The potential for inferring the presence of cancer by the detection of miRNA in human blood has motivated research into the design and operation of DNA-based chemical amplifiers that can operate in bodily fluids. As a first step toward this goal, we have tested the operation of a DNA-based autocatalytic network in human serum and mouse serum. With the addition of sodium dodecyl sulfate to prevent degradation by nuclease activity, the network was found to operate successfully with both DNA and RNA catalysts

D.Y. Zhang

E. Graugnard

L. He

L. Zhang

P.S. Mitchell

R.W. Carthew

X. Chen

Graugnard, Elton

Cox, Amber

Lee, Jeunghoon

Jorcyk, Cheryl

Yurke, Bernard

Hughes, William L.

Boise State University - ScholarWorks

Boise State UniversityScholarWorksMaterials Science and Engineering FacultyPublications and Presentations Department of Materials Science and Engineering1-21-2011Operation of a DNA-Based Autocatalytic Networkin SerumElton GraugnardBoise State UniversityAmber CoxBoise State UniversityJeunghoon LeeBoise State UniversityCheryl JorcykBoise State UniversityBernard YurkeBoise State UniversitySee next page for additional authorsThis is an author-produced, peer-reviewed version of this article. The final publication is available at www.springerlink.com. Copyright restrictions mayapply. DOI: 10.1007/978-3-642-18305-8AuthorsElton Graugnard, Amber Cox, Jeunghoon Lee, Cheryl Jorcyk, Bernard Yurke, and William L. HughesThis conference proceeding is available at ScholarWorks: http://scholarworks.boisestate.edu/mse_facpubs/19Operation of a DNA-Based AutocatalyticNetwork in SerumElton Graugnard1, Amber Cox1, Jeunghoon Lee2, Cheryl Jorcyk3,Bernard Yurke1,4, and William L. Hughes1,∗1 Materials Science & Engineering,2 Chemistry & Biochemistry,3 Biological Sciences,4 Electrical & Computer Engineering,Boise State University, Boise, ID 83725 USA*willhughes@boisestate.eduAbstract. The potential for inferring the presence of cancer by the de-tection of miRNA in human blood has motivated research into the designand operation of DNA-based chemical amplifiers that can operate in bod-ily fluids. As a first step toward this goal, we have tested the operation ofa DNA-based autocatalytic network in human serum and mouse serum.With the addition of sodium dodecyl sulfate to prevent degradation bynuclease activity, the network was found to operate successfully withboth DNA and RNA catalysts.1 IntroductionWorldwide, approximately 1.3 million deaths per year are caused by lung cancer[1]. Early detection and diagnosis of cancer can lead to decreased mortality rates,yet current screening methods require significant resources [2]. Recently, micro-ribonucleic acids (miRNAs) have been detected in human blood serum [3]. Micro-RNAs are small, single-stranded, non-coding RNAs that are 21-23 nucleotidesin length and regulate genes by suppression of messenger RNAs [4, 5]. SeveralmiRNAs are amplified in various cancers [6], and expression profiling revealsthat miRNA signatures can be used for cancer classification and prognosis.Current diagnosis technology requires reverse-transcription polymerase chainreaction (RT-PCR) to detect miRNAs in serum [7]. Developments in DNA com-puting have shown that it is possible to construct metastable DNA-based chem-ical networks that accept DNA as catalytic inputs and generate output DNAstrands whose concentration increases exponentially to produce an easily de-tectable signal [8]. As miRNAs occur in blood in low abundance, such ampli-fication networks would allow for detection without using PCR. In this study,we report the operation of the DNA-based autocatalytic network reported byZhang et al. in human blood serum with sodium dodecyl sulfate (SDS). The au-tocatalytic DNA system accepts the input of either DNA or RNA catalysts andproduces output signal strands that generate an easily detectable fluorescencesignal.2 E. Graugnard et al.2 Autocatalytic NetworkTo assess the feasibility of detecting miRNA in human serum using a DNA-basedcatalytic network, the entropy-driven autocatalytic system developed by Zhanget al. was selected as a test network. Figure 1(a) reproduces the autocatalyticnetwork with the same domain naming convention [8]. In this network, strand4 2bc is the autocatalyst that initiates signal strand production by toehold-mediated strand invasion of the substrate complex via the 2b domain. Withan exposed 3¯ domain, the fuel strand displaces two autocatalyst strands bytwo strand invasion processes and forms the waste complex. In each cycle, theamount of autocatalyst strand is doubled, leading to exponential growth of thesignal strand. In Fig. 1(b), the released signal strand reacts with a reportercomplex to displace a tetrachlorofluorescein (TET) labeled strand. An increasein signal strand concentration is detected by an increase in the TET fluorescenceintensity. Zhang et al. were able to demonstrate exponential behavior of thisautocatalytic network in buffer and in a solution with total mouse liver RNA andrabbit reticulocyte lysate [8], demonstrating successful operation in a complexbiological environment.AutocatalystWasteSubstrate2bc4a4b 432bcFuel2bc4b4 2bcAutocatalyst4b 432bc2b3 42bc4b2b3 44 2bcIntermediate4 2b2c4b2bc4a63 44b2bc4b2b3 4Signal6 3 4(a)Signal6 3 4 6 3 44a3636ReporterTETFQ(b)4a36FQ36TETFluorescenceFig. 