Elsevier

Journal of Biotechnology

Volume 150, Issue 4, December 2010, Pages 466-473
Journal of Biotechnology

ARDesigner: A web-based system for allosteric RNA design

https://doi.org/10.1016/j.jbiotec.2010.10.067Get rights and content

Abstract

RNA molecules play vital informational, structural, and functional roles in molecular biology, making them ideal targets for synthetic biology. However, several challenges remain for engineering novel allosteric RNA molecules, and the development of efficient computational design techniques is vitally needed. Here we describe the development of Allosteric RNA Designer (ARDesigner), a user-friendly and freely available web-based system for allosteric RNA design that incorporates mutational robustness in the design process. The system output includes detailed design information in a graphical HTML format. We used ARDesigner to engineer a temperature-sensitive AR, and found that the resulting design satisfied the prescribed properties/input. ARDesigner provides a simple means for researchers to design allosteric RNAs with specific properties. With its versatile framework and possibilities for further enhancement, ARDesigner may serve as a useful tool for synthetic biologists and therapeutic design. ARDesigner and its executable version are freely available at http://biotech.bmi.ac.cn/ARDesigner.

Introduction

RNA molecules are highly versatile, with roles ranging from genetic information storage and transfer to the catalysis of biochemical reactions. This functional diversity is derived in part from the organization of RNA molecules into specific functional modular domains. RNA molecules frequently undergo conformational or allosteric changes prerequisite to protein synthesis and translocation (Douthwaite et al., 1982, Lodmell and Dahlberg, 1997, Kurland et al., 1975, Schwarz et al., 1976, Wakao et al., 1990, Wagner and Garrett, 1979, Wintermeyer and Gualerzi, 1983, Gowda and Zwieb, 1997), mRNA splicing (LeCuyer and Crothers, 1994, LeCuyer and Crothers, 1993, Michel and Westhof, 1990, Staley and Guthrie, 1999, Wang et al., 1993), viral and cellular gene expression (Du and Babitzke, 1998, Stroynowski and Yanofsky, 1982, Wang et al., 1998), and viral RNA replication and packaging (Tounekti et al., 1992, Mougel et al., 1993, Miller and Silver, 1991). Such structural changes can regulate RNA function, and may be effected by RNA interactions with other cellular machinery components (Du and Babitzke, 1998, Lodmell and Dahlberg, 1997, Stroynowski and Yanofsky, 1982, Staley and Guthrie, 1999). These properties of RNA molecules have inspired synthetic biologists to use molecular cellular biology and biotechnology techniques to design novel allosteric RNAs (ARs) for exploratory (Soukup and Breaker, 1999, Breaker, 2004) or functional purposes (Benner and Sismour, 2005, Hasty et al., 2002, Hasty et al., 2001, McDaniel and Weiss, 2005, Sprinzak and Elowitz, 2005, Wall et al., 2004). For example, ARs may be used as cognate effector sensors or as controllable therapeutics.

Recently, two important studies have reviewed the advances in RNA synthetic biology (Saito and Inoue, 2009, Win et al., 2009). These advances have enhanced our ability to engineer novel RNA motifs. Many structural motifs of RNAs have been engineered for various biological uses (Buskirk et al., 2004, Buskirk et al., 2003, Rackham and Chin, 2005a, Rackham and Chin, 2005b, Saha et al., 2003, Yen et al., 2004, Suess et al., 2004, Grate and Wilson, 2001, Werstuck and Green, 1998, Isaacs et al., 2004, Bayer and Smolke, 2005, Desai and Gallivan, 2004, Thompson et al., 2002). Noncoding RNAs have been designed to regulate bacterial gene expression (Isaacs et al., 2004), and orthogonal ribosome combinations have been generated to program Boolean logic functions in Escherichia coli (Rackham and Chin, 2005b). Suess et al. (2004) designed a system in which the binding of an incorporated theophylline-binding aptamer to a reporter mRNA repressed gene expression through helix slippage and subsequent ribosomal binding site (RBS) occlusion. While designing an RNA gene expression platform in mammalian systems, another study regulated mRNA translation by incorporating the properties of a ligand-responsive ribozyme (Yen et al., 2004). Other researchers have designed transacting riboregulators in Saccharomyces cerevisiae (Bayer and Smolke, 2005). Finally, two separate groups Saha et al. (2003) and Buskirk et al., 2003, Buskirk et al., 2004 independently constructed RNA-based transcription factors in yeast.

