IEEE SENSORS JOURNAL, VOL. 8, NO. 6, JUNE 2008 1011 Towards De Novo Design of Deoxyribozyme Biosensors for GMO Detection Elebeoba E. May, Member, IEEE, Patricia L. Dolan, Paul S. Crozier, Susan Brozik, and Monica Manginell Abstract—Hybrid systems that provide a seamless interface be- properties make molecular beacons effective platforms for tween nanoscale molecular events and microsystem technologies detecting genetically modified targets in biodefense systems. enable the development of complex biological sensor systems that not only detect biomolecular threats, but also are able to determine A. Deoxyribozyme Molecular Beacons and execute a programmed response to such threats. The chal- lenge is to move beyond the current paradigm of compartmental- Molecular beacons are single-stranded oligonucleotide izing detection, analysis, and interpretation into separate steps. We probes that form stem-loop structures. The loop contains a present methods that will enable the de novo design and develop- probe sequence that is complementary to a target sequence. ment of customizable biosensors that can exploit deoxyribozyme Traditional molecular beacons contain a fluorophore and computing (Stojanovic and Stefanovic, 2003) to concurrently per- quencher on each arm of the stem of the beacon. Fluorescence form in vitro target detection, genetically modified organism detec- is achieved by separation of the fluorophore and quencher due tion, and classification. to a conformation change that takes place following target Index Terms—Avian influenza, biosensor, deoxyribozyme, error hybridization to the loop structure [5]. However, traditional control codes, hybridization thermodynamics, molecular beacons, beacons are limited by a 1:1 (target: signal) stoichiometry, and single nucleotide polymorphism. the sensitivity of the detection is linked to the amount of target present. Target amplification (PCR) is required to increase the level of sensitivity. I. INTRODUCTION Though DNA serves primarily as a carrier of the genetic YBRIDIZATION-BASED target recognition and dis- code and no enzymes made of DNA have been found in nature, Hcrimination is central to a wide variety of applications: molecular beacons comprised of single-stranded DNAs can high throughput screening, distinguishing genetically modified be engineered to perform catalytic reactions similar to those organisms (GMOs), molecular computing, differentiating bi- of protein and RNA. Catalytic DNAs or deoxyribozymes are synthesized in the laboratory via an in vitro iterative selection ological markers, fingerprinting a specific sensor response for process. complex systems, etc. The recognition substrate can exist in Like traditional molecular beacons, catalytic molecular solution or be immobilized onto a transducer and hybridiza- beacons also contain a stem-loop structure that undergoes a tion events can be detected optically, electrochemically, or conformational change following target hybridization to the via a mass-sensitive device [2]. The bioreceptor or probe is loop region. Unlike traditional molecular beacons, catalytic critical to the specificity of the biosensor. Although several molecular beacons are modular molecules, containing a de- single-stranded DNA sensor technologies, such as DNA mi- oxyribozyme appended to the stem-loop structure [6]. Catalytic croarrays, are widely used, molecular beacon probes are highly activity is initiated by a target DNA sequence binding to the sensitive and specific bioreceptors [2]–[4]. They can detect loop region that is distinct from the enzymatic active site. This mutations in target sequences and can be multiplexed [3]; these allosterically activates the deoxyribozyme complex to bind and cleave a labeled substrate oligonucleotide molecule, producing Manuscript received March 20, 2008; accepted March 21, 2008. Sandia is a a detectable signal. Once activated, the catalytic molecular multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin beacon will continuously cleave labeled substrate molecules. Company for the United States Department of Energys National Nuclear Secu- rity Administration under contract DEAC0494AL85000. This work was sup- Target amplification is not required since signal amplification ported by Sandia National Laboratories’ Laboratory Directed Research and De- is obtained through repeated processing of excess substrate velopment Program. E.E.M. and P.L.D. contributed equally to this work. The molecules due to the recognition and binding of a single target. associate editor coordinating the review of this paper and approving it for pub- lication was Dr. Dennis Polla. Single and multi-receptor site configurations enable the de- E. E. May is with the Department of Computational Biology, Sandia