1. The DNA-based autocatalytic network reported by Zhang et al. [8]. In (a)the autocatalyst initiates the release of the signal strand. The fuel strand displacesboth autocatalysts, producing the waste complex. Both released autocatalysts can theninitiate new cycles. In (b) the reporter complex consists of a dye-quencher pair in thequenched state. The signal strand from (a) reacts with the reporter complex, displacingthe TET dye labeled strand and producing an increase in fluorescence intensity.3 Network Operation in SerumTo test the autocatalytic system in human serum, the reported DNA sequencesof the network were purchased without modification from Integrated DNA Tech-nologies with the same purification processes. Substrate and Reporter complexeswere prepared in 1× phosphate buffered saline (PBS) and filtered by polyacry-lamide gel electrophoresis to remove excess single-stranded components. BothAutocatalytic Networks in Serum 3DNA and RNA versions of the Autocatalyst strand were used to initiate thenetwork.Whole blood was collected from volunteers and allowed to clot for 30 minutesat room temperature. The clotted solutions were centrifuged at room tempera-ture for 10 minutes leaving the serum as the supernatant, which was extracted toseparate vials for storage at -80 ◦C. In order to ensure successful operation of theautocatalytic network in serum, the use of the ionic detergent sodium dodecylsulfate (SDS) added to serum was used as a means to suppress nuclease activ-ity without disrupting DNA hybridization. SDS is commonly used to denatureproteins, and with 10% SDS, DNA lifetime and hybridization rates in serum areincreased [9]. Figure 2 shows the results for operating the autocatalytic networkin a solution of 50% human serum, 10% SDS and 0.5×PBS with both DNA,Fig. 2(a), and RNA, Fig. 2(b), catalysts. The data represent the normalized flu-orescence intensity of TET dye integrated over one minute intervals with every75th data point marked with a symbol. For the data shown in Fig. 2(a), theSubstrate and Fuel components of the network were present in solution at con-centrations of 100 nM, while the Reporter was present at 200 nM. The networkwas operated with zero added catalyst and with the DNA catalyst added at 10and 100 nM. The times to half completion for the DNA catalyst were 5.5 min.at 100 nM, 15.6 min. at 10 nM, and 21.7 min. with no catalyst added. For thedata shown in Fig. 2(b), the Substrate and Fuel components of the network werepresent in solution at concentrations of 25 nM, while the Reporter was present at50 nM. The network was operated with zero added catalyst and with the RNAcatalyst added at 2.5 and 25 nM. For the RNA catalyst, the times to half com-pletion were 54.7, 82.1, and 101.3 min. for 25, 2.5, and 0 nM, respectively. Theresulting fluorescence versus time data are in qualitative agreement with theresults reported previously [8], exhibiting a similar concentration dependenceand initial exponential intensity increase. Although the times to half completionfor the DNA catalyst are similar to previous results, it should be noted thathere the concentrations of all components is 10 times greater. The longer timesto half completion for the RNA catalyst are expected for the factor of four re-duction in the component concentrations. It should be noted that operation ofthe autocatalytic network without added catalyst indicates a non-zero leak rateof the system, as observed previously [8]. Methods to reduce this leak rate arecurrently being studied.Initial experiments in detecting cancer-related miRNA will be performed inmouse models of lung cancer. To verify that the autocatalytic network can serveas a test system for the detection of miRNA in mouse models, the network wasoperated in mouse serum. Figure 3 shows the results for autocatalytic networkoperation in a solution of 50% mouse serum, 10% SDS, and 0.5×PBS with RNAcatalyst added at concentrations of 10 and 100 nM, as well as operation withno added catalyst. The measured times to half completion were 13.6, 37.8, and47.4 min. for 100, 10, and 0 nM, respectively. These half completion times areonly slightly longer than those for DNA catalysts in human serum at the same4 E. Graugnard et al.0 20 40 60 80 100 1200.00.20.40.60.81.00 50 100 150 200 250 3000.00.20.40.60.81.0(a)DNA catalyst   100 nM 10 nM 0 nMNormalized Intensity (a.u.)Time (minutes)(b)   25 nM 2.5 nM 0 nMNormalized Intensity (a.u.)Time (minutes)RNA catalystFig. 