Although highly promising in theory, the use of synthetic biology to create ARs faces two main complications in its application: an insufficient number of available molecular parts (module units) (Endy, 2005), and a failure of simple molecular designs to incorporate cross-talk between the synthetic genetic circuit and natural systems in vivo. The design efficiency affects the degree to which therapeutic ARs can be developed. Furthermore, mutational robustness, which measures the phenotypic invariance of a system during genetic perturbation, is also an important aspect that should be incorporated in AR design. Indeed, numerous studies have focused on identifying the mechanisms that increase mutational robustness in various biological processes (Bhalla and Iyengar, 2001, Barkai and Leibler, 1997, von Dassow et al., 2000, Alon et al., 1999, Csete and Doyle, 2002, Csete and Doyle, 2004, Kitano, 2004a, Kitano, 2004b, Kitano, 2007a, Kitano, 2007b, Kitano et al., 2004, Kitano and Oda, 2006, Stelling et al., 2004, Savageau, 1985a, Savageau, 1985b, Savageau, 1998). Given these challenges, it is imperative that efficient computational techniques should be developed to design RNA components for synthetic systems.

Given an RNA secondary structure, some studies have designed RNA sequences that fold into the desired structure (Dirks et al., 2004, Hofacker et al., 1994, Andronescu et al., 2004). One approach is implemented in RNAinverse (Hofacker et al., 1994), which is included in the Vienna RNA Package (Hofacker, 2003). There, the strategy of an “adaptive” walk search on the so-called compatible sequences (i.e. sequences that can possibly form a base-pair at the required positions in the desired structure) is used and local optima are found according to two different criteria, namely a structural distance between the MFE structure of the designed sequence and the target structure (MFE-mode) and the probability of folding into the target structure (P-mode). A second algorithm is called RNA-SSD (RNA Secondary Structure Designer) and is developed by Andronescu et al. (2004). RNA-SSD is based on a recursive stochastic local search that also tries to minimize a structure distance. Busch and Backofen (2006) have developed another algorithm for the inverse folding problem, INFO-RNA. It consists of two parts: a dynamic programming method for good initial sequences and a following improved stochastic local search that uses an effective neighbor selection method.

Here, we extend the design of RNA sequences that fold into a desired structure to the design of an RNA molecule that fold into prescribed multiple stable conformations. We show that bistable, and more generally, multiple stable RNA molecules with a variety of additional properties can be designed rather easily. We developed the novel Allosteric RNA Designer (ARDesigner) system to design temperature-sensitive and energy barrier ARs with a variety of additional specified properties, including mutational robustness. We treated the design of ARs that fold into prescribed multiple stable conformations as a combinatorial optimization problem (Flamm et al., 2001), which we solved using simple heuristics. We tested the efficiency of our system through the design of a temperature-sensitive AR.

Section snippets

RNA folding and RNA design

Within the energy model, the RNA folding problem of finding the optimal secondary structures of a given sequence is a direct problem, which can be solved efficiently by means of dynamic programming, such as Mfold (Zuker and Stiegler, 1981), RNAfold in Vienna RNA package (Hofacker, 2003, Hofacker et al., 1994). The folding process can be viewed as the evaluation of the “folding function” Φ, which computes the secondary structure Φ(S) of a given sequence S. Sequence S folds into structure S, that

Overall architecture of ARDesigner

ARDesigner contained three parts: a user web interface, middle-tier Common Gateway Interface (CGI) application running on a web server, and a server part (Fig. 1). The server part designs the AR with specific properties, using one of three software programs depending on the number of prescribed structures: the Mono-Stable, Bi-Stable, or Multi-Stable ARDesigner generates ARs with one, two, or three prescribed structures, respectively. The user provides input via the web interface, which sends

Discussion and conclusion

Designing robustness biological systems that perform human desired functions is one of the long-term goals of synthetic biology. However, researchers currently face several scientific and technical challenges in designing and building biological systems. An important one is that our ability to understand and engineer biological systems is limited by the tools available to design functional molecular exhibiting novel properties. Here, we developed a user-friendly and freely available web-based

Competing interests

The authors declare that there are no competing interests.

Acknowledgements

The authors would like to thank the reviewers and the editors of the paper for their constructive comments which contributed to an improved presentation. The authors would also like to thank the Super Biomed Computation Center at Beijing Institute of Health Administration and Medicine Information for providing computing resources. This work is supported by grant from the National High Technology Research and Development Program of China (No. 2007AA02Z311) and grants from the National Nature

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