Bruce A. Shapiro

RNAJunction: a database of RNA junctions and kissing loops for three-dimensional structural analysis and nanodesign

We developed a database called RNAJunction that contains structure and sequence information for RNA structural elements such as helical junctions, internal loops, bulges and loop–loop interactions. Our database provides a user-friendly way of searching structural elements by PDB code, structural classification, sequence, keyword or inter-helix angles. In addition, the structural data was subjected to energy minimization. This database is useful for analyzing RNA structures as well as for designing novel RNA structures on a nanoscale. The database can be accessed at: http://rnajunction.abcc.ncifcrf.gov/

CyloFold: secondary structure prediction including pseudoknots

Computational RNA secondary structure prediction approaches differ by the way RNA pseudoknot interactions are handled. For reasons of computational efficiency, most approaches only allow a limited class of pseudoknot interactions or are not considering them at all. Here we present a computational method for RNA secondary structure prediction that is not restricted in terms of pseudoknot complexity. The approach is based on simulating a folding process in a coarse-grained manner by choosing helices based on established energy rules. The steric feasibility of the chosen set of helices is checked during the folding process using a highly coarse-grained 3D model of the RNA structures. Using two data sets of 26 and 241 RNA sequences we find that this approach is competitive compared to the existing RNA secondary structure prediction programs pknotsRG, HotKnots and UnaFold. The key advantages of the new method are that there is no algorithmic restriction in terms of pseudoknot complexity and a test is made for steric feasibility. Availability: The program is available as web server at the site: http://cylofold.abcc.ncifcrf.gov.

Protocols for the In Silico Design of RNA Nanostructures

Recent developments in the field of nanobiology have significantly expanded the possibilities for new modalities in the treatment of many diseases, including cancer. Ribonucleic acid (RNA) represents a relatively new molecular material for the development of these biologically oriented nanodevices. In addition, RNA nanobiology presents a relatively new approach for the development of RNA-based nanoparticles that can be used as crystallization substrates and scaffolds for RNA-based nanoarrays. Presented in this chapter are some methodological shaped-based protocols for the design of such RNA nanostructures. Included are descriptions and background materials describing protocols that use a database of three-dimensional RNA structure motifs; designed RNA secondary structure motifs; and a combination of the two approaches. An example is also given illustrating one of the protocols.

Engineered RNA Nanodesigns for Applications in RNA Nanotechnology

Abstract Nucleic acids have emerged as an extremely promising platform for nanotechnological applications because of their unique biochemical properties and functions. RNA, in particular, is characterized by relatively high thermal stability, diverse structural flexibility, and its capacity to perform a variety of functions in nature. These properties make RNA a valuable platform for bio-nanotechnology, specifically RNA Nanotechnology, that can create de novo nanostructures with unique functionalities through the design, integration, and re-engineering of powerful mechanisms based on a variety of existing RNA structures and their fundamental biochemical properties. This review highlights the principles that underlie the rational design of RNA nanostructures, describes the main strategies used to construct self-assembling nanoparticles, and discusses the challenges and possibilities facing the application of RNA Nanotechnology in the future.

Triggering RNAi with multifunctional RNA nanoparticles and their delivery

Abstract Proteins are considered to be the key players in structure, function, and metabolic regulation of our bodies. The mechanisms used in conventional therapies often rely on inhibition of proteins with small molecules, but another promising method to treat disease is by targeting the corresponding mRNAs. In 1998, Craig Mellow and Andrew Fire discovered dsRNA-mediated gene silencing via RNA interference or RNAi. This discovery introduced almost unlimited possibilities for new gene silencing methods, thus opening new doors to clinical medicine. RNAi is a biological process that inhibits gene expression by targeting the mRNA. RNAi-based therapeutics have several potential advantages (i) a priori ability to target any gene, (ii) relatively simple design process, (iii) sitespecificity, (iv) potency, and (v) a potentially safe and selective knockdown of the targeted cells. However, the problem lies within the formulation and delivery of RNAi therapeutics including rapid excretion, instability in the bloodstream, poor cellular uptake, and inefficient intracellular release. In an attempt to solve these issues, different types of RNAi therapeutic delivery strategies including multifunctional RNA nanoparticles are being developed. In this mini-review, we will briefly describe some of the current approaches.

RNA Toehold Interactions Initiate Conditional Gene Silencing

interference pathway. Now, Afonin et al. show in their recent publication that single-stranded (ss) RNA, rather than ssDNA, is preferential for use as the toehold within cognate hybrid duplexes, providing several benefits compared to previous cognate hybrid designs. From the perspective of thermodynamics, the use of RNA toeholds is advantageous as it greatly reduces the length of the singlestranded ends required to unzip the hybrids and generate the functional RNA element. From a design perspective, because RNA is used as the toehold strand, the toehold sequence can inherently be part of the functional Dicer substrate 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. The authors demonstrate that RNA nanorings functionalized with RNA-toeholded hybrids exhibit increased yields from enzymatic co-transcriptional synthesis, as well as reduced overall nanoparticle size, compared to nanorings functionalized with DNA-toeholded hybrid duplexes. In the second publication from Shapiro and colleagues, Bindewald et al. present a novel two-stranded RNA switch that releases a small hairpin RNA (shRNA) upon recognition of a specific cellular mRNA (Figure 1, right). The RNA switch initially exists in an inactive state, with the shRNA strand sequestered in an inactive fold through formation of an inter-strand pseudoknot structure. Interaction of the switch’s ssRNA toehold with a complementary “trigger” RNA unzips the switch and leads to a conformational rearrangement. This conformational change produces a functional shRNA structure able to act as a Dicer substrate and activate the RNAi pathway for knockdown of target gene expression. As a proof of concept, the authors show that the RNA switch is able to silence eGPF expression in response to the presence of connective tissue growth factor (CTGF) mRNA in human breast cancer cells. Notably, any combination of mRNA triggers and silencing targets can theoretically be accommodated by this switch design, as there is no overlap between the sequence region that encodes the DOI 10.1515/rnan-2016-0002 Received April 20, 2016; accepted May 5, 2016

Computational Prediction and Modeling Aid in the Discovery of a Conformational Switch Controlling Replication and Translation in a Plus-Strand RNA Virus

This chapter presents computational tools used to predict the secondary structure and model the 3D structure of a novel translation enhancer element found within the 3′-UTR of the Turnip crinkle virus (TCV). Our Massively Parallel Genetic Algorithm program (MPGAfold) was used to predict the secondary structure, including one H-type pseudoknot, of the translation enhancer element. The results were confirmed and augmented by experiments. The combined secondary structure information was used to create a 3D model of the enhancer element with the aid of our program RNA2D3D. The 3D structure resembles that of tRNA, while its secondary structure is different from canonical tRNAs. It is the first such element found within a 3′-UTR, and it is a part of a conformational switch involved in the control of translation and transcription. It is possible that similar mechanisms may exist in other eukaryotic genomes.