Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Project: Difference between revisions
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:''Main article: [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Domain Level Project Design|Domain Level Project Design]]'' | :''Main article: [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Domain Level Project Design|Domain Level Project Design]]'' | ||
Our goal for the summer is to develop a system that autonomously sorts DNA tagged structures. Our base system involves randomly placed DNA tagged cargo on a rectangular DNA origami [7]. One edge of the origami is tagged with goal strands, and the rest of the origami is filled with track strands. The origami is then populated with random walkers that traverse the origami, picking up cargo and dropping them off at the goal. The motion of the walker and cargos will be examined by atomic force microscopy imaging. Bulk behavior of the system, kinetics of walking, and mechanisms of cargo picking up, and cargo dropping off will be analyzed by SPEX experiment. | |||
[[Image: ProjectOverview.jpg | 700px]] | [[Image: ProjectOverview.jpg | 700px]] | ||
Revision as of 20:55, 2 November 2011
Wednesday, April 24, 2024
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Project DesignOverall Project Design
Our goal for the summer is to develop a system that autonomously sorts DNA tagged structures. Our base system involves randomly placed DNA tagged cargo on a rectangular DNA origami [7]. One edge of the origami is tagged with goal strands, and the rest of the origami is filled with track strands. The origami is then populated with random walkers that traverse the origami, picking up cargo and dropping them off at the goal. The motion of the walker and cargos will be examined by atomic force microscopy imaging. Bulk behavior of the system, kinetics of walking, and mechanisms of cargo picking up, and cargo dropping off will be analyzed by SPEX experiment.
Domain Level Design
To accomplish a cargo-reorganizing-task, a walker is extended to have picking up arm which is complementary to cargos (domain x and l in figure 1). When walker randomly walks and encounters a cargo molecule, it picks up the cargo by strand displacement using toehold l. It continues random walking after picking up, and when a walker gets to the cargo goal, cargo is dropped off at the cargo goal using toehold u/u*, which both cargo and cargo goal share. Therefore, random walking process is purely stochastic, yet a deterministic end result can be achieved by specific recognition between the cargo molecules and their destinations. Another important stand is walker goal. Since walker goal contains both a1* and a2* which are complementary to the both of the toeholds of the walker, walker stays on the walker goal when it gets there. Walker goal will be used in verifying random walking on origami, and its use will be explained in later section. While the system is under construction, (e.g. track being planted), a walker or cargo goal should be deactivated to prevent undesired random walking or cargo sorting. Walker inhibitor and cargo goal inhibitor are thus designed. Later, walker trigger and cargo goal trigger will rip off the inhibitors by strand displacement using toehold wi and cgi. Detacher stands were designed to detach particular strands from samples with origami for the future gel experiments. Probes are the extended part of staples which are complementary to the bottom part of the strands which should be anchored on the origami surface. Different kinds of probes were designed for each strand. Origami will be annealed with certain staples extended with probes at predetermined positions, and some strands, such as tracks or cargo goals, will be planted on those specific positions using interaction between probe regions. Overall domain level design is illustrated in figure 1. Following abbreviation will be frequently used: walker [W], walker inhibitor [WI], track 1 [TR1], probe for track 1 [PTR1], track 2 [TR2], probe for track 2 [PTR2], cargo 1 [C1], cargo attacher [CA], probe for cargo attacher [PCA], cargo goal inhibitor [CGI], cargo goal 1 [CG1], probe for cargo goal [PCG], walker goal [WG], and probe for walker goal [PWG]. 1.3 Sequence Design
With our overall design in mind, we must design DNA sequences, down to the base level, which undergo the interactions that we desire, without forming secondary structures and binding in unintended ways. We approach this through a combination of pre-generated noninteracting sequences, and trial-and-error design using NUPACK simulation software. 2. Experimental Design2.1 Verification of Mechanisms through Gel Experiments
Before constructing our origami and observing how it behaves, we run a large number of experiments observable through Gel Electrophoresis to verify that many of our mechanisms behave as we expect them to. 2.2 Verification of Mechanisms through Fluorescent Spectroscopy
Various DNA strands were tagged with fluorophores and quenchers in order to investigate different mechanisms more directly, both in solution and on origami. 2.3 Verification of Mechanisms through Atomic Force Microscopy
Walkers tagged with biotins were planted onto DNA origami, attempts were made to observe random walking on the origami directly under AFM. References[1] Lulu Qian and Erik Winfree. A simple DNA gate motif for synthesizing large-scale circuits. In International Meeting on DNA Computing, 2008. [2] David Soloveichik, Georg Seelig and Erik Winfree. DNA as a Universal Substrate for Chemical Kinetics. DNA 14, LNCS 5347: 57-69, 2009 [3] David Soloveichik, Matthew Cook, Erik Winfree and Jehoshua Bruck. Computation with Finite Stochastic Chemical Reaction Networks. Natural Computing Feburary, 2008. [4] Ye Tian, Yu He, Yi Chen, Peng Yin, and Chengde mao. A DNAzyme That Walks Processively and Autonomously along a One-Dimensional Track. Angewandte Chemie International EditionVol. 44, 4355-4358, 2005 [5] Kyle Lund, Anthony J. Manzo, Nadine Dabby, Nicole Michelotti, Alexander Johnson-Buck, Jeanette Nangreave, Steven Taylor, Renjun Pei, Milan N. Stojanovic, Nils G. Walter, Erik Winfree, and Hao Yan. Molecular Robots Guided by Prescriptive Landscapes. Nature, 206-210, 2010 [6] Hongzhou Gu, Jie Chao, Shou-Jun Xiao, Nadrian C. Seeman. A proximity-based programmable DNA nanoscale assembly line. Nature 465, 202–205. 2010 [7] Paul W. K. Rothemund. Folding DNA to Create Nanoscale Shapes and Patterns. Nature, 297-302, 2006
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