Biophysically Inspired Rational Design of Structured Chimeric Substrates for DNAzyme Cascade Engineering Matthew R. Lakin 1 , Carl W. Brown III 2 , Eli K. Horwitz 2 , M. Leigh Fanning 1 , Hannah E. West 2 , Darko Stefanovic 1,2 *, Steven W. Graves 2,3 * 1 Department of Computer Science, University of New Mexico, Albuquerque, New Mexico, United States of America, 2 Center for Biomedical Engineering, University of New Mexico, Albuquerque, New Mexico, United States of America, 3 Department of Chemical and Biological Engineering, University of New Mexico, Albuquerque, New Mexico, United States of America Abstract The development of large-scale molecular computational networks is a promising approach to implementing logical decision making at the nanoscale, analogous to cellular signaling and regulatory cascades. DNA strands with catalytic activity (DNAzymes) are one means of systematically constructing molecular computation networks with inherent signal amplification. Linking multiple DNAzymes into a computational circuit requires the design of substrate molecules that allow a signal to be passed from one DNAzyme to another through programmed biochemical interactions. In this paper, we chronicle an iterative design process guided by biophysical and kinetic constraints on the desired reaction pathways and use the resulting substrate design to implement heterogeneous DNAzyme signaling cascades. A key aspect of our design process is the use of secondary structure in the substrate molecule to sequester a downstream effector sequence prior to cleavage by an upstream DNAzyme. Our goal was to develop a concrete substrate molecule design to achieve efficient signal propagation with maximal activation and minimal leakage. We have previously employed the resulting design to develop high-performance DNAzyme-based signaling systems with applications in pathogen detection and autonomous theranostics. Citation: Lakin MR, Brown III CW, Horwitz EK, Fanning ML, West HE, et al. (2014) Biophysically Inspired Rational Design of Structured Chimeric Substrates for DNAzyme Cascade Engineering. PLoS ONE 9(10): e110986. doi:10.1371/journal.pone.0110986 Editor: Mukund Thattai, Tata Institute of Fundamental Research, India Received August 18, 2014; Accepted September 18, 2014; Published October 27, 2014 Copyright: ß 2014 Lakin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This material is based upon work supported by the National Science Foundation (http://nsf.gov/) under grant numbers 1027877, 1028238, and 1318833, which supported M.R.L., C.W.B., D.S., and S.W.G. C.W.B. gratefully acknowledges support from an Integrative Graduate Education and Research Traineeship in Integrating Nanotechnology with Cell Biology and Neuroscience from the National Science Foundation (http://nsf.gov/) under grant number DGE- 0549500. M.R.L. gratefully acknowledges support from the New Mexico Cancer Nanoscience and Microsystems Training Center (http://cntc.unm.edu/) under National Institutes of Health (http://www.nih.gov/) grant number 5R25CA153825. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: graves@unm.edu (SWG); darko@cs.unm.edu (DS) Introduction DNA is a versatile nanoscale engineering material. The sequence-specific nature of DNA hybridization via Watson-Crick complementarity and the predictability of DNA secondary structure [1–3] make it feasible to rationally design DNA nanostructures [4–10], synthetic molecular motors [11–16], and dynamic nanoscale logic devices [17–24]. Rational design is an important concept because the ability to directly apply biophysical principles [25] and straightforward DNA interaction rules [26,27] to molecular design is a key reason for the success of DNA nanotechnology. In contrast, recent efforts in metabolic engineer- ing [28] and protein-based molecular computation [29–32] show promise because of the wide-ranging chemical repertoire of protein chemistry, but the design process is complicated by the complexity of protein structures and the promiscuous nature of amino acid interactions. This paper concerns the application of biophysical principles in the design of structured nucleic acid molecules to implement molecular logic circuits with DNAzymes. This molecular logic architecture can perform nanoscale compu- tations in response to chemical stimuli, with potential applications in pathogen detection and autonomous theranostic devices. DNAzymes [33,34] (also known as deoxyribozymes) are single DNA strands that have been found to catalyze a range of chemical reactions [35–48]. The use of DNAzymes for molecular logic is well reported in the scientific literature [49–59]. RNA-cleaving DNAzymes are the most widely used and best characterized, owing to their potential for therapeutic applications [59,60]. We have previously reported [61] molecular logic gates based on regulating the 8–17 DNAzyme [45,62,63] by toehold-mediated strand displacement (TMSD) reactions [21], which provide a precise means of controlling DNAzyme activation. The 8–17 DNAzyme can cleave a chimeric DNA-RNA substrate at a cleavage site denoted by a single RNA base. We use the 8–17 DNAzyme here because of its compact size and high turnover rate [64]. We refer to these as DNAzyme displacement (DzD) logic gates. Connecting multiple DNAzyme logic gates into signaling circuits is necessary to increase their computational power beyond that of parallel DNAzyme gate arrays [50,51,54,55] and to incorporate non-trivial circuit motifs analogous to cellular PLOS ONE | www.plosone.org 1 October 2014 | Volume 9 | Issue 10 | e110986