2. Autocatalytic network operation in 50% human serum, 10% SDS and 0.5×PBSwith (a) DNA and (b) RNA catalysts. Both the DNA and RNA catalysts successfullyinitiated the autocatalytic network. The observed fluorescence increase and catalystconcentration dependence are in qualitative agreement with the results for networkoperation in buffer as reported previously [8].Autocatalytic Networks in Serum 5component concentrations, Fig. 2(a), which suggests that test results from mousemodels should be readily applicable to human systems.0 20 40 60 80 100 1200.00.20.40.60.81.0  100 nM 10 nM 0 nMNormalized Intensity (a.u.)Time (minutes)RNA catalystMouse SerumFig. 3. Autocatalytic network operation in 50% mouse serum, 10% SDS and 0.5×PBSwith 100, 10, and 0 nM of RNA catalysts. Network operation in mouse serum wassuccessful with times to half completion comparable to operation in human serumwith DNA catalysts.4 ConclusionA DNA-based autocatalytic network was successfully operated in 50% humanserum, 10% SDS, 0.5×PBS using both DNA and RNA catalysts. Network oper-ation was also confirmed in mouse serum using an RNA catalyst. Operation inserum with 10% SDS was shown to be sufficient to prevent rapid degradationof the network. Times to half completion were similar to those for operation inbuffer solution, although the strand concentrations were an order of magnitudehigher in the experiments reported here. In all cases, the network exhibited anapparent exponential increase in fluorescence intensity and clear dependence onthe catalyst concentration. These results clearly support the feasibility of de-tecting miRNA in human serum and mouse serum using a DNA-based catalyticnetwork.Acknowledgments. The authors thank David Y. Zhang, Erik Winfree, LuluQian, andWilliam B. Knowlton for valuable assistance with this work. This workwas supported by NIH Grant No. P20 RR016454 from the INBRE Program ofthe National Center for Research Resources, DARPA Contract No. N66001-01-C-80345, and NSF CCF 0855212.6 E. Graugnard et al.References1. World Health Organization, Cancer,http://www.who.int/mediacentre/factsheets/fs297/en/print.html2. American Cancer Society, Cancer Facts and Figures 2009,http://www.cancer.org/docroot/STT/STT 0.asp3. Chen, X., Ba, Y., Ma, L., Cai, X., Yin, Y., Wang, K., Guo, J., Zhang, Y., Chen, J.,Guo, X., Li, Q., Li, X., Wang, W., Zhang, Y., Wang, J., Jiang, X., Xiang, Y., Xu,C., Zheng, P., Zhang, J., Li, R., Zhang, H., Shang, X., Gong, T., Ning, G., Wang,J., Zen, K., Zhang, J., Zhang, C.-Y.: Characterization of microRNAs in serum: anovel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 18,997–1006 (2008)4. He, L., Hannon, G. J.: MicroRNAs: Small RNAs with a big role in gene regulation.Nat. Rev. Genet. 5, 522–531 (2004)5. Carthew, R. W.: Gene regulation by microRNAs. Curr. Opin. Genet. Dev. 16, 203–208 (2006)6. Zhang, L., Volinia, S., Bonome, T., Calin, G. A., Greshock, J., Yang, N., Liu, C.-G., Giannakakis, A., Alexiou, P., Hasegawa, K., Johnstone, C. N., Megraw, M. S.,Adams, S., Lassus, H., Huang, J., Kaur, S., Liang, S., Sethupathy, P., Leminen, A.,Simossis, V. A., Sandaltzopoulos, R., Naomoto, Y., Katsaros, D., Gimotty, P. A.,DeMichele, A., Huang, Q., Bu¨tzow, R., Rustgi, A. K., Weber, B. L., Birrer, M. J.,Hatzigeorgiou, A. G., Croce, C. M., Coukos, G.: Genomic and epigenetic alterationsderegulate microRNA expression in human epithelial ovarian cancer. Proc. Natl.Acad. Sci. USA 105, 7004–7009 (2008)7. Mitchell, P. S., Parkin, R. K., Kroh, E. M., Fritz, B. R., Wyman, S. K., Pogosova-Agadjanyan, E. L., Peterson, A., Noteboom, J., O’Briant, K. C., Allen, A., Lin, D.W., Urban, N., Drescher, C. W., Knudsen, B. S., Stirewalt, D. L., Gentleman, R.,Vessella, R. L., Nelson, P. S., Martin, D. B., Tewari, M.: Circulating microRNAs asstable blood-based markers for cancer detection. Proc. Natl. Acad. Sci. USA 105,10513–10518 (2008)8. Zhang, D. Y., Turberfield, A. J., Yurke, B., Winfree, E.: Engineering entropy-drivenreactions and networks catalyzed by DNA. Science 318, 1121–1125 (2007)9. Graugnard, E., Cox, A., Lee, J., Jorcyk, C., Yurke, B., Hughes, W. L.: Kinetics ofDNA and RNA Hybridization in Serum and Serum-SDS. IEEE Trans. Nanotechnol.(in press)

Operation of a DNA-Based Autocatalytic Network in Serum

Elton Graugnard

Amber Cox

Jeunghoon Lee

Cheryl Jorcyk

Bernard Yurke

William L. Hughes

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http://scholarworks.boisestate.edu/cgi/viewcontent.cgi?article=1018&context=mse_facpubs

Operation of a DNA-Based Autocatalytic Network in Serum

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