oxyribozyme to function as YES, NOT, or multi-input AND National Laboratories, Albuquerque, NM 87185-1316 USA (e-mail: gates [1]. [email protected]). P. L. Dolan, S. Brozik, and M. Manginell are with the Department of Current high-throughput DNA sensing systems rely heavily Biosensors and Nanomaterials, Sandia National Laboratories, Albuquerque, on silicon-based computing for interpretation of molecular NM 87185-0892 USA (e-mail: [email protected]; [email protected]; recognition events. Complex bioinformatics algorithms are [email protected]). P. S. Crozier is with the Department of Multiscale Computational Materials tasked with de-noising and processing of sensor output sig- and Methods, Sandia National Laboratories, Albuquerque, NM 87185-1322 nals. This approach is error prone and not easily integrated USA (e-mail: [email protected]). into emerging nanoscale sensor architectures for lab-on-chip Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. systems. Molecular computation, although impractical for real Digital Object Identifier 10.1109/JSEN.2008.923945 time computing needs, offers the ability for massively parallel 1530-437X/$25.00 © 2008 IEEE Authorized licensed use limited to: IEEE Xplore. Downloaded on January 12, 2009 at 15:09 from IEEE Xplore. Restrictions apply. 1012 IEEE SENSORS JOURNAL, VOL. 8, NO. 6, JUNE 2008 Fig. 2. Schematic of deoxyribozyme gate catalysis. Fig. 1. i (E6) Gate (DNA mfold server used) http://frontend.bioinfo.rpi. Fluorogenic assays to determine the detection and single nu- edu/applications/mfold/cgi-bin/dna-form1.cgi. cleotide polymorphism (SNP) differentiation capability of the gate were conducted with the FluoDia T70 Microplate Reader using black 384-well microplates. For the detection computation and detection, and is a promising technology limit experiments, a 55 detection volume containing the base for the development of next generation hybrid molecular gate (100 nM), the TAMRA-labeled substrate (2.5 ), sensor systems. In vitro computation using deoxyribozyme and varying concentrations of input DNA in reaction buffer gates enables the design of complex biosensor systems and (HEPES pH 7.0, 1 M NaCl, 2 mM ) was used. For reduces reliance on classical computing for interpretation of the SNP experiments, a 55 detection volume containing sensor response. the gate (100 nM), the TAMRA-labeled substrate In this work, we present methods for the development of a (1.0 ), and input DNA (0.2 ) in reaction buffer was used. deoxyribozyme-based computational biosensor. In Section II, The estimation of substrate turnover assays were conducted in we demonstrate the sensing capability of deoxyribozymes and 55 reaction buffer with the: 1) E6 deoxyribozyme (100 nM) present quantitative methods for predicting the response of and TAMRA-labeled substrate (2.5 )or2) gate the deoxyribozyme to fully complementary and genetically (100 nM), the TAMRA-labeled substrate (1.0 ), and input modified target probes. Results are presented and analyzed in DNA (0.2 ). Section III. We conclude by demonstrating the use of our sensor 1) Equipment, Oligonucleotides, Chemicals: All experi- platform in the de novo design and development of biosensor ments were conducted with the FluoDia T70 Microplate Reader systems for detecting genetic modifications in avian influenza (Photon Technology International, Inc.) using black 384-well followed by a summary discussion. microplates (OptiPlate-384 F, Perkin-Elmer). Oligonucleotides were custom synthesized by Integrated DNA Technologies and purified and reconstituted as follows. II. MATERIALS AND METHODS 1) The E6 deoxyribozyme was HPLC purified and stored as 100 stock solutions in RNase/DNase/Protease-free A. Deoxyribozyme Construction Assays water (Acros Organics) at . E6 sequence [8]: We constructed the (E6) deoxyribozyme gate (Fig. 1; 5-CTCTTCAGCGATGGCGAAGCCCACCCATGTTAG [6]) based upon a previously reported modular design [5] that TGA -3. appended a 15 nucleotide molecular recognition loop to the E6 2) The deoxyribozyme gate was purified by denaturing deoxyribozyme [7]. This one-input gate contains the E6 de- PAGE and stored as 100 stock solutions in oxyribozyme, a ribonucleic acid phosphodiesterase, and a stem- RNase/DNase/Protease-free water or reaction buffer loop structure attached to the 5 end of the enzymatic domain. (HEPES pH 7.0, 1 M NaCl, 2 mM )at . When the structure is in its inactive form, the stem is comple- (E6) gate [8]: 5 -CTGAAGAGTATCCGAT- mentary-bound to the substrate recognition region and inhibits TATAAGCCTCTTC-AGCGATGGCGAAGCCCACCCA hydrolysis of a dual-labeled substrate oligonucleotide. Upon hy- TGTTAGTGA-3 . bridization of a target (input) oligonucleotide complementary 3) The substrate molecule was dual-labeled with