates. The functionality of these particles was confirmed in different human cell lines. 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. From a design perspective, the RNA toehold can be part of the functional DS RNA, or other potential RNA moiety, reducing the size and minimizing the design constraints of the resulting hybrid duplexes. Conditional hybrids that contain ssRNA toeholds also prove advantageous for incorporation into more complex RNA nanoparticles. It was shown that RNA nanorings functionalized with RNA toeholded hybrids exhibited increased yields from enzymatic co-transcriptional synthesis, as well as reduced overall nanoparticle size, compared to nanorings functionalized with DNA-toeholded hybrid duplexes. An additional scheme has also been exploited using RNA-RNA interactions. Here, an RNA strand is designed to interact with specific mRNA strands in cells. The RNA strand contains both therapeutic and trigger components that are designed to dissociate from each other in the presence of a trigger mRNA and form a byproduct as well as a short hairpin-like RNA which can be processed by dicer to form functional siRNA. The conformational change takes place due to the presence of an extended ssRNA toehold in the trigger-binding strand which allows for the specific binding to an mRNA in diseased cells. This approach allows for the conditional activation of therapeutic RNAs only where a designated trigger strand is present. For potential use in treatment of diseases, such as cancer, this can reduce off target silencing and allow for more precise treatment. Because the self-assembly of RNA complexes is an interplay of several RNA strands, a new algorithm, HyperFold was developed that predicts the folding properties of all possible combinations of strand complexes. Also, the general problem of finding the lowest free energy base pairing is difficult for computational algorithms. Because the number of possible base pair combinations grows exponentially with sequence length, searching through all possible base pair combinations quickly becomes prohibitive for nucleic acid structures possessing long strands and pseudoknotted structures. HyperFold solves this problem using a tunable heuristic that depends on a single parameter. By default the search utilizes a middle-ground strategy. for predicting the interactions between multiple RNA and DNA strands with possibly complex knotted structures. Designing self-assembling RNA ring structures based on known 3D structural elements connected via linker helices is a challenging task due to the immense number of motif combinations, many of which do not lead to ring-closure. We developed an in silico solution to this design problem by combinatorial assembly of RNA 3-way junctions, bulges, and kissing loops, and tabulating the cases that lead to ring formation. The solutions found are made available in the form of a web Ring Catalog. As an example of a potential use of this resource, we chose a predicted RNA square structure consisting of five RNA strands and demonstrated experimentally that the self-assembly of those five strands leads to the formation of a square-like complex. A new concept was developed that utilizes cognate nucleic acid nanoparticles which are fully complementary and functionally-interdependent to each other, whereby the physical interaction between sets of designed nanoparticles initiates a rapid isothermal shape change which in turn triggers the activation of multiple functionalities and biological pathways including transcription, energy transfer, functional aptamers and RNA interference. The individual nanoparticles are not active and have controllable kinetics of re-association and fine-tunable chemical and thermodynamic stabilities. Additionally, tunable immunostimulatory properties of the nanoparticles suggest that the particles that do not induce pro-inflammatory cytokines and high levels of interferons can be used as scaffolds to carry therapeutic oligonucleotides, while particles with strong interferon and proinflammatory cytokine induction may qualify as vaccine adjuvants.This dual cognate nanoparticle approach is an outgrowth of our previous work with the activatable hybrid cubes built from RNA or DNA cores with hybrid arms each cube having its own controllable immune responses. Since we are able to control immune response, we have embarked on 2 new collaborations (Joost Oppenheim- CCR, and Chris Jewell, UMD) to take advantage of these properties to activate the immune system for anti-cancer treatment. In addition, we have been working with (Electron Kebebew, Clinical Center, NCI) on the use of our RNA-based nanoparticles to target a lethal form of thyroid cancer. 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 inherent negative charge. To counter some of these issues we have been working with various lipid and polymer formulations. In the case of the lipids we have constructed delivery agents with hyaluronic acid for targeting cancer cells exhibiting CD44 receptors (in collaboration with Esta Sterneck, CCR). Initial experiments look quite positive. In addition, we have working with Jonathan Lovell (U of Buffalo) on the development of photoactivatable polymers for the delivery of our RNA-based nanoparticles. Initial results are also quite encouraging. An invited book on protocols for RNA Nanobiology is now in press, and in addition, several invited review papers and book chapters were also written on the above described subjects.
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